Phd thesis AFJ van Aken
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Phd thesis AFJ van Aken Phd thesis AFJ van Aken Document Transcript

  • Effects of the expression of alternative oxidase on oxidising pathway kinetics in Schizosaccharomyces pombe mitochondria Alexander Frans Johan van Aken Submitted for the degree of Doctor of Philosophy University of Sussex July 2006
  • ii I hereby declare that this thesis has not been and will not be submitted in whole or in part to this or any other University for a degree. Signed……………………………………………
  • iii Acknowledgements I would like to express my gratitude to professor Moore for giving me the opportunity to do a D.Phil. project in his laboratory which has been a bumpy ride at times but was overall a fruitful experience. Although I started working in a different field, having returned to neuroscience I still manage to find the time to further explore my bioenergetic research and being a tutor. I would like to thank the (past) members of the Moore laboratory (Charles, Jane, Paul, Alice, Sarah, Rob, Nick) for their help and support over the years. I would like to particularly thank Dr Mary Albury for her supervision with regards to the yeast expression system. I would also like to thank Dr David Whitehouse for helping me improve my English scientific writing and for having many useful scientific discussions. I would also like to thank professor Derek Lamport for many not so useful scientific discussions. Many thanks also to professor Kros for his scientific support. I would especially like to thank Judita a fellow PhD sufferer over the years for all the support and having spent several years together in suspended animation (hvala lijepa moja učiteljica). Many thanks to my parents and sister for the financial support and for putting up with not seeing me very much over the past four years. Also many thanks to Remon and Angelique for helping out financially at times. Many thanks to all the lovely people I met and am still in contact with after having lived in Kings Road for all the good times and for many visits to Poland, Croatia and Slovakia (Maja, Zuzana, Wojciech, Przemek, Ewa, Basha, Vasek, Marek, Zuzana, Sandra, Tamara, Hidemi, and Gang) hvala, d’akujem and dziękuję.
  • iv Summary The alternative oxidase (AOX) is a non-protonmotive terminal oxidase found in the respiratory chains of higher plants, various fungi and some protists. Its activity results in dissipation of free energy and affects efficiency of energy transduction in mitochondria. A plant AOX has been heterologously expressed previously in Schizosaccharomyces pombe mitochondria in this laboratory. The work presented in this thesis describes the effects of the expression of AOX on the respiratory kinetics of isolated yeast mitochondria. Succinate dehydrogenase (SDH) is the only respiratory complex which is both a component of the electron transfer chain and the citric acid cycle and possible has a strong regulatory role. It is still relatively unknown how this enzyme is regulated exactly. SDH in potato mitochondria can be activated by ADP, ATP and oligomycin. It has been hypothesized that these substances activate SDH indirectly via an effect on the membrane potential. This hypothesis was tested in a series of experiments using a multi- electrode setup. A characterisation of S. pombe mitochondrial respiratory kinetics is given and determinations of the membrane potential are presented for the first time. Oxidising pathway kinetics of S.pombe mitochondria are notably different from what is seen in mitochondria from other tissues. Results indicate that cytochrome bc1 complex activity is probably the underlying mechanism responsible for these kinetics. The expression of AOX in S. pombe mitochondria showed a substrate dependent difference in oxidising pathway kinetics. It was determined that neither cytochrome pathway or alternative pathway activity could account for these differences.
  • v Contents Acknowledgements iii Summary iv Contents v Abbreviations xiii Chapter 1 General Introduction 1.1 General background 1 1.1.1 Mitochondria 1 1.1.2 Energy transducing systems 2 1.2 The electron transfer chain 7 1.2.1 General background 7 1.2.2 Complex I 9 1.2.3 Alternative NAD(P)H dehydrogenases 10 1.2.4 Complex II 11 1.2.5 Ubiquinone / Ubiquinol 15 1.2.6 Complex III 17 1.2.7 Cytochrome c 21 1.2.8 Complex IV 22 1.2.9 Complex V 22 1.2.10 Uncoupling protein 23 1.2.11 Alternative oxidase 24 1.3 Schizosaccharomyces pombe 30 1.3.1 General background 30 1.3.2 The respiratory chain of S. pombe mitochondria 32
  • vi 1.3.3 SDH activation in S. pombe mitochondria 34 1.4 Summary energy transducing systems 35 1.5 A modular representation 36 1.6 Summary 38 1.7 AIMS 38 Chapter 2 Materials and Methods 2.1 Isolation and purification of mitochondria 40 2.1.1 Schizosaccharomyces pombe 40 2.1.1.1 The Expression system 40 2.1.1.2 Yeast transformation 41 2.1.1.3 S. pombe growth 41 2.1.1.4 Isolation of mitochondria from S. pombe cultures 43 2.1.1.5 Spheroplast preparation 43 2.1.1.6 Isolation of mitochondria 43 2.1.1.7 S. pombe media 44 2.1.2 Saccharomyces cerevisiae 46 2.1.3 Potato tuber 46 2.1.3.1 Isolation of mitochondria from potato tubers 47 2.1.3.2 Potato tuber media 47 2.1.4 Arum maculatum 48 2.1.4.1 Isolation of mitochondria from Arum maculatum spadices 48 2.1.4.2 Arum maculatum media 49 2.1.5 Specifics of plant mitochondrial isolation 50 2.2 Polyacrylamide gel electrophoresis & Western analysis 50 2.2.1 SDS-PAGE 50 2.2.2 Blotting to nitrocellulose 50 2.2.3 Immuno-detection of proteins 51 2.3 Protein estimations 51 2.4 Electrochemical techniques 52
  • vii 2.4.1 The oxygen electrode 53 2.4.2 The Q-electrode 54 2.4.3 The TPP+ -electrode 57 2.4.3.1 The TPP+ -electrode setup 59 2.4.3.2 Detection of [TPP+ o] 60 2.4.3.3 Construction of the TPP+ -electrode membrane 60 2.4.3.4 Conditioning of the TPP+ -electrode 61 2.4.3.5 Calibrating the TPP+ -electrode 62 2.4.3.6 TPP+ -electrode correction 63 2.4.3.7 TPP+ -electrode sensitivity 63 2.4.3.8 Durability of TPP+ -electrodes 64 2.4.3.9 TPP+ -electrode response time 64 2.4.4 Respiratory measurements 65 2.4.4.1 Preparation of respiratory effectors 65 2.4.4.2 Nomenclature 66 2.4.4.3 Basic bioenergetic parameters 67 2.4.4.4 Q-pool kinetics 67 2.4.5 Modelling of Q-pool data 68 2.6 Other methods 70 2.6.1 Spectroscopy 70 2.7 Bioinformatic resources 70 Chapter 3 New insights into the regulation of plant succinate dehydrogenase - revisited 3.1 INTRODUCTION 71 3.1.1 General background and aims 71 3.1.2 The membrane potential in mitochondria 73 3.1.3 Regulation of SDH 75 3.2 RESULTS 77
  • viii 3.2.1 General characterization 77 3.2.2 Stimulation of SDH by adenine nucleotides 81 3.2.3 Are the effects of ATP on , Qr/Qt and vO2 simultaneous? 86 3.2.4 Stimulation of SDH by adenine nucleotides in the presence of uncoupler 89 3.2.5 Stimulation of SDH by oligomycin 91 3.2.6 Adenine nucleotides and oligomycin inhibit succinate dependent respiration in potato mitochondria 93 3.3 DISCUSSION 99 Chapter 4 Respiratory characteristics of Schizosaccharomyces pombe mitochondria 4.1 INTRODUCTION 112 4.2 RESULTS 113 4.2.1.1 Respiratory rates with different substrates 113 4.2.2 Schizosaccharomyces pombe - cytochrome pathway kinetics 122 4.2.2.1 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under ADP limited conditions with NADH as a substrate 122 4.2.2.2 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under state 3 conditions with NADH as a substrate 124 4.2.2.3 The relationship between Qr/Qt vs. vO2 under uncoupled conditions with NADH as a substrate 127 4.2.2.4 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 and ∆ vs. vO2 relationships under ADP limited conditions 130 4.2.2.5 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 relationships under state 3 conditions 131 4.2.2.6 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 relationships under uncoupled conditions 132 4.2.3 Schizosaccharomyces pombe - reducing pathway kinetics 134 4.2.3.1 SDH reducing pathway kinetics in sp.011 wt mitochondria under state 2 and uncoupled conditions 134
  • ix 4.2.3.2 External NADH dehydrogenase reducing pathway kinetics in sp.011 wt mitochondria under state 2 and uncoupled conditions 136 4.2.4 Are the biphasic patterns due to cytochrome bc1 complex kinetics? 137 4.2.5 Are biphasic respiratory kinetics a characteristic of yeast mitochondria? 141 4.3 Discussion 142 4.3.1 Respiratory characteristics of S. pombe mitochondria 142 4.3.2 Are the biphasic patterns due to an experimental artefact? 146 4.3.3 Are the biphasic patterns due to cytochrome bc1 complex kinetics? 147 4.3.4 Are biphasic respiratory kinetics a characteristic of yeast mitochondria? 148 Chapter 5 Functional expression of the alternative oxidase in Schizosaccharomyces pombe mitochondria 5.1 INTRODUCTION 150 5.2 RESULTS 153 5.2.1 General characterisation of sp.011 AOX, AOX + T, pREP and wt respiratory kinetics 153 5.2.2 Oxidising pathway kinetics with NADH as substrate 157 5.2.2.1 Comparing sp.011 pREP and wt cytochrome pathway kinetics with NADH as substrate 158 5.2.2.2 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway kinetics with NADH as a substrate 163 5.2.3 Oxidising pathway kinetics with succinate as substrate 170 5.2.3.1 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway kinetics with succinate as substrate 170 5.2.4 Oxidising pathway kinetics in sp.011 AOX mitochondria 176 5.2.4.1 Comparing sp.011 AOX oxidising pathway kinetics with either NADH or succinate as a substrate 176 5.2.5 Cytochrome pathway kinetics in sp.011 AOX+T mitochondria 179 5.2.5.1 Comparing sp.011 AOX + T cytochrome pathway kinetics with either NADH or succinate as a substrate 179 5.2.6 Alternative pathway kinetics in sp.011 AOX mitochondria 181
  • x 5.2.6.1 Comparing sp.011 AOX alternative pathway kinetics with either NADH or succinate as substrate 181 5.2.7 Oxidising pathway kinetics in Arum maculatum mitochondria 183 5.2.7.1 Investigating substrate dependent differences in oxidising pathway kinetics in Arum maculatum mitochondria 183 5.3 DISCUSSION 191 5.3.1 Differences between the various S. pombe mitochondria used 191 5.3.2 Does transformation of S. pombe mitochondria lead to changes in respiratory kinetics? 192 5.3.3 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics with NADH as a substrate 192 5.3.4 Does AOX activity affect  in S. pombe mitochondria? 193 5.3.5 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics with succinate as a substrate 195 5.3.6 Why are the oxidising pathway kinetics obtained in this study different from Affourtit’s study? 196 5.3.7 Are there any substrate dependent differences in sp.011 AOX oxidising pathway kinetics? 196 5.3.8 Are the substrate dependent differences a characteristic of the cytochrome pathway? 197 5.3.9 Are the substrate dependent differences a characteristic of the alternative pathway? 197 5.3.10 Are the substrate dependent differences a characteristic of the expression system used? 198 5.3.11 Can the alternative pathway compete with the cytochrome pathway in S. pombe mitochondria expressing AOX? 198 5.3.12 Does expression of the alternative oxidase lead to a change in Q-pool behaviour? 198 5.4 CONCLUSION 199
  • xi Chapter 6 Modelling of oxidising pathway kinetics in Schizosaccharomyces pombe mitochondria expressing the alternative oxidase 6.1 INTRODUCTION 200 6.1 RESULTS 201 6.1.1 Modelling of sp.011 AOX oxidising pathway kinetics 201 6.1.2 Are the oxidising pathway activities additive? 204 6.1.3 sp.011 AOX mixed titration studies 207 6.1.4 Applying Q-pool kinetics to fit sp.011 AOX mixed substrate oxidising pathway data 212 6.3 DISCUSSION 215 6.4 CONCLUSION 222 Chapter 7 General Discussion 7 General Discussion 223 7.1 Characterisation of the wild type S. pombe mitochondria 224 7.1.1 How does cytochrome bc1 complex activity lead to biphasic cytochrome pathway kinetics in S. pombe mitochondria? 225 7.1.2 Future work suggestions pertaining to the biphasic patterns in S. Pombe cytochrome pathway kinetics 230 7.2 Functional expression of AOX in S. pombe mitochondria yields substrate dependent differences in oxidising pathway kinetics 231 7.2.1 Does AOX activity affect  in S. pombe mitochondria ? 232 7.2.2 Substrate dependent differences in oxidising pathway kinetics of S. pombe mitochondria expressing AOX 232 7.2.3 What causes the substrate dependent differences in oxidising pathway kinetics in S. pombe mitochondria expressing AOX? 233 7.2.4 Are the substrate dependent differences in oxidising pathway kinetics 234
  • xii in S. pombe mitochondria expressing AOX due to dehydrogenase characteristics? 7.2.5 Future work suggestions pertaining to the substrate dependent oxidising pathway kinetics in S. pombe mitochondria expressing AOX 236 7.3 Conclusion 237 Appendix 1 238 Appendix 2A 241 Appendix 2B 242 References 244
  • xiii Abbreviations  H ~  proton electrochemical gradient  membrane potential G Gibbs free energy p protonmotive force AA antimycin A ADH alcohol dehydrogenase ADP adenosine 5'-diphosphate AK adenylate kinase AMP adenosine 5'-monophosphate ANC adenine nucleotide carrier AOX alternative oxidase ATP adenosine 5'-triphosphate BCA bicinchoninic acid BSA bovine serum albumin CAT carboxyatractyloside CCCP carbonyl cyanide m-chlorophenylhydrazone DCCD N,N’-dicyclohexylcarbodiimide DCIP 2,6-dichlorophenolindophenol DNP 2,4-dinitrophenol E° standard redox potential ETC electron transfer chain G6P Glucose-6-Phosphate G6PD Glucose-6-Phosphate dehydrogenase F Faraday constant (9.65104 C mol-1 ) FAD flavin adenine dinucleotide FMN flavin mono-nucleotide ISP iron-sulfur protein IMM inner mitochondrial membrane
  • xiv IMS intermembrane space KCN potassium cyanide NADH nicotinamide adenine dinucleotide, reduced form OAA oxaloacetate OG octyl gallate OMM outer mitochondrial membrane MK menaquinone Pi inorganic phosphate pmf protonmotive force PmitoKATP plant mitochondrial K+ ATP channel PMS phenazine methosulfate Q ubiquinone QH2 ubiquinol Qr/Qt Q-redox poise R gas constant (8.31 J mol-1 K-1 ) RCR respiratory control ratio ROS reactive oxygen species SDH succinate dehydrogenase SHAM salicyl hydroxamic acid SMP submitochondrial particle SQOR succinate:quinone oxidoreductase T absolute temperature (K) TPP+ tetraphenyl phosphonium UCP uncoupling protein vO2 oxygen consumption rate z valence number
  • 1 Chapter 1 General introduction 1.1 General background 1.1.1 Mitochondria—Mitochondria are double walled organelles found in eukaryotic cells (Figure 1.1). The innermost compartment, the matrix, is separated from the intermembrane space (IMS) by the inner mitochondrial membrane (IMM) which is relatively impermeable to ions and large solutes. The outer mitochondrial membrane (OMM) on the other hand is relatively permeable to most solutes with a molecular weight less than 10 kDa [1] and because of this the intermembrane space is assumed to be continuous with the cytosol. The IMM shows numerous invaginations (cristae). Whether or not the cristae are continuous with the intermembrane space is still under investigation [2]. In this study it is assumed that under in vitro conditions (isolated mitochondria in solution) there are only two compartments, the matrix and outside of the matrix. Figure 1.1 A schematic representation of a mitochondrion. (Source: courtesy of Dr. Michael W. Davidson, Florida State University). Mitochondria are typically depicted in this ‘sausage’ form but the shape can vary dramatically depending on tissue and/or developmental state.
  • 2 Mitochondria are known as the powerhouses of cells responsible for the generation of ATP, the cellular energy currency. ATP is used to drive many thermodynamically unfavourable processes in the cell and a continuous supply is needed in order for the cell to survive. For instance, to continuously maintain a resting membrane potential most cells expend as much as 30% of cellular ATP to keep the Na+ /K+ exchanger active, neurons in the central nervous system have to expend as much as 70% [3]. A healthy complement of mitochondria is therefore vital for cellular functioning. The energy released upon hydrolysis of the terminal anhydride bond of the ATP molecule is used to drive the uphill process to which ATP hydrolysis is coupled. Hydrolysis of one mole of ATP under normal cellular conditions, i.e. where the mass action ratio of the ATP synthesis reaction is kept away 10 orders of magnitude from equilibrium, can release 57 kJ of energy. The ratio of ATP to ADP concentration in the cytosol is typically maintained at a value of 1000 [1]. Mitochondria do not just generate ATP at a constant rate, ATP synthesis is tightly regulated and mitochondrial activity is highly flexible depending on energetic conditions. Given that cells rely heavily on the efficient generation of ATP by the mitochondria it is curious that respiratory chains of many organisms contain respiratory complexes which actively reduce this efficiency by dissipating energy. Two of these complexes are the uncoupling protein (UCP) (see section 1.2.10) and the alternative oxidase (AOX) (see section 1.2.11). In order to understand how these complexes can reduce the efficiency with which ATP is synthesized an understanding of energy transducing systems is needed. 1.1.2 Energy transducing systems—Returning to the example of neurons, the brain needs a continuous supply of oxygen and glucose; temporary shortages of either of them (e.g. ischaemia) can lead to disastrous results in which cells may go into apoptosis. Upon restoring the supply of oxygen and glucose things may get even worse as happens under the conditions of reperfusion injury [4] and excitotoxicity [3], during which mitochondrial processes set off a series of unfortunate events which lead to cell death. During oxygen deprivation mitochondria become ‘highly reduced’; when oxygen becomes available again this increased reduction level leads to the formation of reactive oxygen species (ROS) which leads to the breakdown of membranes jeopardizing cellular integrity.
  • 3 In order to understand how the synthesis of ATP, the consumption of glucose and oxygen and the formation of ROS are related a brief description of energy transducing systems will be given. All organisms need a continuous energy supply in order to prevent a state of thermodynamic equilibrium (death). Energy is readily available in the form of electromagnetic rays from the sun for those organisms which can trap this form of energy to subsequently transduce it into another form. Most other organisms derive energy from the breakdown of ‘energy rich’ compounds, such as glucose. The breakdown of glucose under standard conditions yields 2870 kJ mol-1 . Most energy utilising reactions in the cell require between 10 to 50 kJ mol-1 [5] so there is a need to partition the energy released during breakdown of glucose. ATP, releasing 57 kJ mol-1 upon hydrolysis (under cellular conditions) is used predominantly. Some ATP is generated through substrate level phosphorylation (about 5% [6]), in the presence of molecular oxygen however the bulk of cellular ATP is generated via energy transducing reactions. Basically all energy transducing systems operate along the same principles: two proton pumps, located in the same membrane (which is relatively impermeable to protons and other ions) their activities coupled to each other via a proton current. In Figure 1.2 the situation as it occurs in mitochondria is shown schematically. By convention the matrix is considered the N side (N for negative) and the intermembrane space the P side (P for positive). The so called primary pump utilises electrons1 to drive the transport of protons against their concentration gradient from the matrix to the intermembrane space. This creates a protonmotive force (pmf) which is subsequently utilised by the secondary pump to drive the synthesis of ATP via the influx of protons from the intermembrane space to the matrix. 1 It would be more correct to use the term ‘reducing equivalents’ as will be explained further in section 1.2.
  • 4 Figure 1.2 A schematic representation of an energy transducing membrane containing two proton pumps communicating with each other via a proton circuit. N: negative P: positive. The pmf, or p, is a driving force with units of V, which consists of two components: a concentration gradient of protons (pH) and an electrical potential difference (). Displacement of ions across a membrane generates an electrochemical potential which is expressed in kJ mol-1 (units of energy). The change in free energy (G) upon the transport of 1 mol of protons across a membrane (in the absence of a ) is given by the following equation: [1.1] R: gas constant (8.31 J mol-1 K-1 ) T: absolute temperature (K) i: inside o: outside          o i H H RTmolkJG ][ ][ log3.2)1(
  • 5 The free energy change associated with the separation of 1 mol of univalent ions across a membrane (in the absence of a concentration gradient) is given by:  zFmolkJG )1( [1.2] z: valence number (1 in this case) F: Faraday constant (9.65104 C mol-1 ) Protons in the matrix and the intermembrane space normally will be affected by both a concentration gradient and an electrical gradient which gives:            o i H H RTzFmolkJG ][ ][ log3.2)1( [1.3] This Gibbs energy difference is generally referred to as the proton electrochemical gradient:  H ~  And with the definition for pH (pH = - log [H+ ]) the equation can be further simplified: pHRTFmolkJH    3.2)( ~ 1  [1.4] To facilitate comparison with redox potential differences in the electron transfer chain (ETC) Mitchell [1] defined the term protonmotive force (p) which is: FmVp H /)( ~         [1.5]
  • 6 p is expressed in units of V and substituting values for R and T at 25 C the equation simplifies to: pHmVp  59)( [1.6] A good understanding of these basic equations is necessary to appreciate the method with which membrane potentials were determined in this study. This topic will be discussed in detail in chapter 2.
  • 7 1.2 The electron transfer chain 1.2.1 General background—Figure 1.3 shows the ETC as it is organised in the mitochondria of mammals. The various components within the chain are organised according to their redox potentials in order of increasing value. Substrates (e.g. NADH or succinate) can be oxidised at specific locations where they donate reducing equivalents (a reducing equivalent can be defined as 1 mole of hydrogen atoms, one proton and one electron per H atom [6]). The red arrows indicate the transfer of electrons through the chain, which eventually reduce oxygen to water at complex IV. At three sites (complexes I, III and IV) the transfer of electrons is coupled to the translocation of protons from the matrix to the intermembrane space (blue arrows), this generates the aforementioned pmf, the matrix being negative with respect to the IMS. Protons can re-enter the matrix via complex V, a process which is coupled to the synthesis of ATP from ADP and Pi. Another inward pointing arrow indicates the passive leak of protons back into the matrix, it is postulated that protons can traverse the IMM via the junctions between lipid and protein [1]. Apart from leak and ATP synthesis there are many transporters (symporters and antiporters) which utilise the proton electrochemical gradient to drive the translocation of metabolites across the IMM (not shown in the figure). Overall, oxygen is consumed and substrates are oxidised, as a result of this, energy is stored in a pmf, which can be utilised by the ATP synthase to drive the reaction of ATP synthesis, this process is known as oxidative phosphorylation. The components of the mammalian ETC are: complexes I to V, the Q pool and cytochrome c. With respect to the relative abundance of complexes within the ETC the following stoichiometry is currently accepted: complexes I : II : III : IV : cytochrome c : ubiquinone = 1 : 2 : 3 : 7 : 14 : 63 [7]. The plant ETC contains the same components but is more complicated than its mammalian counterpart due to the presence of some extra respiratory proteins, see Figure 1.4. The plant ETC contains several alternative NAD(P)H dehydrogenases, which are non-protonmotive, two of them located on the inner leaflet of the IMM and two on the outer leaflet. Another component is the alternative oxidase, which like complex IV catalyses the reduction of molecular oxygen to water [8].
  • 8 Figure 1.3 Schematic representation of the mammalian ETC. I : complex I (NADH dehydrogenase), II : complex II (succinate dehydrogenase), III: complex III (ubiquinol:cytochrome c oxidoreductase), IV: complex IV (cytochrome c oxidase), V: complex V (ATP synthase), c: cytochrome c, Q: the Q-pool (ubiquinone + ubiquinol). Blue arrows: proton flow. Red arrows: electron flow. Also indicated is the non specific leak of protons across the IMM. The route taken by electrons transferred from QH2 to complex III (and subsequently to cytochrome c to complex IV) is referred to as the cytochrome pathway. Electrons transferred to AOX are said to use the alternative pathway. The main difference between these two pathways is that the alternative pathway is non-protonmotive [8]. Figure 1.4 Schematic representation of the plant ETC which contains several additional components compared to the mammalian system (see Figure 1.3). NDH (non-protonmotive NADH dehydrogenase), AOX (alternative oxidase).
  • 9 A physical description of the components of the ETC will be given in the remainder of this section. The alternative oxidase will be discussed in detail given its importance in this study. Also complexes II and III will be discussed in somewhat more detail because a thorough understanding of the functioning of these respiratory proteins is necessary in order to interpret the acquired experimental results. 1.2.2 Complex I (NADH:quinone oxidoreductase, NADH dehydrogenase): Complex I catalyses the transfer of two electrons to ubiquinone in a reaction coupled to proton translocation across the IMM. Currently the proton translocation stoichiometry is believed to be 4H+ /2e- [1]. Of all the complexes involved in oxidative phosphorylation, complex I is by far the largest. In mammalian mitochondria it consists of 43 subunits with a total molecular weight in the range of 750-1000 kDa. Not all of these subunits are required for electron transfer as it was found that bacteria contain a minimal functional unit of just 14 subunits [1]. Complex I is normally taken to be L-shaped with a hydrophilic and a hydrophobic part. The hydrophilic part contains a flavin mono-nucleotide (FMN) moiety which is reduced by NADH, electrons subsequently are transferred through 8 or 9 iron sulfur clusters (FeS) where a molecule of ubiquinone (Q) accepts the electrons. Complex I is both nuclear (nDNA) and mitochondrial (mtDNA) encoded and is potently inhibited by rotenone, piericidin A [9] and rhein [10]. Recently, complex I defects caused by pathogenic mutations in mtDNA and nDNA have been linked to various neurodegenerative diseases such as Parkinson’s disease [9]. Defective complex I functioning leads to a decreased H+ and a concomitant decrease in ATP production whereas ROS formation is stimulated.
  • 10 1.2.3 Alternative NAD(P)H dehydrogenases [11, 12] : In mammalian mitochondria the only ETC complex able to accept reducing equivalents from NADH is complex I. In the mitochondria of plants and fungi (including S. pombe) one or more alternative NAD(P)H dehydrogenases can be found. Like complex I these dehydrogenases catalyse the transfer of two electrons to ubiquinone, however this reaction is not coupled to proton translocation across the IMM, therefore no energy is conserved. Another difference is the use of a flavin adenine dinucleotide molecule (FAD) as a redox prosthetic group instead of FMN. The external NADH dehydrogenase (Ext. NDH) and the external NAD(P)H dehydrogenase (Ext. N(P)DH) are situated at the outer leaflet of the IMM facing the IMS. The internal NADH dehydrogenase (Int. NDH) and the internal NAD(P)H dehydrogenase (Int. N(P)DH) are situated at the inner leaflet of the IMM facing the matrix. Unlike complex I all the alternative NADH dehydrogenases are believed to be relatively small with only one to four subunits. Complex I inhibitors have no effect on the alternative NAD(P)H dehydrogenases and any inhibitors which do affect these complexes are rare and mostly unspecific. It is hypothesized that alternative NADH dehydrogenases can be employed as a dynamic response to changing metabolic needs. Given their small size they can be made readily available as opposed to complex I which requires 43 subunits to be expressed. Varied expression and activity of the alternative NAD(P)H dehydrogenases and the alternative oxidase provides flexibility in regulating the redox state of cytoplasmic and mitochondrial matrix NAD(P)H pools. Mitochondria of some organisms lack complex I completely (e.g. S. cerevisiae and S. pombe [13]) and they are dependent on alternative NADH dehydrogenases to oxidise matrix generated NADH. External NADH dehydrogenase: The Ext. NDH dependent oxygen uptake can be stimulated by the presence of divalent cations which electrostatically screen negative membrane charges. Also Ext. NDH has a high affinity for calcium binding which is believed to affect the interaction with ubiquinone. Early work done on external NADH oxidation gave ADP/O* values between 1.2 and 1.4 whilst NADH oxidation could be inhibited with antimycin A (AA, complex III inhibitor) and cyanide (complex IV inhibitor). These observations indicate that electrons enter the ETC just before complex III. Its molecular weight is estimated to be 32 kDa. * the amount of ADP molecules converted to ATP molecules per atom of oxygen , see section 2.4.4.3.
  • 11 External NAD(P)H dehydrogenase: The Ext. N(P)DH has similar ADP/O values as the Ext. NDH and it is also inhibited by AA and cyanide indicating a point of entry in the ETC just before complex III. The Ext. NDH and Ext. N(P)DH have different pH profiles. Also Ext. N(P)DH is more calcium dependent than Ext. NDH. A protein doublet with molecular weight 58 kDa, localized to the outer surface of the IMM, was found to oxidize both NADH and NADPH. Internal NADH dehydrogenase: Internal NADH oxidation, in the presence of rotenone, showed ADP/O values of 1.5 in plant mitochondria. It was also found that the Int. NDH had a ten times lower affinity for NADH than complex I. No calcium activation has been found so far. In S. cerevisiae mitochondria a single polypeptide with a weight of 53 kDa was identified as an internal NADH dehydrogenase. Internal NAD(P)H dehydrogenase: Unlike the Int. NDH the Int. N(P)DH is activated by calcium. Apart from NAD(P)H it possibly also oxidizes NADH. Its molecular weight is estimated to be 43 kDa. A complex identified as an NADPH dehydrogenase in one species may be found in another species where it can only oxidise NADH, this illustrates that the alternative NADH dehydrogenases still require a lot of research. 1.2.4 Complex II (succinate dehydrogenase) [14, 15]: Succinate dehydrogenase (SDH) is the only ETC complex which is also a component of the citric acid cycle and fulfils therefore a dual role, being active in both the process of energy transduction and the generation of carbon intermediates for biosynthetic metabolism. SDH is a member of the succinate:quinone oxidoreductases (SQOR, EC 1.3.5.1). SQORs couple the oxidation of succinate to fumarate to the reduction of quinone to quinol [16]: succinate  fumarate + 2H+ + 2e- quinone + 2H+ + 2e-  quinol
  • 12 This oxidoreduction reaction is not coupled to proton translocation therefore complex II does not contribute to the conservation of energy. In vitro, SQORs can catalyse both succinate oxidation and fumarate reduction, be it at different rates. By providing substrate in excess, directionality is achieved under experimental conditions. SQORs consist of four subunits referred to as A, B, C and D, see Figure 1.5. Figure 1.5 Succinate dehydrogenase. The hydrophilic subunits A and B are exposed to the matrix (negative side). The hydrophobic subunits C and D are situated within the IMM. The SDH shown here is a type C SQOR (class 3) which is normally found in eukaryotic mitochondria. Adapted from Figure 2C in [14]. The presence of a single heme group (indicated by the rectangle within subunits C and D) and the presence of two hydrophobic subunits are indicative of an eukaryotic SDH. Other classes show variations in the amount of heme groups and hydrophobic subunits. Another way of classifying SQORs is on the basis of quinone substrate. In this study the respiratory activity of yeast and plant mitochondria was investigated therefore no description of archeal and bacterial SQORs will be given here, for more information on these complexes see Refs 14 and 15. Although most bacteria express SDH of a form different from what is found in eukaryotic species, recently acquired X-ray structures show that SDH in E. coli would be classified equivalent to the mammalian complex [16, 17], see Figure 1.6.
  • 13 Figure 1.6 Three dimensional structure of the E. coli SDH taken from Figure 1C in [17]. Subunits A and B are coloured teal and purple respectively. Subunits C and D are shown in orange and yellow respectively. Prosthetic groups shown are covalently bound FAD (subunit A), [2Fe-2S], [4Fe-4S] and [3Fe-4S] iron-sulfur centers (subunit B). Subunits C and D display bound quinone (black) and heme b556 (magenta). The E. coli SDH is equivalent to the SQOR type normally found in eukaryotic mitochondria. Subunit A (also known as the flavoprotein Fp or CII-1) contains a covalently bound FAD prosthetic group and the dicarboxylate binding site; its molecular weight is 70 kDa. Subunit B (also known as the iron-sulfur protein or CII-2) contains three iron-sulfur clusters, [2Fe- 2S], [4Fe-4S] and [3Fe-4S] (also known as Centers 1-3) and weighs 27 kDa. Subunits C and D (also referred to as anchor proteins or CII-3 and CII-4 respectively) contain the quinone reduction and oxidation sites and one heme group (in eukaryotes), their molecular
  • 14 weights are 15 and 13.5 kDa respectively. Subunits A and B have a high sequence homology amongst species, whereas this is much lower for subunits C and D. All subunits are nuclear encoded making complex II unique in the sense that the other main ETC complexes (I, III, IV and V) are all partially encoded by mitochondrial DNA. Subunits A and B are hydrophilic and extend into the matrix. Subunits C and D are hydrophobic and span the IMM, i.e. parts of subunits C and D are accessible from the cytosolic side. Electron transfer though complex II is linear and experimental data have shown that electron transfer through SDH is not sensitive to uncouplers [17]. Succinate binds to the Fp unit where it subsequently donates two electrons and two protons to the FAD group reducing it to FADH2. From there electrons are transferred to the IP unit where they pass through the three iron-sulfur centres. The electrons end up reducing ubiquinone to ubiquinol where two protons are taken up from the matrix. At present the role of heme in the electron transfer from succinate to ubiquinone is unclear. Inhibitors: Malonate and oxaloacetate (OAA) are potent competitive inhibitors of SDH. Several inhibitors interfere with quinone binding such as 2-thenoyltrifluoroacetone (TTFA) and 3- methyl-carboxin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). These inhibitors do not interfere with the activity of the solubilised enzyme. The site of action is between Center 3 and the Q-pool. Apparently cyanide disrupts Center 3 in SDH [18]. Intracellular oxidation of succinate is inhibited by fluoride [19]. Regulation of SDH: SDH in isolated mitochondria is in a partially deactivated state due to bound OAA [15]. The slowness of the reaction and the high energy of activation (35.6 kcal/mol* ) of SDH was interpreted as a conformational change in the enzyme [20]. SDH can be activated by ATP and ADP [21]. Chapter 3 deals with SDH activation and regulation of SDH is discussed more in-depth in its introduction, see section 3.1.3. * 149 kJ/mol
  • 15 1.2.5 Ubiquinone / Ubiquinol (Q-pool): The electron transfer chain has two mobile pools of electron carriers: the cytochrome c pool (see section 1.2.7) and the Q-pool. The Q-pool consists of both oxidised and reduced forms of ubiquinone. Ubiquinone (Q) undergoes an overall 2H+ + 2e- reduction to form ubiquinol (QH2). Because of its long hydrocarbon side chain both Q and QH2 are highly hydrophobic [1]. Figure 1.7 shows the chemical structure of Q. The side chain can vary in length depending on species, n = 10 in mammalian mitochondria [1], whereas plant mitochondria contain a mixture of ubiquinone molecules with n = 9 and 10 [22]. In yeast mitochondria n = 6 [1]. Figure 1.8 shows the two step reduction of Q. After the first step a highly reactive intermediate (semiquinone) is formed, if allowed to react with molecular oxygen it would lead to ROS formation. Hence the necessity to firmly bind semiquinone during reduction of Q to QH2 as will be discussed in 1.2.6. It is generally assumed that the Q-pool functions as a homogenous pool of electron carriers which forms the linkage between dehydrogenases (complexes I and II and the alternative NADH dehydrogenases) and ubiquinol oxidases (complex III and AOX). In mammalian submitochondrial particles (SMP) the activity of both complex I and II were found to be linearly dependent on the Q redox poise (QH2 / (Q+QH2)). A linear relationship was also found between the dependency of complex III activity on Q redox poise. These linear dependencies from both dehydrogenases and ubiquinol oxidases on the level of Q reduction are commonly referred to as quinone pool behaviour [23, 24]. Figure 1.7 Structure of ubiquinone as adapted from Figure 5.6 in [1]. The length of the side chain (R) can vary, n = 10 in mammalian mitochondria, in plant mitochondria a mixture of n=9 and n=10 is found. n = 6 in yeast mitochondria2 and n = 9 in amoebal mitochondria. 2 This figure does not hold for all yeasts, e.g. Candida utilis mitochondria contain both UQ7 and UQ9 [25].
  • 16 Quinone Semiquinone Quinol Figure 1.8 Two step reaction of quinone reduction to ubiquinol. The homogeneity of the Q-pool was deduced from the observation that antimycin A titrations (which inhibit complex III) do not result in a linear response. Figure 1.9 shows a plot with fictitious data representing two situations: one in which every Q molecule is in a fixed relationship with a bc1 complex () and one in which all Q molecules are free to engage with different bc1 complexes (). In the first situation inhibition titrations with AA would lead to a linear relationship. In the second situation Q molecules can still donate electrons to uninhibited bc1 complexes and overall electron flux, expressed as respiratory activity is seen to be ‘antimycin resistant’. The antimycin resistant respiratory kinetics are normally seen in mitochondria [24, 26]. In an article on pool behaviour of Q (and cytochrome c) in S. cerevisiae mitochondria Boumans et al. [27] reported non-homogenous pool behaviour and a linear relationship between respiratory activity and complex III inhibition was found; from this it was concluded that in yeast mitochondria the respiratory components of the ETC are arranged as an ordered macromolecular assembly which does not allow for diffusion based collisions between components. Another study done in the same year by Rigoulet et al. [28] showed that S. cerevisiae mitochondria do show homogenous pool behaviour (cf. Figure 3B in [28]). Several studies showed that AOX (from either Candida albicans or Lycopersicon esculentum (tomato)) expressed in S. cerevisiae mitochondria [29] and [30] respectively, could utilise QH2 as a substrate, which implies Q-pool behaviour in S. cerevisiae. e- e- + 2H+
  • 17 Figure 1.9 Theoretical antimycin A titration data points illustrating homogenous () and non-homogenous Q pool () behaviour in mitochondria, see text for details. 1.2.6 Complex III (ubiquinol:cytochrome c oxidoreductase, bc1 complex): The bc1 complex is a protonmotive homodimer catalysing the oxidation of ubiquinol to the reduction of cytochrome c. In bovine heart and S. pombe mitochondria each monomer consists of 11 subunits of which 8 do not have a catalytic role in the oxidation of ubiquinol [31]. Complex III in S. cerevisiae mitochondria contains 10 subunits. A presequence targeting the Rieske protein is cleaved from the protein; in bovine heart and S. pombe mitochondria this cleaved presequence is retained as a subunit whereas in S. cerevisiae [31] and in potato [32] it is degraded. The redox groups consist of a 2Fe/2S centre which is located on the iron-sulfur protein (ISP), two B-type heme groups (bL and bH) located on a single polypeptide and the heme of cytochrome c1 [1] (see Figure 1.10). In many bacteria a functionally similar but structurally simpler version of the bc1 complex is found in the plasma membrane. These complexes have the same electron transfer and proton translocation functionality as their mitochondrial counterparts. Paracoccus for instance
  • 18 only has a basic three subunit enzyme similar to the protein complex in Figure 1.10. This indicates that the supernumerary subunits are not required for electron transfer or proton translocation [31]. Crystal structures of the bc1 complex have become available in recent years, see Figure 1.11 for the S. cerevisiae bc1 complex structure. Figure 1.10 Schematic representation of the bc1 complex. Only the subunits containing redox groups are shown. The iron-sulfur protein (ISP) also referred to as the Rieske protein. The b-type hemes containing polypeptide and the cytochrome c1 subunit. The midpoint potentials at pH 7 for the redox centres in the yeast bc1 complex are: ISP +280 mV, cytochrome c1 +240 mV, bL –30 mV and bH +120 mV [33]. In order to understand the pathway of electron flow through the bc1 complex an understanding of the Q-cycle [32] is needed, see Figure 1.12. In a complete turnover of the Q-cycle two molecules of ubiquinol are oxidised, one molecule of ubiquinone is reduced, 2 protons are taken up from the matrix, 4 protons are released in the IMS and two cytochrome c1 groups are reduced [1]. The Q-pool in the IMM exists in large molar excess over the bc1 complexes with a ratio of 21:1 [7]. In stage 1 a molecule of ubiquinol diffuses to the binding site Qp (p for positive as it is situated near the positive site of the IMM) where it is oxidised in several stages: One electron is transferred to the ISP, two protons are released to the cytosol and a semiquinone molecule (see Figure 1.7) remains temporarily bound at Qp. The second electron is transferred to bL. The electron transferred to the ISP passes down the ETC to ISP cyt c1 bL bH IMS matrix
  • 19 cytochrome c1, cytochrome c and cytochrome c oxidase. The electron on bL passes onto bH. This electron is used to reduce a molecule of ubiquinone, at another binding site Qn, to semiquinone which remains bound there until a next molecule of ubiquinol comes along in the second part of the cycle. Figure 1.11 Structure of the S. cerevisiae bc1 complex taken from Figure 1A in [34]. The bc1 complex shown in its homodimeric form. Cytochrome c1 is shown in red, the Rieske protein in green, cytochrome b in blue, the hinge domain in cyan. Antibodies binding to the bc1 complex are shown in orange. Cytochrome c bound to one monomer is shown in yellow. All redox prosthetic groups are shown in black. IMS: intermembrane space. IM: inner membrane. MA: matrix.
  • 20 Figure 1.12 The Q-cycle in mitochondria, adapted from Figure 5.14 in [1]. P: positive N: negative See text for explanation.
  • 21 When a second molecule of ubiquinol binds to Qp some of the steps in stage 1 are repeated. One electron is transferred to ISP, again 2 protons are released into the cytosol. One electron is transferred to bL. The electron on ISP is passed down the ETC to cytochrome c1, cytochrome c and cytochrome c oxidase. The electron on bL is transferred to bH. The semiquinone molecule still bound at Qn is reduced by bH and to complete the full reduction of semiquinone to ubiquinol 2 protons are taken up from the matrix. This completes the Q- cycle [1]. Inhibitors: Figure 1.12 shows two inhibition sites in the bc1 complex indicated as red bars in stage 1. Myxothiazol blocks events at Qp and stigmatellin inhibits electron transfer to the Rieske protein. Antimycin A acts at Qn, preventing reduction of ubiquinone by bH [1]. 1.2.7 Cytochrome c: Cytochrome c, a mobile redox carrier, is a peripheral protein located on the P-face of the IMM which transfers electrons between complex III and IV and can be readily solubilised from intact mitochondria. Electrons can be donated artificially from molecules such as tetramethyl-p-phenylene diamine (TMPD) and electrons can leave the ETC via cytochrome c through reduction of ferricyanide (Fe(CN)6 3- ) [1]. Cytochrome c is nuclear encoded and has a molecular weight of about 13 kDa [35]. Apart from being an electron carrier, cytochrome c plays a role in the process of apoptosis. Upon induction of cell death cytochrome c leaves the confinement of the IMM and diffuses to the cytosol where it initiates the activation of caspases, a family of cysteine proteases [35].
  • 22 1.2.8 Complex IV (cytochrome c oxidase): Cytochrome c oxidase in mitochondria consists of up to 13 subunits in mammalian mitochondria (11 in yeast) [36] of which only two (subunits I and II) are involved in electron transfer and proton translocation. Complex IV is present in the IMM as a homodimer. The complex catalyses the complete reduction of oxygen to water and pumps protons across the IMM with a stoichiometry of 2H+ /2e- . Four electrons are transferred sequentially from the cytochrome c pool to complex IV. Subunit II contains a copper centre (CuA) which has two copper ions in a cluster with sulfur atoms. This complex accepts electrons from cytochrome c one at a time. Subunit I contains two heme groups (heme a and heme a3) and another copper centre (CuB). Electrons from CuA are transferred to heme a onto heme a3 and finally onto CuB where oxygen is reduced to water. Heme a3 is also the binding site for several complex IV inhibitors: cyanide, azide, nitric oxide and carbon monoxide [1]. A regulatory effect of adenine nucleotides on complex IV is well known, addition of ATP to yeast mitochondria leads to an increase in enzymatic capacity of cytochrome c oxidase but does not stimulate respiration rate [36]. 1.2.9 Complex V (ATP synthase, F1.Fo-ATPase): Complex V is a proton pump which couples the hydrolysis of one molecule of ATP to ADP and Pi to the translocation of three protons across the IMM. The name F1.Fo-ATPase indicates the two mayor components of this complex, the hydrophobic Fo complex (160kDa) which translocates protons and the F1 complex (370 kDa) which contains the catalytic and regulatory sites. The Fo complex is located in the IMM whereas the F1 part extends into the matrix. The electrochemical gradient of protons generated through ETC activity can be used to drive the synthesis of ATP and the ATP synthase is seen to operate in reverse. The influx of protons drives the thermodynamically
  • 23 unfavourable reaction of ATP synthesis [1] and complex V can be considered a p consumer [37]. The Fo and F1 complexes can be targeted directly by specific inhibitors. Oligomycin (hence the o in Fo) and venturicidin bind at the Fo complex. Aurovertin and efrapeptin bind at the F1 complex. Dicyclohexylcarbodiimide (DCCD) has inhibitory effects on both complexes [1]. The ATP synthase is regulated by the natural inhibitor protein IF1 which binds to a - subunit from the F1 complex [38]. The binding interferes with the cooperative ATP synthesis process of complex V. Binding of the protein inhibits hydrolytic activity and it is suggested that it can also inhibit ATP synthesis [39, 40]. High p induces release of IF1 [40] and the off-rate (rate of release) is high. High concentration of ATP induces binding of IF1 and the on-rate (rate of binding) is high under these conditions [39]. The equilibrium between the on-rate and off-rate determines the steady state inhibition of the ATP synthase by IF1. The IF1 protein was found in mammalian [39, 40], plant [41] and yeast mitochondria [42]. Inhibitory proteins can cross-react with mitochondria from other sources [38, 42]. 1.2.10 Uncoupling protein: The uncoupling proteins (UCP) are a subfamily of the mitochondrial carriers (MC) [43] of which 5 types have been identified so far: UCP1-5. The first UCP to be found was UCP1 in brown adipose tissue where it functions to drive non-shivering thermogenesis in hibernators, cold-adapted rodents and newborn mammals [1]. The activity of the protein is stimulated by fatty acids and inhibited by nucleotides. By catalysing proton transport into the matrix it dissipates the p by increasing the proton conductivity across the IMM [43]. The p dissipation would lead to an increase in body temperature, another beneficial purpose of stimulating UCPs would be to control ROS production [43, 44].
  • 24 1.2.11 Alternative oxidase (AOX): General background: Plant mitochondria exhibit cyanide resistant respiration to various degrees, potato tuber mitochondria show only a little resistance to inhibition with cyanide whereas mitochondria isolated from aroid spadix tissues seem to be fully resistant [8]. This cyanide resistant respiration is associated with the presence of an extra terminal oxidase (apart from complex IV) which functions as an ubiquinol:oxygen oxidoreductase and is referred to as the alternative oxidase (AOX). It catalyses the complete reduction of oxygen to water [45]. Although generally referred to as cyanide resistant, AOX is insensitive to cytochrome pathway inhibitors in general (e.g. antimycin A, azide and carbon monoxide). AOX is sensitive to hydroxamic acid derivatives (such as salicylhydroxamic acid (SHAM) which interferes with Q-binding [46]) and alkyl gallates (such as octyl gallate and dodecyl gallate [47]). AOX accepts electrons from the Q-pool, bypassing the cytochrome pathway. Given the non-protonmotive nature of AOX no energy is conserved during this step which is reflected in a decreased ADP/O ratio. In a situation where reducing equivalents are donated to either complex II (or any of the alternative NAD(P)H dehydrogenases) and if the alternative pathway is the only available oxidising pathway, all energy freed by the oxidation reactions is dissipated as heat. Apart from higher plants, AOX is found in several other species which have branched respiratory pathways, such as various fungi (e.g. Neurospora crassa and Pichia anomala [48]) and protists (e.g. Trypanosoma brucei and Chlamydomonas reinhardtii [48]). Quite recently (2004) the occurrence of AOX encoding genes was found to extend into the animal kingdom as well. Sequences coding for AOX were found in the genomes of a mollusc (Crassostrea gigas), a nematode (Meloidogyne hapla) and in chordates (Ciona intestinalis and Ciona savignyi) [49]. The belief that AOX only occurs in eukaryotes has been challenged recently by reports on the occurrence of AOX in prokaryotes such as Novosphingobium aromaticivorans [50]. A recent search in a metagenomic dataset from
  • 25 marine microbes in the Sargasso Sea uncovered 69 different AOX genes [51] which indicates that AOX may be widespread in aquatic environments. Cyanide resistant respiration in higher plants has been reported since the early 1900’s [52] but only with the advent of antibodies raised against the partially purified alternative oxidase from Sauromatum guttatum [53] was it demonstrated positively that AOX was a genuine component of the ETC of cyanide-resistant mitochondria. The plant alternative oxidase is nuclear encoded and the first identified gene was named aox1 [54], importantly this gene encodes for a protein which includes a pre-sequence targeting it to mitochondria with an approximate weight of 39 kDa. Cleaving the target sequence yields a protein of approximately 32 kDa [55]. Several other genes (aox2a, aox2b) have been identified since [56]. In plants, AOX is found as a homodimeric enzyme whereas in fungi it is a monomer [57]. Apart from a structural difference both types of AOX are also very different with respect to activation mechanisms [58]. In this study a plant alternative oxidase from S. guttatum was heterologously expressed in S. pombe mitochondria [59], therefore the remainder of this section will focus on plant AOX and only significant differences between plant AOX and non-plant AOX will be discussed. Contrasting values for the plant AOX apparent KM for oxygen have been reported from ~1.7 M to 10-20 M [26]. In any case plant AOX affinity for oxygen is lower than that of complex IV (0.14 M [60]). The partitioning of reducing equivalents between the alternative and the cytochrome pathway will be discussed in-depth in chapter 5. Structure of AOX: Several models of the AOX structure have been proposed over the years [61]. In this section only the latest consensus model will be discussed. The current consensus model is the Andersson Nordlund (AN) model [62]. The alternative oxidase is believed to be an interfacial di-iron carboxylate protein [63] attached to the inner leaflet of the IMM facing the matrix space, see Figure 1.13. Getting structural information has been notoriously difficult and continuous efforts at spectroscopic detection were not successful until Berthold et al. in 2002 managed to get an EPR signal from a membrane fraction of E. coli expressing the Arabidopsis thaliana AOX
  • 26 [52]. Their results were the first experimental evidence supporting the hypothesis that AOX is indeed a member of the di-iron carboxylate proteins [64], a group of nonheme iron proteins that contain a coupled binuclear iron center. Figure 1.13 Structure of the plant AOX according to the AN model, adapted from Figure 1 in [63]. Regulation of AOX: Regulation of the alternative oxidase can be divided into two categories, regulation through expression and post-translational regulation. Regulation of the plant AOX is quite different from its counterpart in fungi. For this study a plant alternative oxidase (S. guttatum) was expressed in S. pombe, therefore in this section emphasis will be on plant AOX regulation. Regulation through expression: AOX expression can be increased in many ways. Stress conditions such as chilling, wounding, injury and osmotic stress are all known to increase expression [65]. In many fruits alternative pathway activity is known to increase during the ripening process. In
  • 27 mango fruit alternative pathway activity, amounts of protein and mRNA levels all increase in parallel [66]. In Hansenula anomala (now Pichia anomala) incubation with cytochrome pathway inhibitors, such as antimycin A or KCN, led to increased transcription of AOX, similar behaviour was seen in tobacco cells [66]. How inhibition of the cytochrome pathway is perceived by and transmitted to the nucleus to activate AOX expression is unclear. One suggested mechanism is via generation of ROS. In tobacco suspension cells it was found that upon addition of H2O2 within the span of two hours the level of AOX mRNA was increased [67]. In S. guttatum it was found that application of salicylic acid led to an increase in AOX mRNA levels [68]. AOX expression is also shown to be developmentally regulated [69]. Post-translational regulation: As mentioned previously, the plant AOX is a homodimer whereas the fungal AOX is a monomer. The plant AOX is subject to two interrelated post-translational mechanisms of regulation. In plants the alternative oxidase can be in an oxidised or a reduced form [61]. Plant AOX can be activated by reduction of a dimer-forming disulphide bridge. The reduced (active) form is a non-covalently linked dimer whereas the oxidised (inactive) form is covalently linked [70]. In transgenic tobacco plants expressing AOX it was found that certain TCA intermediates (citrate, isocitrate and malate) could reduce AOX [71]. It was hypothesized that the aforementioned intermediates may be involved in NADP reduction in plants and that NADPH mediates reduction of plant AOX in vivo. This could be a means of regulating AOX in response to changing matrix reduction levels. The second mechanism is direct activation of AOX by certain organic acids. A study by Millar et al. [72] showed that the plant AOX can be activated by a range of organic acids, most of them -keto acids: pyruvate, hydroxypyruvate, glyoxylate, -ketoglutarate, oxaloacetate, L-malate and succinate. It was determined that from these acids L-malate and succinate did not activate AOX directly. It was found that in the absence of malic enzyme (in SMP’s) succinate and malate could no longer activate AOX, which implies that it is in fact generation of pyruvate via malic enzyme which causes activation. It was concluded that this type of plant AOX activation is restricted to -keto acids. It was also determined that pyruvate activation is not
  • 28 dependent on pyruvate metabolism which implies that pyruvate has a direct effect on AOX [66]. As opposed to activation of AOX via pyruvate formation an alternative mechanism was hypothesised by Wagner et al. [73] where addition of succinate or malate led to changes in membrane fluidity which could facilitate the diffusion of QH2 from dehydrogenase to AOX. A substrate dependent difference in AOX activity is commonly seen where succinate dependent cyanide resistant respiratory rates are higher than NADH ones [8, 73-76]. This could be explained by a change in membrane fluidity, however it is more generally accepted that the higher cyanide resistant respiration rate with succinate is due to production of endogenous pyruvate [77]. The activating effect of pyruvate on AOX initially was assumed to change the affinity of AOX for QH2. In the absence of pyruvate, plant AOX is known to activate only at relatively high levels of Q-reduction (between 35- 50% reduced) [78, 79]. Addition of pyruvate was seen to reduce the threshold level of Q- pool reduction at which AOX becomes engaged [80] whilst pyruvate showed no effect on the redox status of the AOX protein disulfide bond. The two mechanisms are interrelated because pyruvate can only significantly activate AOX when the dimer is in the reduced form [80]. Conversely, if pyruvate is present, significant AOX activation can only be achieved when the dimer is reduced [81]. Interestingly enough many studies indicate that pyruvate can activate AOX activity with succinate as a substrate [79, 80] although it has been reported that succinate itself can activate AOX. This suggests that the amount of pyruvate generated indirectly from succinate is not sufficient to fully activate AOX and that further addition of pyruvate is required to fully activate AOX. It was concluded from experiments in which malic enzyme was inhibited that differences in the generation of intramitochondrial pyruvate can explain differences in AOX activity between tissues and substrates [77]. A pH effect on AOX mediated respiration is seen in certain plant species [82], for instance with external NADH as substrate S. guttatum mitochondria displayed a pH optimum for cyanide-resistant respiration [83]. Activation of fungal and protist AOX is quite different from their plant counterpart. AOX in fungi and protists are generally found as monomers and are not subject to organic acid stimulation [61]. Interestingly, in recent work where the protist AOX from Ciona
  • 29 intestinalis was expressed in human cells, pyruvate was found to activate the AOX protein [84]. Purine nucleotides, such as AMP, GMP and IMP are reported to have an activating effect on fungal and protist AOX [58]. Also an effect of pH on AOX activation in Acanthamoeba castellanii mitochondria was found [82]. Despite differences between plant and non-plant AOX at the level of regulation, monoclonal antibodies raised against Sauromatum guttatum AOX cross-react with fungal and protist AOX proteins Other factors regulating AOX are the amount of ubiquinone present [85], the Q-pool redox poise [86] and the amount of AOX protein present [85, 87]. AOX Function: The only function of AOX which has been commonly accepted is that of heat generation in order to volatilise odiferous compounds in order to attract insects during pollination in thermogenic plants [88]. Its function in non-thermogenic plants, let alone in non-plant species to date is still a matter of debate. Several, not mutually exclusive, hypotheses have been proposed. Continued turnover of the TCA cycle during any condition that inhibits or decreases the activity of the cytochrome pathway, such as under ADP limited conditions or during stress (wounding, chilling, drought etc.) will allow continuous production of biosynthetic precursors [89]. Another possible function of AOX is to scavenge harmful ROS produced under conditions (limited ADP, stress conditions) where components within the respiratory chain become highly reduced [65]; by keeping the ETC relatively oxidised AOX activity could prevent synthesis of ROS. Both hypotheses have in common that inhibition of the cytochrome pathway leads to activation of AOX. A more recent hypothesis suggests that AOX activity in plants serves as a means to keep plant growth relatively stable under variable environmental conditions [90]. AOX in relationship to UCP: Both AOX and UCP dissipate free energy as heat. It is interesting to note that some organisms express both enzymes in their mitochondria [91, 92]. Affourtit et al. raise the question as to what the physiological need could be for having two energy dissipating
  • 30 enzymes in one system [37]. It has been demonstrated in Acanthamoeba castellanii mitochondria that combined activity of both AOX and UCP leads to stronger reduction in ROS formation than with either of the complexes being active alone [91]. Furthermore it has been shown in plant mitochondria that activity of UCP appears to be coordinated with AOX activity [92]. These observations suggest that when both energy dissipating mechanisms are present in the same system their coordinated activities are involved in reducing ROS concentration. 1.3 Schizosaccharomyces pombe 1.3.1 General background: In this study S. pombe mitochondria were used as a model system to heterologously express a plant alternative oxidase [59] in order to investigate its respiratory characteristics within the respiratory chain. The same system has been used previously in our laboratory to investigate structure-function relationships [26, 45, 46, 59, 63, 70, 93, 94]. Yeast systems are a useful tool to investigate protein characteristics, it is relatively easy to express a foreign gene and within a short time a large amount of the protein of interest can be harvested. The yeast Schizosaccharomyces pombe also referred to as ‘the other yeast’ [95] is increasingly the preferred model system to investigate a wide range of processes such as the cell cycle [96], DNA repair [97], microtubule formation, meiotic differentiation, cellular morphogenesis and stress response mechanisms [98] over the traditionally used Saccharomyces cerevisiae (which recently has also been used to heterologously express a plant alternative oxidase [30]). S. pombe divides by fission and is one of the few free-living eukaryotic species whose genome has been completely sequenced [99] at the time of writing. The S. pombe genome is haploid and contains three chromosomes 13.8 Mb in size [98]. The mitochondrial genome is 20 kb in size [99]. It has been reported that S. pombe resembles mammalian cells more closely than does S. cerevisiae [100], for instance, S. pombe recognizes various mammalian promoters, splices mammalian introns and shares the same polyadenylation signals with mammalian cells, unlike S. cerevisiae [101]. It is also reported
  • 31 that S. pombe genes have longer upstream regions on average than those of S. cerevisiae which may mean that they are more complex and possibly more like those of higher eukaryotes [98]. S. pombe has proportionally more genes conserved in metazoans than does S. cerevisiae which is another argument in favour of the claim that S. pombe as an organism is more closely related to higher eukaryotes than S. cerevisiae. On the other hand each yeast shares genes with metazoans which the other lacks [102]. Furthermore a significant number of chromosome associated proteins are absent in S. cerevisiae but shared between S. pombe and metazoans making S. pombe the preferred system to study chromosome dynamics [102]. In 2002 a total of 172 S. pombe proteins were found to have similarities to human disease proteins whereas 182 such proteins were identified in S. cerevisiae. Most of the genes coding for these proteins are shared between the two yeasts [99]. Therefore both yeasts appear to be similarly useful as model organisms for the study of human disease gene function although given their different biologies one organism could be preferred for certain genes over the other and vice versa. Although S. pombe has been extensively used to investigate the cell cycle and genome repair mechanisms, in comparison to S. cerevisiae, relatively little work has been done on S. pombe metabolism [103] and even less has been done on the respiratory characteristics of its mitochondria [104, 105]. Recently however S. pombe mitochondria have been used to investigate several bioenergetic processes. S. pombe is the preferred system to investigate F1-ATPase (complex V) catalysis. The F1 part of complex V has several nuclear encoded subunits (the  and  units). S. cerevisiae cannot produce mutants of these subunits, leading to the production of “petite” colonies, i.e. cells with impeded oxidative phosphorylation. Hence F1 mutants cannot be studied in S. cerevisiae [106]. It has been a long held belief that S. pombe does not express a mitochondrial alcohol dehydrogenase (ADH), recent work done in this laboratory however indicates otherwise [26]. Mitochondrial ADH couples the oxidation of ethanol to the reduction of endogenous NAD+ to NADH, which can subsequently be oxidised by an internal NADH dehydrogenase, as happens in S. cerevisiae [107]. The presence of a gene encoding for ADH in S. pombe was confirmed as far back as 1983 [108], it was concluded that the protein was a cytosolic one [26]. The current hypothesis in this laboratory is that having both a cytosolic and a mitochondrial ADH can function as a means to equilibrate
  • 32 NAD+ /NADH ratios on both sides of the IMM in a way similar to the situation in S. cerevisiae [26]. The S. pombe gene SPAC5H10.06c was identified as a likely candidate encoding the mitochondrial ADH isozyme. Because of a recent discovery of a homolog of this protein in human liver [109] the discovery of a S. pombe mitochondrial ADH may have potential medical implications [26]. Another typical protein involved in bioenergetic processes is the adenylate kinase (AK) which catalyses the reaction: ATP + AMP  2 ADP [110]. In potato mitochondria its activity was shown to be responsible for the relatively high respiratory rate under ADP limited conditions [110]. Work done in this laboratory showed that continuous regeneration of ADP (from either endogenous or added nucleotides) led to constant activity of the ATP synthase affecting both membrane potential and oxygen consumption rate [38, 110, 111]. A gene coding for AK in S. pombe has been identified [112] and results suggested that the enzyme was found both in the cytosol and in the mitochondria. 1.3.2 The respiratory chain of S. pombe mitochondria: The respiratory chain of S. pombe mitochondria is rather similar to the mammalian one, see Figure 1.14: Figure 1.14 Schematic representation of the S. pombe ETC. See legends of figures 1.3 and 1.4.
  • 33 The S. pombe ETC, just like S. cerevisiae [27] does not contain complex I [13], therefore the only means of generating a pmf is via the cytochrome pathway. Work done in this laboratory [104] showed that isolated S. pombe mitochondria could respire on either succinate or NADH (in a rotenone-insensitive manner) indicating the presence of complex II and an external NADH dehydrogenase (which is nuclear encoded [113]) respectively. The aforementioned findings on the S. pombe mitochondrial ADH indicate the presence of an internal NADH dehydrogenase [26]. To the best of our knowledge the membrane potential across the IMM in isolated S. pombe mitochondria had not been measured prior to this study, but results by Moore et al. showed the occurrence of protein import into the matrix. This process could be abolished by addition of valinomycin which confirmed the presence of a membrane potential [104]. Respiration in S. pombe mitochondria can be completely inhibited by cytochrome pathway inhibitors which indicates the absence of an alternative oxidase3 [26, 104]. Unlike the yeast Hansenula anomala (now Pichia anomala) [116] AOX expression cannot be induced in S. pombe by incubation of the cells with antimycin A [59]. It has been proposed that cyanide resistant respiration (due to the presence of AOX) is found only in non-fermentative and Crabtree-negative yeasts (capable of fermentation but not under aerobic conditions) [117]. It was found, in general, that yeasts which do not display cyanide resistant respiration also do not express complex I in their ETC [118]. Non-fermentative yeasts under conditions where the cytochrome pathway is inhibited can still generate an electrochemical gradient of protons via complex I using the alternative oxidase as a terminal oxidase. The pmf generated could be utilised by complex V to drive the synthesis of ATP. It was hypothesized by Veiga et al. [117] that non-fermentative yeasts express both complex I and AOX as an alternative to cytochrome pathway respiration, whereas yeasts such as S. cerevisiae and S. pombe (which do not express complex I [13]) use fermentation as an alternative to cytochrome pathway respiration. In section 1.2.5 it was mentioned that in S. cerevisiae mitochondria the Q-pool displayed non-pool behaviour [27]. Given the similarities between the make up of the 3 Within older literature [114] but also in recent textbooks [115] (page 213) S. pombe mitochondria are reported to display cyanide resistant respiration, which is incorrect.
  • 34 respiratory chains of both yeasts this had implications for S. pombe. In this laboratory experiments were done, employing the same techniques which were used in the S. cerevisiae study and it was found that in S. pombe mitochondria the Q-pool does show pool behaviour [26]. Also, it was found previously that S. pombe mitochondria display antimycin resistant respiratory kinetics (see section 1.2.5) during NADH dependent respiration, cf. Figure 2A in [119]. It was reported that in addition to the respiratory components described so far S. pombe contains genes for several other respiratory linked proteins, namely: Gut2 encoding a glycerol-3-phosphate dehydrogenase, hmt2 encoding a sulphide dehydrogenase and ura3 encoding a dihydroorotate dehydrogenase. All three proteins can donate electrons to the Q- pool [26]. Upon performing a BLAST search4 (Basic Local Alignment Search Tool [120]) through the S. pombe genome another gene coding for a respiratory linked protein was found. SPAC20G8.04c codes for an electron transfer flavoprotein-ubiquinone oxidoreductase (ETF) which is a water-soluble matrix based complex that contains a FAD moiety and can accept electrons from several dehydrogenases containing flavin [1]. In this study S. pombe is used to functionally express AOX [59]. The alternative oxidase is non-protonmotive and its activity dissipates free energy. It was described in section 1.2.11 that AOX and UCP have the capacity to act in synergism. The presence of an uncoupling protein in yeast has been demonstrated [121]. It is therefore relevant to know whether or not S. pombe expresses an uncoupling protein. It was found by Stuart et al. [122] that the S. pombe genome did not contain any UCP homologs5 . However, at the time of that study (1999) the S. pombe genome was only partially sequenced (55%). At present the whole S. pombe genome is known [99] and a recent BLAST search did not reveal any UCP homologs. 1.3.3 SDH activation in S. pombe mitochondria: Comparable to plant mitochondria, activation of SDH in yeast requires the addition of ATP [123]. It is known that the mechanism of ATP activation is not due to unbinding of OAA 4 http://www.genedb.org/genedb/pombe/index.jsp 5 The same study also showed that the S. cerevisiae genome does not contain any UCP homologs.
  • 35 [124]. And addition of ATP to S. pombe mitochondria only partially activates SDH. For full activation the addition of glutamate (which leads to removal of OAA) is required [93]. Also an inhibitory effect of the uncoupler CCCP (which leads to dissipation of the pmf) on succinate dependent respiration in S. pombe mitochondria was found [26] in a way similar to plant succinate dependent respiration [21]. 1.4 Summary energy transducing systems To recapitulate; energy transducing systems can be defined in terms of the chemiosmotic theory put forward by Peter Mitchell [125]. An energy transducing system: 1) has a set of membrane located components which reversibly couples the translocation of protons to oxido- reduction reactions which generates an electrochemical potential of protons. (the ETC being an example of such a set of components). 2) has a membrane located ATP hydrolysing proton pump which can work in reverse driven by the aforementioned electrochemical potential of protons which leads to the catalysis of the thermodynamically unfavourable reaction of ATP synthesis. (complex V). 3) can use the aforementioned electrochemical potential of protons to directly or indirectly drive the transport of substrates across the membrane. (e.g. succinate via the dicarboxylate carrier [43] or ADP exchanged for ATP by the adenine nucleotide carrier (ANC) [126]). 4) the systems of postulates 1,2 and 3 are located in a specialised coupling membrane which has a low permeability to protons and to other ions in general. (e.g. the IMM).
  • 36 The chemiosmotic theory is generally accepted in the field of bioenergetics and it therefore is considered paradigmatic in this work. Although generally accepted, as recently as 2005, the theory is still questioned, see references [127-129]. 1.5 A modular representation: In order to study oxidative phosphorylation in this study we used a modular approach in which ETC components are lumped into Q-pool reducing pathways and Q-pool oxidising pathways [37], see Figure 1.15. The external NADH dehydrogenase and the combined activity of both SDH and the dicarboxylate carrier are the reducing pathways. The cytochrome pathway and the alternative pathway are the oxidising pathways. The Q-pool can be viewed as a reservoir which can accept electrons from the reducing pathways and can donate electrons to the oxidizing pathways. When all ubiquinone is reduced to ubiquinol the reservoir is ‘full’ and when all ubiquinol is completely oxidized it is ‘empty’. Under steady state conditions the rate with which electrons flow into the Q-pool equals the rate with which they leave. The overall electron flux through the respiratory chain can then be assessed by measuring the oxygen consumption rate. The steady state Q redox poise and oxygen consumption rate values are dependent on the interplay between the activities of the reducing and oxidising pathways. Activity of the cytochrome pathway leads to the formation of a pmf, due to the backpressure of this proton gradient the activity of this pathway can be limited. If now an uncoupler were added, the backpressure would be relieved and the activity of the cytochrome pathway increases, which is reflected in an increased steady state oxygen consumption rate with a concomitant oxidation of the Q pool. Some reducing pathways are not fully active upon addition of substrate, e.g. SDH becomes more active upon addition of ATP, which is reflected also in an increased steady state oxygen consumption rate, but in this situation the Q pool would become more reduced. Things are not as straightforward when a condition changes which affects reducing and oxidising pathway activities simultaneously, as will be discussed in chapter 3. But for now these examples illustrate clearly the general idea of the modular approach that is used in this study and which has been used successfully in previous studies [76, 93, 130, 131]. This approach has two main tenets:
  • 37 1) the Q-pool is homogenous, i.e. ubiquinone (ubiquinol) molecules can freely interact with different dehydrogenases and oxidases. 2) Reducing pathways only interact with oxidising pathways via the Q-pool as an intermediate, i.e. there is no direct interaction between these pathways. Figure 1.15 A modular representation of the ETC with the Q-pool as the central intermediate between pathways. Reducing pathways: external NADH dehydrogenase and the combined activities of SDH and the dicarboxylate carrier. Oxidising pathways: the cytochrome pathway (complexes III, IV and cytochrome c) and the alternative pathway consisting of the alternative oxidase only. A different modular approach called ‘top-down’ metabolic control analysis using p as the central intermediate has been successfully applied to both mammalian and plant mitochondria [132, 133]. In that approach energy transducing processes are classified as either p producers or p consumers. Three components are defined which communicate with one another via p: the ‘respiratory chain’ (dicarboxylate carrier + ETC), the ‘proton leak’ (proton leak, cation cycles etc.) and the ‘phosphorylating system’ (ATP synthase, the phosphate carrier and the adenine nucleotide carrier) [132]. The approach used in this study assumes the pmf to be constant [37].
  • 38 1.6 Summary Hopefully this introduction has managed to illustrate that mitochondria are a bit more than just little cellular batteries, they are indeed very important in regulating cellular physiology. Not only in plants or yeasts but as much in mammalian cells. Another trend of recent years, it appears, is the realisation that mitochondria are more and more involved in many human medical afflictions ranging from diabetes [134] to hearing disorders [135]. The alternative oxidase is quite often considered a typical plant protein and therefore according to current funding body standards maybe not so ‘fashionable’ however given the recent finding that AOX is also found in the animal kingdom and the fact that it has been expressed in human cells [84] could place the alternative oxidase back in the picture as a clinical tool for investigating human diseases. 1.7 Aims: To set up a three electrode system which allows for simultaneous determination of oxygen consumption rate (vO2), Q-redox poise (Qr/Qt) and membrane potential () in isolated mitochondria (chapter 3). This was achieved and successful recordings were made. SDH activation in potato mitochondria by ADP, ATP and oligomycin was hypothesized to occur indirectly using  as an intermediate. This was investigated (chapter 3) and it was determined that SDH activation did not occur indirectly via . S. pombe respiratory kinetics have previously been characterised in terms of oxygen consumption rate and Q-redox poise under various energetic conditions, but to the best of our knowledge the membrane potential had not yet been determined. A further characterization of the S. pombe respiratory kinetics was undertaken (chapter 4). This yielded some interesting oxidising pathway kinetics which have not been seen previously in mitochondria from other species.
  • 39 S. pombe has been used previously in this laboratory to heterologously express AOX. It was found then that AOX expression led to a change in respiratory kinetics under various energetic conditions. Kinetic curves were fitted to data obtained from malonate titrations done on mitochondria respiring on succinate, which has yielded a limited amount of data points. In this study a novel titration method (an NADH regenerating system) was used to obtain a larger dataset and a possible effect of AOX expression on  generation was studied (chapter 5). This approach demonstrated that S. pombe mitochondria expressing AOX display substrate dependent differences in oxidising pathway kinetics. Furthermore an effect of  on AOX activity could not be demonstrated.
  • 40 Chapter 2 Materials and Methods 2.1 Isolation and purification of mitochondria In this study mitochondria were isolated from (transformed) Schizosaccharomyces pombe cultures, Saccharomyces cerevisiae cultures, fresh potato tubers and Arum maculatum spadices. 2.1.1 Schizosaccharomyces pombe—The S. pombe strain used in this study is the so called sp.011, ade6-704, leu1-32, ura4-D18,h- [104]. From this strain three types of yeast cultures were grown, a wild-type (sp.011 wt) and two transformants. One type of transformant had the S. guttatum AOX expressed, depending on the presence of thiamine in the growth medium AOX was either expressed or repressed in the mitochondria (sp.011 AOX and sp.011 AOX+T respectively). Another type of transformant had an empty vector expressed (sp.011 pREP). Therefore a total of four different types of S. pombe mitochondria were investigated in this study. All media used for yeast transformation, yeast growth and yeast mitochondrial isolation are described in section 2.1.1.7. 2.1.1.1 The Expression system—Functional expression of a plant alternative oxidase in S. pombe was achieved by using a system developed by Albury et al. [59]. A Sauromatum guttatum cDNA clone (pAOSG81 [136]) which represents the nuclear gene aox1 [54] was cloned into the expression vector pREP [137] in which it is under the control of the nmt1 promoter6 . A transformed S. pombe culture will not express AOX when grown in the presence of thiamine. Apart from aox1 several other genes are present on the plasmid. The LEU2 gene (from S. cerevisiae) codes for a protein involved in leucine biosynthesis. Using a S. pombe strain in 6 no message in thiamine
  • 41 which the equivalent gene (leu1- ) is disrupted the plasmid becomes essential for growth in a medium lacking leucine. The ars1 gene (autonomously replicating sequence) is required for initiation of replication in yeast, whereas the ori gene is required for initiation of replication in bacteria. The AMP gene (resistance against amphicillin) provides a method to select for the plasmid in bacteria. When expressed in S. pombe (grown in the absence of thiamine and leucine) AOX is targeted to and incorporated into the IMM as a functional enzyme [59]. 2.1.1.2 Yeast transformation—S. pombe cells were transformed with pREP-AOX (coding for the S. guttatum AOX) or with pREP (just the vector) using a modified lithium acetate method [101]. A single sp.011 wt colony was inoculated in 5 ml YES medium and incubated overnight (150 rpm, 30 C) which normally grew to ~2.5x107 cells/ml. 800 l of this culture was used to inoculate 100 ml of minimal medium, which was grown overnight (150 rpm, 30 C). The cells were harvested by bench-top centrifugation (2 minutes at 3000 rpm at room temperature) and washed in distilled water. The cells were then resuspended in 0.1 M lithium acetate / Tris-EDTA (LiA/TE) in 10 0.1 ml aliquots at a density of ~1x109 cells/ml. The cells were then incubated at 30 C (waterbath) for one hour with occasional mixing. To each aliquot 1-2 g DNA (in ~10 l) and 290 l 50% polyethylene glycol (PEG) dissolved in LiA/TE was added. The cells were then again incubated for one hour in the waterbath at 30 C with occasional mixing. This was followed by a heat shock step of 15 minutes at 43 C (waterbath) for 15 minutes. Cells were then pulsed to a pellet and the supernatant removed. The pellet was gently resuspended in 100 l 0.1 M LiA/TE. The cells were then plated on minimal medium agar plates and grown for 3-5 days at 30 C. Colonies grown on these plates were subsequently used to inoculate larger cultures. 2.1.1.3 S. pombe growth—A starter culture was set up by picking a colony from a yeast plate and adding it to a 200 ml solution of minimal media. Supplements were added depending on yeast type. 0.4 mM adenine and 0.7 mM uracil were added to all cultures. In the case of sp.011 AOX+T 0.5 mM thiamine was added to repress expression of AOX. The wild type sp.011 WT requires addition of 1.1 mM leucine. A culture was incubated under
  • 42 aerobic conditions for three days at 30 C (150 rpm) for the cells to reach stationary phase. Cell concentration was assessed spectrophotometrically (light scattering at A595 [93]) using a 15-fold dilution of the cell culture in distilled water. This value was used to calculate the volume of culture required to inoculate each of four fresh 1 litre culture flasks (minimal medium with the appropriate supplements) to give cell concentrations ~ 40% of the stationary phase density at the anticipated time of harvesting. For this the following exponential growth equation was used: [2.1] Where  is the growth rate, N0 is the starting concentration, Nx the final concentration and t the period of growth. After rearranging this gives: [2.2] In this form N0 represents the starting cell density which gives the required Nx density after t time (hours) with cells growing at a rate of . In a previous study values of  of ~0.14 and 0.12 h-1 for non-transformed and transformed cells were determined [26]. In this study similar values were obtained. The inoculated 1 litre flasks were typically incubated for 19- 21 hours under aerobic conditions at 30 C and 150 rpm. t NNx    )log(log303.2 0  ) 303.2 (log 0 10 t Nx N    
  • 43 2.1.1.4 Isolation of mitochondria from S. pombe cultures—On the day before isolation four 1 litre flasks were sub-cultured by adding a certain volume of starter culture, the amount of which calculated as described in 2.1.1.3. The isolation of mitochondria from S. pombe cultures can be broadly divided into two stages. 1: Spinning down of the yeast cells from the four 1 litre cultures and treatment of the cells with lysing enzymes to remove the outer membranes and induce spheroplast formation. 2: Inducing spheroplast lysis by osmotic shock and subsequent harvesting of the mitochondria through differential centrifugation. The isolation protocol used here is based on the method of [104]. 2.1.1.5 Spheroplast preparation—Cells were harvested by centrifugation (10 minutes, 7000 rpm). Cells were washed by resuspension in distilled water at 4 C and again spun down (10 minutes, 7000 rpm). The wet weight was recorded (typically between 15-20 g). Cells were resuspended in 200 ml spheroplast buffer (SB) and incubated at 30 C, 150 rpm for 15 minutes in the presence of the cell wall-digesting enzyme preparation Zymolyase 20T7 (5 mg / g wet weight). Upon addition of a second preparation, ‘lysing enzyme’8 (15 mg / g wet weight) the suspension was incubated for a further 45 minutes at 30 C, 150 rpm. Spheroplast formation was subsequently assessed spectrophotometrically by diluting a suspension aliquot 100x in distilled water. The level of scattering (A800) due to cells and intact spheroplasts compared to cells not treated with digestive enzymes was used to indicate the proportion of osmotically sensitive spheroplasts in the sample. Also, a 5 l aliquot of cell suspension was osmotically shocked by addition of 5 l distilled water, the effect of which was observed using a light microscope. Due to removal of the cell wall this led to lysis. 2.1.1.6 Isolation of mitochondria—All procedures described from here on were performed on ice to minimize enzymatic activity. The spheroplast suspensions were diluted 2-fold in spheroplast wash (SW) and spun down for 10 minutes at 1600 rpm at 4 C. As a washing step the pellets were resuspended in SW and again spun down for 10 minutes at 1600 rpm at 4 C. Pellets were resuspended in ~2 ml mannitol wash (MW) and transferred to a glass 7 Seikagaku corporation, code number: 120491 8 Sigma, code number: L1412
  • 44 homogeniser. With two gentle strokes the resuspended pellets were homogenised, the total volume was subsequently increased to 600 ml with MW to lyse the spheroplasts (osmotic shock). Cell debris and unlysed cells were removed by centrifugating at 3500 rpm for 15 minutes at 4 C. The supernatant was subsequently centrifuged at 13000 rpm for 10 minutes at 4 C. Pellets highly enriched with mitochondria were pooled and centrifuged at 10000 rpm for 10 minutes at 4 C to yield a final mitochondrial pellet which was resuspended in a small volume of ~1-2 ml of MW and kept on ice throughout the remainder of the experimental day. 2.1.1.7 S. pombe media—The following media were used for the transformation of S. pombe cells, the growth of S. pombe cultures and the isolation of mitochondria from these cultures. YES medium (Yeast Extract with Supplements): amt component final conc 5 g/l yeast extract 0.5% w/v 30 g/l glucose 3.0% w/v Supplements: 225 mg/l adenine, histidine, leucine, uracil and lysine hydrochloride. Solid media (for plates) was made by adding 2% Difco Bacto Agar. Transformation media: 0.1 M LiA/TE : 0.1 M lithium acetate in Tris-HCl (10 mM, pH 7.6) and EDTA (1 mM). 50% PEG in 0.1 M LiA/TE made up fresh on the day of transformation, not sterilised.
  • 45 Minimal medium [138]: 0.11 M glucose 19 µM FeCl3.6H2O 93 mM NH4Cl 0.9 µM Na2MoO4.H2O 15 mM Na2HPO4 0.6 µM KI 15 mM KH-Phthalate 0.2 µM CuSO4.5H2O 5.2 mM MgCl2.6H2O 5 µM citric acid 0.1 mM CaCl2 1 µM Na pantothenate 14 mM KCl 80 µM nicotinic acid 0.3 mM Na2SO4 55 µM inositol 8.0 µM H3BO3 40 nM biotin 1.8 µM MnSO4.4H2O 1 mM NaOH 1.4 µM ZnSO4.7H2O Solid media (for plates) was made by adding 2% Difco Bacto Agar. Spheroplast buffer (pH 5.8) 1.35 M sorbitol 1 mM EGTA 10 mM Citrate/phosphate Citrate/phosphate: 100 mM citric acid and 100 mM Na2HPO4 pH 5.8 mixed in ratio ~50:200 Spheroplast wash (pH 6.8) 0.75 M sorbitol 0.4 M mannitol 10 mM MOPS
  • 46 Mannitol wash (pH 6.8) 0.65 M mannitol 2 mM EGTA 10 mM MOPS Yeast reaction medium (pH 6.8) 0.65 M mannitol 1 mM MgCl2 5 mM Na2HPO4 10 mM NaCl 20 mM MOPS 2.1.2 Saccharomyces cerevisiae—The S. cerevisiae strain used in this study was ordinary baker’s yeast ‘Carrs – breadmaker yeast’ purchased at a local supermarket. A small quantity of dried yeast was dissolved in distilled water and subsequently plated on YES medium (section 2.1.1.7) based agar plates to grow S. cerevisiae colonies. The same protocols used for growing S. pombe cultures and isolating S. pombe mitochondria were used with S. cerevisiae with some minor alterations. The starter culture was grown for one day only, as opposed to three days and in the degradation step only Zymolyase was used (5 mg / g wet weight), the lysing enzymes were omitted. For electrochemical experiments the yeast reaction medium was used (section 2.1.1.7). 2.1.3 Potato tuber—Fresh potato tubers were bought at a local supermarket. The protocol to isolate and purify mitochondria from this tissue is based on [139]. Media used in this protocol are described in section 2.1.3.2. All operations were performed on ice to minimize enzymatic activity.
  • 47 2.1.3.1 Isolation of mitochondria from potato tubers—Potatoes (~1.5 kg) were peeled thickly, cut into large chip-sized pieces and homogenised in grinding medium using a juice extractor (Moulinex type 140). The juice was collected directly in 1.3 liter of grinding medium (GM). The homogenate was then filtered through a moistened muslin (to remove large starch particles). The pH was adjusted to 7.4. Subsequently, three centrifugation steps were performed, all at 4 C. First the homogenate was centrifugated for 5 minutes at 1500 rpm (to completely get rid of starch). The supernatant was centrifugated for 10 minutes at 4000 rpm. The supernatant was then centrifugated for 15 minutes at 10000 rpm. The pellet was resuspended in 2-5 ml washing medium (WM). The suspension was transferred to two 50 ml centrifuge tubes and WM was added to fill the tubes. This was followed by another centrifugation step of 10000 rpm for 10 minutes at 4 C. The pellets were resuspended in a small volume (2-5 ml) of WM and pipetted on top of a self-forming PercollTM gradient in 25 ml of purification medium (PM). The tubes were centrifugated at 18000 rpm for 30 minutes at 4 C. Using a pastette the mitochondria were removed from the gradient and diluted in WM (at least 1:10). Purified mitochondria were pelleted by a centrifugation step of 10000 rpm for 10 minutes at 4 C. The mitochondrial pellet was then resuspended in a small amount of WM (1-2 ml) and kept on ice for the remainder of the experimental day. 2.1.3.2 Potato tuber media: Grinding medium (pH 7.4) 0.3 M mannitol 0.1% w/v BSA 40 mM MOPS 2 mM EDTA 0.6% w/v PVP 40 10 mM cysteine
  • 48 Washing medium (pH 7.4) Identical to grinding medium apart from the fact that cysteine is omitted. Purification medium (pH 7.4) 21% v/v PercollTM 0.3 M sucrose 5 mM MOPS 0.1% w/v BSA Potato reaction medium (pH 7.2) 0.3 M mannitol 1 mM MgCl2 5 mM K2HPO4 10 mM KCl 20 mM MOPS 2.1.4 Arum maculatum—The protocol to isolate and purify mitochondria from this tissue was based on [88]. Media used in this protocol are described in section 2.1.4.2. All operations were performed on ice to minimize enzymatic activity. 2.1.4.1 Isolation of mitochondria from Arum maculatum spadices—Spadices from local Sussex woods were isolated from the leaf tissue and chopped into small ~1 cm3 slices and added to ice-cold grinding medium (GM). The slices were homogenised in a WaringTM blender in 2x3 s bursts. The homogenate was filtered through a wetted muslin and centrifugated at 4000 rpm for 10 minutes at 4 C. The supernatant was then centrifugated at 10000 rpm for 10 minutes at 4 C. The pellet was resuspended in washing medium (WM) which was then centrifugated at 12000 rpm for 10 minutes at 4 C. The pellet was resuspended in a minimal amount of WM and loaded onto a 21% PercollTM self-forming
  • 49 gradient. The gradient was centrifugated at 14000 rpm for 30 minutes at 4 C. The mitochondrial band was removed using a pastette and transferred to WM. The mitochondrial suspension was then centrifugated at 12000 rpm for 10 minutes at 4 C. Mitochondria were gently resuspended in a small volume (1-2 ml) of WM and kept on ice during the remainder of the experimental day. Mitochondria were isolated either immediately after picking of the spadices or after storing the spadices overnight at 4 C. 2.1.4.2 Arum maculatum media: Grinding medium (pH 7.5) 0.3 M mannitol 0.2% w/v BSA 20 mM MOPS 2 mM EDTA 2 mM pyruvate 7 mM cysteine Washing medium (pH 7.5) Identical to grinding medium apart from the fact that cysteine is omitted. Purification medium (pH 7.5) 21% v/v PercollTM 0.3 M sucrose 5 mM MOPS 0.1% w/v BSA 2 mM pyruvate Reaction medium The same as for potato, see section 2.1.3.2.
  • 50 2.1.5 Specifics of plant mitochondrial isolation [38, 140]: Plant cells have a rigid cell wall which requires the use of shearing forces to disrupt, in order to liberate cytoplasmic organelles. This leads inevitably to the rupture of the cell vacuole thereby releasing harmful compounds, such as hydrolytic enzymes, phenolic compounds, tannins, alkaloids and terpenes, which can interact with the mitochondrial membranes. In order to minimise interaction of these compounds with the mitochondria several precautionary measures can be taken. Phenolic compounds and their oxidation products (quinones) being highly reactive can react strongly with mitochondrial membranes. Bovine serum albumin (BSA) is routinely used, not only to bind free fatty acids, but also because it can bind to quinones. Cysteine which is added to the grinding medium of potato and arum preparations is another protective agent preventing quinone interactions. Polyvinylpyrrolidone acts as a scavenger of phenols and tannins. pH is kept between 7.2-7.5 as alkaline pH will increase phenol autooxidation and acid pH will increase interaction between phenols and protein functional groups. 2.2 Polyacrylamide gel electrophoresis & Western analysis 2.2.1 SDS-PAGE—Proteins were separated using 1-D SDS PAGE. Mitochondrial samples (stored at –80 C) were defrosted on the day of electrophoresis. Mitochondrial protein (15 µg per lane) was separated on 0.75 mm thick 10% SDS-polyacrylamide gels according to the method of [141]. Electrophoresis was performed for ~ 1 hour at 150 V. Samples were run under non-reducing conditions through the omission of -mercaptoethanol in the gel. 2.2.2 Blotting to nitrocellulose—Separated proteins were transferred from SDS- polyacrylamide gels to nitrocellulose membranes using standard electrophoretic methods [142]. Transfer was carried out in ice cold transfer buffer (25 mM Tris-192 mM glycine, 10% v/v methanol, pH 8.8) for 1 hour at 100 V.
  • 51 2.2.3 Immuno-detection of proteins—After blotting, nitrocellulose membranes were washed in Tris-buffered saline (TBS; 140 mM NaCl, 20 mM Tris-HCl pH 7.6) and gently agitated overnight at 4 °C in blocking solution (BS; 2% w/v milk powder 3% w/v BSA and 0.1% v/v Tween 20 in TBS). This was followed by 6 x ~ 5 min washes in TBS, filters were incubated for 1 hour at room temperature in BS containing mouse monoclonal, anti-AOX (from Sauromatum guttatum) antibodies [53] (1:2000 dilution). Filters were washed in TBS, as before, and then incubated for 1 hour in BS containing a 1:1000 dilution of secondary antibody (linked to horseradish peroxidase). Antibodies were detected on light- sensitive film using an enhanced chemiluminescence kit (Amersham International plc). S. guttatum AOX antibodies were a gift from Dr. Tom Elthon (University of Nebraska). 2.3 Protein estimations Due to the time consuming nature of the isolation of mitochondria and subsequent experiments; protein estimations were normally done on a different day. Therefore isolated mitochondria were kept frozen at –80 C and defrosted on the day of protein estimation. Protein concentrations were determined using a bicinchoninic acid (BCA) assay [143] in the form of a kit (BCA, Pierce, Rockford, UK) with bovine serum albumin (BSA) as a standard. In this assay, a solution of protein is incubated with a solution containing cupric sulfate and BCA. The cupric ion (Cu2+ ) is reduced to the cuprous ion (Cu+ ) by proteins in an alkaline medium. This reaction is often referred to as the Biuret reaction. The cuprous ion then forms a purple-colored complex with BCA that strongly absorbs light at 562 nm. All samples were kept for 30 minutes at 37 C before determining absorbances in a spectrophotometer (CARY 400 Scan). A calibration curve (absorption vs. mg protein) was made using a series of BSA standards by diluting a stock solution of BSA of 2 mg/ml with distilled water, including one cuvette filled with distilled water as a blank. This was done in duplicate and the absorption values were measured in a spectrophotometer. The acquired values were plotted in Kaleidagraph (version 3.02) and fitted using a linear fit. The mitochondrial samples with unknown protein weight were diluted 20 and 50 times (both in duplicate) and the acquired absorbance values were inserted into the equation which was derived from fitting the calibration curve (also done in Kaleidagraph).
  • 52 2.4 Electrochemical techniques All electrochemical experiments were performed in a setup as shown in Figure 2.1. A perspex reaction chamber was constructed by the University of Sussex mechanical workshop which could accommodate a volume of 2.2 ml (C). After cleaning the vessel with pure ethanol9 and rinsing with distilled water, reaction medium was added. The chamber sits atop a magnetic stirrer (A) which drives a stirring bean present in the chamber to keep the reaction medium homogenous. The vessel can be airtight sealed with a stopper (D) which contains a 1 mm channel through which chemicals can be added via a micro syringe. An oxygen electrode (described in 2.4.2) is mounted in the bottom of the reaction chamber. On the sides of the chamber a total of 4 plugs are present which can be removed in order to add more electrodes to the setup (the Q-electrode and the TPP+ electrode). Figure 2.1 The reaction chamber used in this study. A: magnetic stirrer. B: oxygen electrode. C: reaction chamber. D: stopper. 9 With yeast mitochondria propan-2-ol was used as ethanol is a substrate for these mitochondria. With all other tissues ethanol was used to clean the vessel. A D C B
  • 53 2.4.1 The oxygen electrode—The concentration of dissolved oxygen was measured using a Rank oxygen electrode [144]. The electrode consists of a Ag/AgCl reference anode and a platinum cathode, see Figure 2.2. The electrodes are immersed in a saturated KCL solution and separated from the reaction medium by an oxygen-permeable Teflon membrane. A potential difference of ~0.7 V is set up between the anode and the cathode using a potentiostat (Rank Brothers Ltd. Bottisham, Cambridge UK). The following reactions occur at the electrodes: 4 Ag + 4 Cl-  4 AgCl + 4 e- (anode) 4 H+ + 4 e- + O2  2 H2O (cathode) At the platinum electrode electrons reduce oxygen molecules to water whilst chloride ions migrate to the anode where they release electrons. The overall result is a transfer of electrons from anode to cathode causing a current flow between the two electrodes which is proportional to the concentration of oxygen in the reaction chamber. As oxygen is consumed in this process it can be appreciated that oxygen concentration near the electrodes will decrease over time therefore it is necessary to have a stirring bean present to keep the solution homogenous. As the current between the electrodes is proportional to the concentration of oxygen and the response is linear only two calibration points are necessary [144]. Air saturated reaction medium gives the signal corresponding to maximum oxygen concentration in aqueous solution (250 M at 25C [145]) and sample-induced anaerobiosis gives the signal representing zero oxygen. A more conventional way of achieving anaerobiosis through the addition of sodium dithionite (Na2S2O4) is not used to calibrate the oxygen electrode in this study as it severely affects the functioning of the Q-electrode.
  • 54 Figure 2.2 Oxygen electrode mounted in the bottom of the reaction chamber (see Figure 2.1). Platinum electrode in the middle is the cathode, the annular silver electrode the anode. 2.4.2 The Q-electrode—The Q-electrode allows for the continuous measurement of the Q- redox poise in mitochondria and has been employed successfully in isolated mitochondria from plant [21, 22, 76, 85, 86, 146-149], yeast [70, 93, 104] and mammalian systems [21]. The Q-electrode has also been used to determine the redox state of the plastoquinone pool in thylakoids [150] and the redox state of the ubiquinone pool in bacterial membrane fragments [151]. This apparatus consists of glassy carbon, platinum and Ag/AgCl electrodes mounted in a sample vessel which allows amperometric measurement of the redox poise of the endogenous Q-pool of isolated mitochondria (devised by Prof. Peter Rich, Imperial College, London; see European patent No. 85900699.1 for details [152]). Endogenous Q10 is water-insoluble and therefore bound to the IMM where it cannot be detected by an electrode. Measurements therefore rely on a Q-mimic, Q1 (2,3-dimethoxy-5- methyl-6-isoprenyl-p-benzoquinone) that is sufficiently soluble in both membrane and aqueous environments to allow redox-equilibration with Q10 in the membrane as well as
  • 55 interaction with an electrode surface in aqueous solution to communicate the relative poise. Although the exact nature of Q1:Q10 redox-equilibration is unclear, it has been suggested that it involves Q10H2:Q1 transhydrogenase activity at the Qn site of the mitochondrial bc1- complex (P.G. Crichton D. Phil thesis University of Sussex [26] [Prof. Peter Rich, University College London, personal communication]). The advantage of using this technique is that it allows continuous measurements of the Q-redox poise whilst simultaneously recording the oxygen consumption rate. Another technique which is used at times is the determination of the Q-redox poise via Q extraction [76, 82] where mitochondria in steady state are chemically quenched and subsequently have their quinones extracted. Analysis of redox state is then determined using HPLC. Experiments where both techniques were used on the same mitochondrial preparation gave similar results corroborating the notion that Q-redox poise determinations obtained via the Q-electrode are accurate [22, 76]. Due to limitations in the way that electron transfer occurs between quinones and electrode surfaces (for mechanistic reasons the reaction is electrochemically irreversible [152]), the redox poise of Q1 cannot be measured potentiometrically. Instead, the relative concentration of Q1 or Q1H2 specifically, is measured using an amperometric method. In the electrical setup (see Figure 2.3) an external voltage is applied to poise and maintain a glassy carbon working electrode at –360 mV with respect to a Ag/AgCl reference electrode. This is achieved using a potentiostat (University of Sussex Workshops) where a third ‘auxiliary’ electrode (platinum) along with the working electrode accepts the current in order to avoid polarisation of the reference electrode (which would otherwise result in a potential that varies with current). The combination of electrodes constituting the Q-electrode is typically introduced into an oxygen electrode chamber to allow simultaneous determination of Q-redox poise and oxygen consumption (see Figure 2.4). Importantly, the measured current that flows between the auxiliary and working electrode is proportional to the Q1 concentration. Changes in the Q10 redox poise, and consequently the Q1 concentration, will therefore be reflected in a change in the measured current. During respiratory measurements the system is calibrated by measuring the signal associated with mitochondria in the absence of respiratory substrates where the endogenous pool of Q10 is assumed to be fully oxidised.
  • 56 The signal gained following sample-induced anaerobiosis is used to represent a fully reduced Q-pool. The experimental values relative to these parameters can then be estimated. Figure 2.3 Schematic representation of the Q-electrode as adapted from [153]. Figure 2.4 Q-electrode combined with oxygen electrode. Working electrode (glassy carbon) blue wire. Auxiliary electrode (platinum) red wire. Reference electrode (Ag/AgCl) green wire (at the back).
  • 57 Several substances have been found to interfere with the Q-electrode: NADH, malonate, TMPD, ascorbate, octyl gallate and ammonium sulfate. Precautions have to be taken when using these substances during experiments. 2.4.3 The TPP+ -electrode—In this study a tetraphenyl phosphonium (TPP+ ) electrode was constructed based on the method of [154]. The TPP+ electrode is an ion-selective electrode [155]. The membrane potential across the IMM is negative inside with respect to the outside, therefore a positive lipophilic ion must be used. In this study the selected ion was TPP+ (see Figure 2.5) a lipophilic cation which can readily permeate the IMM. Due to extensive -orbital systems the charge of the TPP+ ion is delocalised. Therefore TPP+ ions can move freely in both aqueous and hydrophobic environments [1]. Other ions which have been used with ion-selective electrodes are triphenyl-methylphosphonium (TPMP+ ) [110] and dibenzyl dimethyl ammonium (DDA+ ) in combination with tetraphenyl boron (TPB- ) [156]. TPP+ has some advantages over other ions, e.g. it permeates the IMM 15 times faster than DDA+ [154]. Some ions such as DDA+ require the presence of a lipid-soluble anion such as tetraphenyl boron (TPB- ) for permeation through the IMM [154] whereas TPP+ does not require any other lipid soluble ions to be present in order to permeate the IMM. Figure 2.5 Structure of tetraphenyl phosphonium. Due to the ease with which lipophilic ions such as TPP+ can permeate through the IMM in response to changes in  they have been successfully employed as indicator ions to determine the magnitude of  [155]. Ion selective electrodes do not measure the  directly. Instead the TPP+ electrode measures the concentration of TPP+ ions in the medium. Upon energization of mitochondria, TPP+ ions in the medium start migrating into the matrix, attracted by the negative electrical potential on the matrix side of the IMM. This
  • 58 results in a decrease in TPP+ ions outside of the mitochondria, [TPP+ o] and an increase of matrix TPP+ ions, [TPP+ i]. By using the Nernst equation one can calculate from the steady state [TPP+ i] and [TPP+ o] the value of the  across the IMM:            ][ ][ log zF RT 2.303(mV) i o TPP TPP [2.3] At room temperature this equation simplifies to:            ][ ][ log95(mV) i o TPP TPP [2.4] This means that for every tenfold difference in the TPP+ concentration ratio across the IMM an electrical potential difference of 59 mV will be detected. Or alternatively, for every 59 mV increase in  a tenfold accumulation of TPP+ i occurs. Given the ionic nature of the TPP+ molecules, migration of these ions across the IMM could possibly lead to a change in . To avoid a possible uncoupling effect (as TPP+ is a positive ion) a low concentration of TPP+ was chosen to work with in this study. A TPP+ concentration of 1 M was used. Comparable studies where the TPP+ -electrode was employed report values for TPP+ concentration up to 10 M without any effect on  [154, 157, 158]. Addition of TPP+ up to 2.5 M did not have an effect on the respiration rate in the mitochondria used in this study, therefore no uncoupling was induced through the addition of TPP+ . In order to determine [TPP+ i] a conversion factor was used where the matrix volume was deduced from its protein content according to [159] with a value of 2 l mg-1 for potato mitochondria and with a value of 1.02 l mg-1 for yeast mitochondria [160]. Membrane potentials were calculated using equation 2.4. The TPP+ -electrode can only determine the concentration of TPP+ in the medium (i.e. any TPP+ molecules present in the matrix cannot be detected). From [TPP+ o] the amount of TPP+ molecules in the medium can
  • 59 be calculated. Combined with the total amount of TPP+ molecules added (2 nmole) [TPP+ i] can be calculated: [2.5] 2.4.3.1 The TPP+ -electrode setup—Figure 2.6 shows the TPP+ -electrode setup. The TPP+ -electrode consists of a chlorated silver wire which is inserted into a PVC tube (see Figure 2.7) filled with a solution of TPP+ (10 mM) dissolved in distilled water. A Ag/AgCl reference electrode makes contact with the same solution as the TPP+ -electrode. The difference in potential between these two electrodes is detected by a Voltmeter (University of Sussex Workshops). Figure 2.6 TPP+ -electrode setup. The TPP+ -electrode is inserted into the reaction chamber on the side. In this configuration  and oxygen consumption rate can be determined simultaneously. umematrix vol TPPTPP TPP mediumtotal i    ][
  • 60 2.4.3.2 Detection of [TPP+ o]: Detection of TPP+ ions in the medium by the TPP+ electrode is based on ion exchange. As mentioned previously a chlorated silver wire is inserted into a PVC tube which is sealed on one end by a membrane that acts as a surface for ion exchange on both sides. The membrane is faced with two solutions, the medium in the reaction chamber with a variable concentration of TPP+ (depending on mitochondrial energy status) and an internal solution of fixed TPP+ concentration. Before inserting the silver wire, the PVC tube is filled with a 10 mM TPP+ solution. The membrane is made out of PVC with tetraphenyl boron (TPB, Na+ salt) imbedded as an ion exchanger (construction of the membrane will be discussed in the next section). The following ion exchange occurs: TPB-Na (membrane) + TPP+ (solution) = TPB-TPP (membrane) + Na+ (solution) Formation of the TPB-TPP complex is called conditioning (discussed in 2.4.3.4). After conditioning the membrane presents a surface of TPP+ to both the internal solution and the medium. As there is TPP+ in solution and on the membrane surface a boundary potential will form due to activity (concentration) difference between membrane surface and solution. That means there are two boundary potentials, the inner boundary potential is constant since the internal TPP+ concentration is also constant, this forms a stable reference. On the outer surface the boundary potential will be a function of the TPP+ concentration in the medium. The overall potential of the electrode will therefore be governed by the concentration of TPP+ in the medium. 2.4.3.3 Construction of the TPP+ -electrode membrane: A 3 ml 10-2 M Na-tetraphenylboron (TPB) solution (dissolved in tetrahydrofuran(THF)) was mixed with a 10 ml solution of 0.5 g polyvinylchloride (PVC) dissolved in THF. This solution was mixed with 1.5 ml of dioctylphtalate (a plasticiser). The solution was poured into a fat free glass Petri dish (60 cm2 ) and was left evaporating overnight in a flowhood avoiding any contamination with dust particles.
  • 61 Construction of the PVC sleeve: PVC tubing 5 mm outer diameter and 3 mm inner diameter was used. With a heated scalpel a length of about 4-5 cm was cut off making sure the cut-off area had a smooth surface. Construction of the TPP+ -electrode: A circle of the same outer diameter as the PVC tubing was stamped out of the membrane and positioned on one of the ends of a 4-5 cm long PVC sleeve. The end to which the membrane was attached was dipped in THF in order to ‘glue’ the membrane to the PVC sleeve. The sleeve with glued on membrane was left to dry overnight. Then the sleeve was for ¾ filled with a solution of 10-2 M TPPCl (in distilled water). The silver wire was chlorated in a bath of 0.1 M HCl in which the silver wire and a platinum wire were inserted. Both wires were connected to an electrical circuit over which a potential of 50 mV was imposed. Electron flow through this circuit was from the silver wire to the platinum wire. A current of 50 mA for the duration of 3 minutes was enough for proper ‘whitening’ of the silver wire, i.e. homogenous depositing of AgCl. After rinsing with distilled water the wire was carefully inserted into the sleeve. Figure 2.7 shows the components of the TPP+ -electrode. 2.4.3.4 Conditioning of the TPP+ -electrode: TPP+ electrodes sleeves are not instantly usable after the membrane is glued to it. After several hours of drying (evaporation of THF) the PVC sleeve was filled with a 10 mM TPP+ solution. The sleeve was soaked in a solution of the same concentration for several hours during which ion exchange could occur on both sides of the membrane.
  • 62 Figure 2.7 Components of the TPP+ -electrode setup. A: Ag/AgCl reference electrode (same type is used with the Q-electrode) B: PVC TPP+ -electrode sleeve C: AgCl coated silver wire. See text for details. 2.4.3.5 Calibrating the TPP+ -electrode: Potential differences between the TPP+ electrode and the reference electrode were recorded using a MacLab/200 (Macintosh Operating Systems, AD Instruments Pty Ltd) and chart analysis software (Chart v3.6/s, AD Instruments Pty Ltd) on an iMac desktop computer. As explained in section 2.4.3 the TPP+ -electrode does not measure the membrane potential directly and although the recorded signals are in units of Volt they do not bear a direct relationship to the membrane potential across the IMM. The TPP+ -electrode detects changes in TPP+ concentration in the medium and therefore the recorded signals in V need to be related to concentration of TPP+ . In order to do this; at the beginning of each experiment a calibration trace is made, see Figure 2.8.
  • 63 Figure 2.8 Calibration of the TPP+ -electrode. The arrows indicate additions of TPP+ to the medium (prior to addition of mitochondria) which leads to downward deflections of the signal. A final concentration of 1 M is reached, equal amounts of TPP+ (0.25 M) are added. 2.4.3.6 TPP+ -electrode correction: No correction for binding was used. During anoxia, upon addition of CCCP (or valinomycin) the TPP+ signal went back to its original value (before mitochondria were added) indicating that no significant binding had occurred. 2.4.3.7 TPP+ -electrode sensitivity: Upon calibration it was found that the TPP+ -electrode had Nernstian slope values of 59 mV at concentrations of TPP+ as low as 0.25 M. Throughout the experiments in this study a concentration of 1 M was used. In the literature it is reported that the TPP+ -electrode responds linearly with a TPP+ concentration of 0.5 M and upwards [154]. It has been reported that at TPP+ concentrations greater than 5-10 M respiration is inhibited [161].
  • 64 2.4.3.8 Durability of TPP+ -electrodes: TPP+ -electrodes (in this context the PVC sleeves with glued on TPP+ membrane) had a variable durability. One electrode lasted for months whereas some electrodes had to be thrown away after only one or two experiments. There are two factors which indicate that an electrode needs to be replaced: - Decrease in response time - Upon calibration the slope of the relationship between electrode deflection signal and concentration of TPP+ in the medium no longer has a Nernstian value (i.e. a value different from 59 is determined) The approach taken in this thesis work was to make several TPP+ -electrodes at a time and to dispense of an electrode at the first sign of trouble, as opposed to trying to repair leaky electrodes by resealing the membrane to the sleeve with THF. Throughout this thesis work between 15 to 20 electrodes were used. 2.4.3.9 TPP+ -electrode response time: It can be seen from Figure 2.8 that upon addition of TPP+ to the medium there is a steep voltage drop. The rate with which the TPP+ signal changes during calibration of the electrode was always quicker than the fastest physiological response measured (uncoupling of the mitochondria by addition of CCCP). Therefore the electrode response is both kinetically competent and not rate limiting.
  • 65 2.4.4 Respiratory measurements—The simultaneous measurement of oxygen uptake and redox poise or oxygen uptake and membrane potential was achieved using an oxygen electrode in combination with the Q-electrode or with the TPP+ -electrode respectively. A combined setup of all three electrodes will be discussed in chapter 3. All signal outputs were recorded using a MacLab/200 (Macintosh Operating Systems, AD Instruments Pty Ltd) and chart analysis software (Chart v3.6/s, AD Instruments Pty Ltd) on an iMac desktop computer. Per assay 0.4-1 mg mitochondrial protein was added to 2.2 ml reaction medium. Reaction media for yeast and plant mitochondria are osmotically different, see section 2.1. Where the Q-electrode was employed, 1 µM ubiquinone-1 (Q1) was included in the reaction medium. Where the TPP+ -electrode was employed 1µM of TPP+ was included in the reaction medium. All experiments were done at room temperature. In order to uncouple mitochondria the chemical carbonyl cyanide m- chlorophenylhydrazone (CCCP) was added. With potato and arum a concentration of 1 M was sufficient to completely uncouple the mitochondria; with yeast mitochondria however it was found that in the presence of 1 M CCCP upon addition of substrate there was still an upward deflection of the TPP+ signal which indicates the formation of . To completely prevent the formation of  the standard concentration of CCCP used with yeast mitochondria was 2 M, twice the concentration used in previous work [93, 104]. 2.4.4.1 Preparation of respiratory effectors—Unless otherwise stated respiratory substrates and effectors were prepared in reaction medium (see section 2.1). For the yeast experiments Ubiquinone-1, CCCP, antimycin A, octyl gallate and salicylhydroxamic acid were dissolved in isopropanol which, unlike the more usual solvent ethanol, is not metabolised by isolated S. pombe mitochondria and does not affect respiratory activity. For the plant experiments ethanol was used.
  • 66 2.4.4.2 Nomenclature—To indicate energetic conditions the conventions of Chance and Williams [1] were used: State 1: mitochondria alone in solution (S1) State 2: mitochondria present + substrate (S2) State 3: a limited amount of ADP added, leading to an increased vO2 (S3) State 4: all ADP converted to ATP, vO2 decreases again (S4) State 5: anoxia (S5) Figure 2.9 shows a schematic representation of an oxygen trace with the different bioenergetic states indicated. Mitochondria in vivo are believed to be in a state intermediate between states 3 and 4 [162, 163]. Figure 2.9 Schematic representation of an oxygen uptake trace as recorded with an oxygen electrode.
  • 67 2.4.4.3 Basic bioenergetic parameters—For characterisation of mitochondria the respiratory control ratio (RCR) and the nmole of ADP molecules consumed per nmole of oxygen atoms (ADP/O) were determined. A measure for the quality of the inner mitochondrial membrane (IMM) in isolated mitochondria is the RCR. Usually the ratio of the state 3 respiration rate (S3) divided by the state 4 respiration rate (S4) or the uncoupled respiration rate divided by the state 2 respiration rate (S2) is used (steady state rates only). The underlying idea being that under state 2 or state 4 conditions the IMM conductivity is low. Upon addition of ADP or uncoupler the conductivity increases. If during isolation the IMM is damaged, the state 2 or state 4 rate is relatively high due to the 'leakiness' of the membrane which will result in a low RCR value. ADP/O values were determined according to [164]. 2.4.4.4 Q-pool kinetics—In this study a modular approach was used where components of the ETC were lumped in two groups of modules: Q-pool reducing and Q-pool oxidising pathways. The external NADH dehydrogenase or the combination of complex II with the dicarboxylate carrier are the reducing pathways. The combination of complexes III and IV combined with cytochrome c (cytochrome pathway) or the alternative pathway (which consists of the alternative oxidase) are the oxidising pathways. Importantly, each unit interacting with the Q-pool exhibits a kinetic dependence on the Q-redox poise. Since a given steady state respiratory rate and corresponding level of Q-reduction represents the situation where both ubiquinone reducing and ubiquinol oxidising pathway activities are equal, it will reflect the respective kinetic dependencies of each of the Q(H2)-interacting pathways involved. By careful modulation of steady state respiratory activity, the kinetic dependence of a particular pathway on the level of Q-reduction can be determined. This is done by specifically altering the activity of one Q-interacting pathway at a time during respiratory measurements so that the change in overall electron transfer (which is dependent upon the activities of both Q-reducing and Q-oxidising pathways) will therefore depend on how the opposing pathway changes with respect to the altered level of its substrate-product ratio (the Q-redox poise). This was done by modulation of succinate dehydrogenase by titrating with malonate (an inhibitor of SDH [165], titration range 0 – 5 mM) to obtain steady states
  • 68 describing Q-oxidising pathway kinetics. Alternatively, respiratory activity was titrated with small additions of subsaturating amounts of NADH (to a maximum of ~ 75 µM with yeast mitochondria, to a maximum of ~100 M with potato mitochondria and to a maximum of ~50 M with Arum mitochondria) which step-wise increased the activity of the external NADH dehydrogenase [146] in the presence of ~ 10 units/ml of Glucose-6- Phosphate dehydrogenase (EC 1.1.1.49) and 10 mM Glucose-6-Phosphate to ensure steady state levels of NADH were maintained, as described in [146]. Additions of aliquots of NADH had small or negligible effects on the Q-electrode (depending on the experimental day). The Q-recordings were corrected for these effects according to [146]. Another method which was employed to obtain Q-oxidising pathway kinetics was through the use of standard traces. In order to activate SDH in yeast mitochondria, both ATP and glutamate are added. When done in sequence (as opposed to pre-incubation) this yields three consecutive data points which are relatively spaced apart, i.e. they are not bundled together in a small cluster. Finally, the modulation of the cytochrome pathway by inhibiting respiratory activity using antimycin A (0 – 40 nM) was employed to obtain Q-reducing pathway kinetics. 2.4.5 Modelling of Q-pool data—The fits used to describe the Q-pool kinetics presented throughout this thesis were modelled according to van den Bergen et al. [76]. In this model, each of the Q(H2)-interacting pathways is assumed to exhibit reversible Michaelis-Menten kinetics in accordance with the following scheme: where the forward reaction represents the oxidation of quinol substrate (S) to give quinone product (P) catalysed by enzyme (E). This reaction scheme can be described by a simple equation (for full derivation see appendix 1): [2.6] PEESES 3 4 1 2  k k k k v    s     s  1
  • 69 When using this simplified rate equation in a v versus s plot, the  value dictates the shape of the kinetic curve. A positive value will give a convex curve indicating that the enzyme has a higher affinity for substrate (QH2) than product (Q). Conversely, a negative value will give a concave plot indicating that the enzyme has a higher affinity for product than substrate. A straight line indicates that the affinity of the enzyme for substrate and product is the same. This equation has been used to model steady state rates of oxygen consumption and Q- reduction levels that represent either Q-reducing or QH2-oxidising kinetics. In all of the above expressions, the forward rate is defined as QH2-oxidation, therefore Q-reducing enzymes will exhibit a negative value of v. For graphical convenience, all rates are plotted in absolute terms, therefore, to model dehydrogenase kinetic data the equations sign is reversed. When fitting QH2-oxidising activity, the  parameter is fixed at zero to reflect that there will be no rate when s = 0 (the Q-pool is fully oxidised). In the case of Q-reducing activity  is fixed at – to reflect that there will be no rate when s = 1 (the Q-pool is fully reduced). A respiratory steady state is reached when the rates through the reducing and oxidising pathways are equal. The steady state Q-redox poise can readily be calculated by solving: [2.7] Where i indicates a summation over all involved enzymes. This model has been applied with success in both plant [76] and yeast systems [70, 93]. Kinetic fits were made with Kaleidagraph version 3.02 which uses the least squared error method (see chapter 9 in [166]). 0 1         i i ii s 
  • 70 2.6 Other methods 2.6.1 Spectroscopy—Reduction of ferricyanide (Fe(CN)6 3− ) was determined using a spectrophotometer (CARY 400 Scan). Ferricyanide can accept electrons from cytochrome c [1]. In the presence of KCN (1 mM), to inhibit complex IV, electron transfer chain activity in isolated mitochondria can be measured spectroscopically by measuring the reduction of ferricyanide (molar extinction coefficient = 1.02 x 103 M-1 cm-1 [167]). Absorption was followed at 420 nm [168] (cuvette filled with reaction medium, mitochondria plus substrates and inhibitors) from this value the signal of a ‘blank’ (cuvette filled with reaction medium) was subtracted. Rates were expressed as nanomoles of ferricyanide reduced per minute per milligram mitochondrial protein. The contents of the cuvette were kept homogenous through the presence of a small stirring bean. Experiments were done at 25C. 2.7 Bioinformatic resources The Wellcome Trust Sanger Institute http://www.genedb.org/ The Center for Biological Sequence Analysis http://www.cbs.dtu.dk/services/NetPhos/ (Technical University of Denmark DTU)
  • 71 Chapter 3 New insights into the regulation of plant succinate dehydrogenase - revisited 3.1 INTRODUCTION 3.1.1 General background and aims—Complex II, otherwise known as succinate dehydrogenase (SDH), is the only component of the electron transfer chain in mitochondria which is also a component of the citric acid cycle and fulfils therefore a dual role, being active in both the process of energy transduction and the generation of carbon intermediates for biosynthetic metabolism [21] (see section 1.2.4 for a description of SDH). It is a well- established fact that ATP can activate this protein [123, 124, 169-171], the mechanism of which, at present, is still unclear. In previous work done in this laboratory it was determined that ATP, ADP and oligomycin had a stimulatory effect on SDH in fresh potato tuber mitochondria [21]. The stimulatory effect of the adenine nucleotides was suggested to occur at the cytosolic side of the IMM. It was found that the effects of ATP and ADP were not additive, suggesting that both nucleotides activate SDH in a similar fashion. It was hypothesized that SDH was activated by these compounds indirectly, using the membrane potential (∆) as an intermediate. On the basis of indirect evidence it was suggested that inhibition of the plant mitochondrial K+ ATP channel (PmitoKATP) [172] upon addition of adenine nucleotides could affect  [21]. Activity of this channel, which is selective for K+ , can decrease the magnitude of . Both ADP and ATP have been shown to inhibit this channel [172]. It was hypothesized that both ATP and ADP could potentially increase the magnitude of  through their inhibitory actions on PmitoKATP [21]. Addition of oligomycin to potato mitochondria respiring on succinate is known to increase the magnitude of  [110]. A stimulatory effect of oligomycin on SDH could be reversed by addition of uncoupler, which also implies a role for  in the regulation of SDH [21]. Based on these observations it was suggested that SDH can be stimulated by an increase in
  • 72 the magnitude of . This however was not demonstrated conclusively as  was not determined in that study (SDH regulation was investigated by simultaneous measurement of oxygen consumption rate (vO2) and Q-pool redox poise (Qr/Qt) [21]). In this study a TPP+ - electrode [154] was used to determine ∆ under various energetic conditions. The hypothesis that SDH in potato tuber mitochondria is activated by adenine nucleotides or oligomycin indirectly via the membrane potential was investigated. Fresh potato tuber mitochondria have been investigated extensively in the field of bioenergetics and the respiratory kinetics of these mitochondria are well understood [76, 133, 139, 157, 162, 173]. Studies done with potato mitochondria focussed either on the relationship between oxygen consumption rate and the membrane potential [111, 133, 139, 157, 173-178] or on the relationship between oxygen consumption rate and the Q-redox poise [21, 76, 131]. One of the aims of this study was to simultaneously determine the oxygen consumption rate, membrane potential and the Q-redox poise in isolated mitochondria. In order to achieve this; an experimental set-up was used in which an oxygen, a Q- and a TPP+ -electrode were combined. The experimental approach in this chapter is novel in the sense that three bioenergetic parameters were determined in isolated mitochondria, either simultaneously by using a 3-electrode set-up or in parallel by doing duplicate experiments with two set-ups: an oxygen and Q-electrode set-up and an oxygen and TPP+ -electrode set-up. Affourtit et al. found that addition of adenine nucleotides or oligomycin to potato mitochondria respiring on succinate led to increased oxygen consumption rates [21]. These results are at variance with findings obtained in an earlier study by Fricaud et al. in the same laboratory [110, 111] where a decrease in oxygen consumption rates was observed. Both findings were reproduced in the present study and an explanation will be given in terms of the interplay between reducing and oxidising pathways showing that the results obtained in these studies [21, 110, 111] are in agreement with each other. In light of recent developments the involvement of the protonmotive force in the process of SDH regulation will be reassessed. In order to appreciate the work presented in this chapter the membrane potential in mitochondria and the regulation of SDH will be discussed in the next two sections.
  • 73 3.1.2 The membrane potential in mitochondria—In vitro, the magnitude of the membrane potential in plant mitochondria is larger than in mammalian mitochondria. Typical state 4 values in plant mitochondria range between -200 to -240 mV [111, 139, 163, 175, 179- 182] although in some studies smaller values ~ -190 mV have been reported [172]. In mammalian mitochondria the state 4 values are considerably smaller, in the range of –150 to -190 mV [132, 154, 163, 183-186]. State 4 membrane potential values in yeast mitochondria are intermediate between those of mammals and plants with values in the range of –180 to -200 mV [160, 187]. A common feature of all types of mitochondria is a 20 to 40 mV depolarisation which is seen upon addition of ADP (state 3) [132, 154, 160, 172, 175, 176, 181, 186]. It has been established that the protonmotive force in potato mitochondria mainly consists of a membrane potential and that the pH component is negligible since addition of nigericin, an ionophore catalysing the electroneutral exchange of H+ for K+ [1] does not lead to an appreciable change in  [110, 139, 157]. It has also been reported that pH values in plant mitochondria are very low [163]. The explanation for the lack of effect by nigericin lies in the fact that plant mitochondria express a K+ /H+ exchanger [188] in the IMM [157, 172]. Addition of nigericin to potato mitochondria isolated in this study did not lead to any noticeable change in TPP+ signal and therefore p was assumed to be equivalent to . Under ADP limited conditions it is found that mitochondrial respiration can be substantially inhibited with only a small concomitant change in p [165, 189]. Figure 3.1 shows a typical  vs. respiration rate relationship under ADP limited conditions. The data in the plot are fictitious with respiration rate given in arbitrary units. In a typical experiment, isolated mitochondria respiring on succinate are titrated with the inhibitor malonate (cf. Figure 3 in [183]). It can be seen that the relationship between electrical potential difference and current (respiration rate represents proton current across the IMM [1]) is non-linear, which is usually referred to as being ‘non-ohmic’ [183]. A small increase in  at high  values leads to a disproportional increase in respiration rate. It is now commonly accepted that this behaviour is due to a non-ohmic increase in proton permeability at high p [190-192] and that the non-ohmic relationship also holds for other cations, such as K+ , tetramethylammonium+ and choline+ [183]. Non-ohmic current/voltage relationships have been determined in mammalian mitochondria [183, 189,
  • 74 190, 193], plant mitochondria [139, 158], chromatophores [194] and bovine heart submitochondrial particles [195]. Figure 3.1 Typical  vs. respiration relationship in mitochondria under ADP limited conditions. A non- ohmic relationship (see text) is seen. The data points are fictitious and respiration rate is given in arbitrary units. Membrane potential values are given in absolute values as is done customarily in bioenergetics literature. Figure adapted from Figure 3 in [183]. To initiate respiration in this study either succinate or NADH were added to the medium containing the isolated potato mitochondria. Therefore  is generated via cytochrome pathway activity only (see section 1.2.1). Plant mitochondria express an alternative oxidase (which is non-protonmotive, see section 1.2.11) and reducing equivalents from succinate or NADH could bypass the cytochrome pathway which might have an effect on . However, fresh potato tuber mitochondria do not display cyanide resistant respiration [8, 76, 176] therefore the respiratory chain of the mitochondria used in this chapter is identical to Figure 1.4 minus the alternative oxidase.
  • 75 3.1.3 Regulation of SDH—SDH is a homotropic enzyme, succinate not only serves as a substrate but also as a positive modifier of the protein. This activation was found in intact mitochondria, membrane preparations and in the soluble, purified enzyme [10]. In this study SDH regulation was assessed in well coupled, intact mitochondria, in which oxygen consumption rate, the Q redox poise and the membrane potential were determined. The regulation of succinate dehydrogenase has been studied for over 50 years [20] and a vast amount of literature is available (see [124, 196] and references therein). Interpreting and comparing data from some of the older publications to the data obtained in this study is not straightforward given the different types of mitochondrial preparations used several decades ago when compared to contemporary standards. In the early literature which focussed on mammalian SDH, Keilin-Hartree particles (using beef heart as source material) were used [20, 197]. These particles were obtained by grinding heart tissue with sand resulting in a mixture of damaged mitochondria and everted vesicles of inner mitochondrial membrane [18], membrane fragments of other types of organelles are present in this type of preparation as well [196]. SDH activity has been routinely determined spectrophotometically and via determination of the oxygen consumption rate. A source of possible confusion is the widespread use of malonate to activate SDH [20, 196, 198]. Malonate is known to be an inhibitor of SDH [86, 133, 139] and has been used in studies to decrease the membrane potential [133, 183] or the Q redox poise [76, 86, 104] in mitochondria respiring on succinate. The binding of malonate to complex II, although it is a substrate competitor, induces a conformational change from an inactive to an active form of the protein [10, 197]. Another inhibitor of SDH, oxaloacetate (OAA) [199] can tightly bound to SDH, preventing the binding of substrate, leaving the protein inert [200]. Therefore when SDH is inactive (SDH-OAA complex) it cannot be reduced by succinate [20]. Several agents can aid in the displacement of oxaloacetate such as succinate, inosine di- and triphosphate and malonate [200]. One widely used method to asses SDH activity is the phenazine methosulfate (PMS) assay [201]. In this method SDH is reduced by succinate and oxidised by PMS. Reoxidation of PMS is accomplished by 2,6-dichlorophenolindophenol (DCIP). Reduction of DCIP is followed spectrophotometically at 600 nm. If SDH needs to be activated prior to
  • 76 the PMS assay it can be incubated with malonate to dislodge OAA. Subsequently, an aliquot of the solution is taken and transferred to the cuvette used in the PMS assay. To prevent excessive inhibition upon carry over, a range between 1-4 mM malonate is normally used [200]. A drawback of using the PMS assay in intact mitochondria is that the IMM needs to be permeabilised which results in deenergized mitochondria [201]. Another disadvantage is the use of artificial electron acceptors, which makes it difficult to compare activity assays done spectrophotometically with assays done using an oxygen electrode. It is well known that isolated SDH complexes have a lower turnover number compared to SDH in its native environment. Upon reconstituting isolated SDH into the IMM turnover numbers are restored [202, 203]. Extraction of quinones from inner membranes results in a decreased turnover number of SDH whereas reinserting quinones leads to restoration of the turnover number [204]. This agrees well with the observation that ubiquinol is an activator of SDH [10]. Addition of externally added QH2 leads to activation whereas addition of Q does not [124]. It has been reported that QH2 has a different binding site than Q, binding of which leads to conformational change [205]. It was also found that QH2 is not oxidised upon activation of SDH [124]. It is a well-established fact that SDH is activated by anions (ClO4 - > I- > Br- > Cl- ) [170, 206], this effect can be counteracted by OAA. It was determined that the anions activate SDH directly, dislodging OAA, as opposed to an indirect chaotropic effect [124]. The ATP activation effect is well known [169] but its mechanism is still poorly understood. Stimulation of SDH by ATP was shown to occur in the presence of cysteine sulfinate (which transaminates OAA very effectively) in Baker’s yeast mitochondrial particles. From this it was concluded that SDH activation by ATP does not occur through removal of OAA [123]. ATP was found to activate SDH within 2-4 minutes, whereas full activation by succinate was achieved only after 15-20 minutes [124]. The activation was found to be not energy dependent as it was insensitive to oligomycin or uncoupler. ATP activation is not seen in several types of enzyme preparations [124]. Whether or not ATP interacts directly with SDH or if it exerts an effect on SDH indirectly via the membrane potential will be investigated in this chapter.
  • 77 3.2 RESULTS 3.2.1 General characterization—In this section the quality and general bioenergetic characteristics of the isolated potato tuber mitochondria used in this study are assessed. Figure 3.2 shows typical oxygen consumption (black) and  (red) traces from an experiment where both parameters were measured simultaneously. ATP (0.2 mM) was incubated to fully activate SDH, the concentration needed to half maximally activate SDH in potato mitochondria was determined to be 3 M [21]. Upon addition of succinate (5 mM) a full membrane potential (217 mV)10 is established (state 2)11 reflected by the upward deflection of the TPP+ signal which indicates that TPP+ molecules disappear from the medium, drawn into the matrix, due to the build up of an electrical potential (the matrix side of the IMM being negative with respect to the cytosolic side). Addition of ADP (0.1 mM) leads to a decrease in  and a increase in oxygen consumption rate (vO2) which demonstrates that respiration is coupled to phosphorylation (state 3). A downward deflection of the TPP+ signal reflects expulsion of TPP+ molecules from the matrix back into the medium. When all ADP is depleted (state 4)  and vO2 return to values comparable to state 2. It can be seen that the amount of ADP added for the first state 3 is not enough to generate a steady state , subsequently twice the amount of ADP is added, which results in a proper state 3. After ADP is depleted again,  and vO2 return to state 4 values. A final addition of ADP (0.2 mM) results in a state 3, which coincides with anoxia.  dissipates slowly, addition of an uncoupler, CCCP (1 M) leads to complete dissipation and the TPP+ signal returns to the value before mitochondria were added. The TPP+ axis does not show units for the membrane potential, as this parameter is not measured directly, see section 2.4.3. It has been reported that upon isolation, plant mitochondria are relatively deenergized and do not generate a membrane potential in the absence of added substrate [139] as opposed to rat liver mitochondria which display an initial low, rotenone sensitive, respiration rate which runs out shortly after the start of an experiment [21]. This observation was confirmed 10 All membrane potential values are given in absolute values as is customarily done in the literature. 11 See section 2.4.4.2 for nomenclature used.
  • 78 in our experiments (not shown) and it is concluded that potato mitochondria upon isolation are depleted of endogenous substrates. With NADH as a substrate (oxidised by the external NADH dehydrogenase, see section 1.2.3) traces similar to the ones in Figure 3.2 were obtained, in contrast to SDH the external NADH dehydrogenase is fully active in the absence of ATP. The membrane potential values obtained in this study agree well with results previously obtained in this laboratory [38, 110, 111, 139] and with those of others [157, 181]. Oxygen consumption rates with succinate as a substrate were found to be higher than with NADH as a substrate which reflects differences in dehydrogenase kinetics [133]. Figure 3.2 Representative traces of oxygen consumption (black) and  (red) measured in isolated potato tuber mitochondria incubated with ATP (0.2 mM) to activate SDH. Additions: succinate (5 mM), ADP (0.1 mM), ADP (0.2 mM), ADP (0.2 mM) and CCCP (1 M). Amount of mitochondrial protein 0.7 mg. Numbers in italics indicate steady state membrane potential values in mV, numbers in regular font indicate steady state oxygen consumption rates in nmol O2 / min / mg protein. Without ATP incubated a state 3 to state 4 transition is required to activate SDH. Figure 3.3 shows typical oxygen consumption (black) and Qr/Qt (red) traces from an experiment where both parameters were measured simultaneously. Upon addition of succinate (5 mM) SDH does not become fully activated which is reflected in the low Qr/Qt value (0.36) 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 0 100 200 300 400 500 0 2 4 6 8 10 12 68 202 63 220 61 217 192 217 176 215 TPP+ signal(V) oxygen(nmol) succinate ADP ADP ADP CCCPanoxia time (min)
  • 79 attained. This value agrees well with previous work done in this laboratory where a 10-35% reduction of the Q-pool was found when succinate was added to potato mitochondria in the absence of ATP [21, 207]. With ATP incubated, addition of succinate leads to a Q- reduction level of 80-90% (data not shown). Addition of ADP (0.2 mM) leads to an increase in vO2 (state 3) and a transient decrease followed by a slow increase in Qr/Qt as SDH is seen to activate throughout state 3 due to the build-up of ATP that is being formed. When all ADP is depleted, vO2 decreases (due to build up of p) and the Q-pool becomes 89% reduced, a value that agrees well with previously obtained results [21, 207]. Upon addition of ADP (0.2 mM) a second state 3 is brought about which coincides with anoxia leading to complete reduction of the Q-pool. NADH at high concentrations (2 mM) reacts strongly with the Q-electrode therefore only representative traces with succinate are shown. Figure 3.3 Representative traces of oxygen consumption (black) and Qr/Qt (red) measured in isolated potato tuber mitochondria. Additions: succinate (5 mM), ADP (0.2 mM), ADP (0.2 mM). Amount of mitochondrial protein 0.5 mg. Numbers in italics indicate steady state oxygen consumption rates in nmol O2 / min / mg protein, numbers in regular font indicate Q redox poise expressed as the fraction QH2 in the pool. 0 100 200 300 400 500 600 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 oxygen(nmol) Qr/Qt time (min) 0.36 0.43 0.89 0.55 80 260 66 238 succinate ADPADP ADP anoxia
  • 80 The RCR values, using the ratio of state 3 and state 4 oxygen consumption rates (see 2.4.4.3) for potato mitochondria respiring on succinate in this study were between 3 and 4 which agrees well with values obtained previously in this laboratory [21, 38, 207] and with those obtained by others [157]. The RCR values indicate that the mitochondria are coupled and that the isolation method yields intact mitochondria. The IMM is shown to function as an isolating membrane, which is a requirement for an energy transducing system (see postulate 4 on page 35). It can be seen in figures 3.2 and 3.3 that vO2 values decrease throughout the trace upon successive state 3 to state 4 transitions, an explanation for this will be offered later. Mitochondria under ADP limited conditions show a non-ohmic  vs. vO2 relationship, see section 3.1.2. Figure 3.4 shows the relationship between ∆ and vO2 under state 4 conditions in potato mitochondria respiring on succinate () or NADH (). Succinate respiration was inhibited with malonate and NADH respiration was titrated upwards using the NADH regenerating system (see section 2.4.4.4). It can be seen that both sets of data overlap which indicates that there are no substrate dependent differences in oxidizing pathway kinetics (see section 2.4.4.4 for a description of reducing and oxidising pathway kinetics). The shape of the relationship between  and vO2 is non-linear and looks similar to the non-ohmic current/voltage relationships found previously in mitochondria, cf. Figure 3.1. Concluding, the isolation method used in this study yields well coupled potato mitochondria of a quality comparable to that of previous studies done in this laboratory. The respiratory kinetics agree well with what has been reported in the literature.
  • 81 Figure 3.4 Combined state 4 cytochrome pathway kinetics of potato mitochondria respiring on either NADH or succinate,  vs. vO2. NADH () data were obtained using the NADH regenerating system. Succinate () data were obtained with the malonate titration method. With succinate as a substrate a state 4 was induced by adding an aliquot of ADP (50M) after the mitochondria were energised with succinate (5mM) + ATP (0.2 mM), when ADP was depleted respiratory activity was titrated with malonate (up to ~ 4 mM). Succinate titration data were obtained from one mitochondrial isolation, data points were taken from three traces. NADH data were obtained from one mitochondrial isolation, data points were taken from two traces. Respiration was initiated by addition of a sub-saturating amount of NADH (10 µM), an aliquot of ADP (50 µM) was added to induce a state 3 to state 4 transition, when ADP was depleted NADH was titrated using sub-saturating amounts up to ~100 µM. Mitochondrial protein used per experiment was ~ 0.8 mg. 3.2.2 Stimulation of SDH by adenine nucleotides—Previous work done in this laboratory demonstrated that SDH was activated by ATP in potato tuber mitochondria [21]. An experiment was done to verify that the mitochondria used in this study displayed the same behaviour. Figure 3.5 shows that upon addition of succinate the Q-pool redox poise (red trace) increases up to 53% with a concomitant vO2 value of 77 nmol O2 / min / mg protein. Addition of ATP (0.2 mM) leads to a further increase in Qr/Qt up to 85% with a concomitant increase in vO2 to 107 nmol O2 / min / mg protein. Upon subsequent addition of ADP (0.2 mM) a state 3 to state 4 transition is seen. These results are comparable to those obtained previously in this laboratory [21, 207]. 0 20 40 60 80 100 150 160 170 180 190 200 210 220 230 succinate NADH vO2 nmolO2 /min/mgprotein membrane potential mV
  • 82 Having reproduced the original observation showing that ATP has a stimulatory effect on SDH (reflected in both vO2 and Qr/Qt) in a next series of experiments the effect of adenine nucleotides on the membrane potential in potato mitochondria respiring on succinate was investigated. Figure 3.5 ATP induced activation of SDH. Representative oxygen concentration (black) and Q-reduction (red) traces illustrate the activation of SDH by ATP. At the points indicated by the arrows succinate (9 mM), ATP (0.2 mM) and ADP (0.2 mM) were added. Mitochondrial protein used was 1 mg. Numbers in italics indicate Qr/Qt values; numbers in standard font indicate vO2 values (nmol O2 / min / mg protein). ATP was hypothesized to stimulate SDH indirectly, via a change in  [21]. Upon hydrolysis of ATP by complex V a membrane potential can be generated [1]. One possibility is that the addition of ATP itself leads to the generation of  which subsequently activates SDH. This is hard to envisage under ADP limited conditions where the high back pressure of p would prevent the hydrolysis of ATP [162]. However, as it was found that incubation of ATP leads to full activation of SDH prior to addition of succinate [207] it could be the case that  generated by ATP hydrolysis activates SDH 0 100 200 300 400 500 600 0 0.2 0.4 0.6 0.8 1 0 0.8 1.6 2.4 3.2 4 4.8 5.6 6.4 oxygen(nmol) Qr/Qt time (min) succinate ATP ADP anoxia 0.53 0.84 0.71 0.94 77 106 324 113
  • 83 prior to the initiation of respiration by addition of succinate. It has been reported that addition of ATP alone (no substrate present) to intact potato mitochondria cannot establish a measurable membrane potential [162, 178]. This was confirmed in an experiment where ATP was added to potato mitochondria in the reaction medium before addition of substrate, see Figure 3.6. Upon addition of ATP (0.2 mM) a transient  is generated with a peak value of 166 mV. No concomitant changes were observed in vO2 or Qr/Qt, which indicates that the transient  was not generated due to ETC activity. Two minutes after dissipation of the ATP induced , succinate (5 mM) is added, SDH is fully activated (as further addition of ATP does not result in an extra increase in  or vO2 , not shown). ATP does not react with the TPP+ electrode and the transient  seen in Figure 3.6 is not a measuring artifact. With carboxyatractyloside (CAT), an inhibitor of the adenine nucleotide carrier [126], or oligomycin incubated, no transient  was seen upon addition of ATP which indicates that the transient  is indeed generated through ATP hydrolysis. Figure 3.6 Addition of ATP to potato mitochondria in reaction medium leads to a transient . Additions: ATP (0.2 mM) succinate (5 mM). Membrane potential values in mV. Mitochondrial protein used was 0.6 mg. It can be concluded from this experiment that SDH is fully activated by incubation with ATP, as previously shown [207] and that the continuous presence of a  is not a 1.2 1.3 1.4 1.5 1.6 1.7 0 1 2 3 4 5 6 7 8 TPP+ signalV time (min) ATP succinate 166 225
  • 84 requirement to keep the complex activated until the moment of succinate addition. One interpretation of this would be that the transient  activated SDH and that the protein remained in an active configuration after  was dissipated. This however is unlikely as it was found by Affourtit et al. that deenergization of potato mitochondria respiring on succinate (in the absence of ATP) led to deactivation of SDH [21]. It was hypothesized by Affourtit that addition of ATP leads to an increase in  stimulating SDH activity which is reflected by an increase in Qr/Qt and vO2, see Figure 3.5 and compare figures 1 and 2 from [21]. The experiment from Figure 3.5 was repeated, this time determining oxygen consumption rate and the membrane potential simultaneously, see Figure 3.7. Upon addition of succinate a 224 mV membrane potential is generated with a concomitant vO2 rate of 77 nmol O2 / min / mg protein. Addition of ATP (0.2 mM) leads to an increase in ∆ up to 230 mV with a concomitant increase in vO2 to 83 nmol O2 / min / mg protein. Upon subsequent addition of ADP (0.2 mM) a state 3 to state 4 transition is seen. This experiment shows the assumption made by Affourtit et al. [21] to be correct. Addition of ATP to potato mitochondria respiring on succinate leads to an increase in . When ATP was pre-incubated a maximum  (~220-230 mV) upon addition of succinate was attained, further additions of ATP had no effect on the membrane potential (data not shown). It was also found by Affourtit et al. that ADP had a stimulatory effect on SDH (cf. Figure 2A in [21]). Addition of ADP to mitochondria in the absence of CAT leads to a decrease in  due to phosphorylation (cf. figures 3.2 and 3.7). To avoid this, potato mitochondria were incubated with CAT12 and under these conditions the addition of ADP to mitochondria respiring on succinate was found to stimulate SDH to an even higher extent than ATP [21] (Qr/Qt and vO2 measured simultaneously). Through inhibition of the adenylate kinase ((by incubating with Ap5A) ADP was prevented from being converted into ATP and it was concluded that the stimulatory effect was due to ADP per se. The experiment was repeated, this time measuring the membrane potential and oxygen consumption rate simultaneously, see Figure 3.8. 12 Addition of CAT to potato mitochondria respiring on succinate does not lead to a change in  (data not shown).
  • 85 1.6 1.7 1.8 1.9 2 2.1 2.2 0 80 160 240 320 400 480 0 2 4 6 8 10 TPPsignal(V) oxygen(nmol) time (min) succinate ADPADPADPADPATP 224 23077 83 anoxia Figure 3.7 ATP induced activation of SDH. Representative oxygen concentration (black) and membrane potential (red) traces illustrate the activation of SDH by ATP. At the points indicated by the arrows succinate (9 mM), ATP (0.2 mM) and ADP (0.2 mM) were added. Membrane potential values (italic) in mV, oxygen consumption rate values (regular font) in nmol O2 / min / mg protein. Mitochondrial protein used was 0.8 mg. Figure 3.8 ADP induced activation of SDH in the presence of CAT (10 M). Representative membrane potential trace illustrating the activation of SDH by ADP. At the points indicated by the arrows succinate (9 mM) and ADP (0.2 mM) were added. Membrane potential values in mV. Mitochondrial protein used was 0.6 mg. 1.1 1.2 1.3 1.4 1.5 1.6 1.7 0 1 2 3 4 5 6 7 TPP+ signal(V) time (min) 204 220 ADPsuccinate
  • 86 With CAT (10 M) incubated, upon addition of succinate (5 mM) a membrane potential of ~204 mV was established, addition of ADP (0.2 mM) led to a further increase up to 220 mV. This result again confirmed Affourtit’s assumption [21] that addition of adenine nucleotides (CAT incubated when ADP is used) leads to an increase in . 3.2.3 Are the effects of ATP on , Qr/Qt and vO2 simultaneous?—The experiments shown thus far were done with either a combination of an oxygen with a Q-electrode or with a combination of an oxygen with a TPP+ -electrode. One of the aims of this thesis work was to set up a combined system of an oxygen, a Q and a TPP+ -electrode in order to determine three parameters simultaneously, vO2, Qr/Qt and . The reaction vessel used allows for multiple electrodes to be fitted, see Figure 2.1. The Q-electrode (platinum and glassy carbon electrodes) and the TPP+ -electrode were fitted in the reaction vessel. To the same vessel two reference electrodes (see Figure 2.7A) were fitted. The presence of the TPP+ -electrode did not lead to any interference with the Q-electrode. However the TPP+ - electrode baseline signal shifted by about 3 V (increase) when the Q-electrode was switched on. Apart from displacing the baseline signal by a fixed value no interference with the TPP+ -electrode was seen. In control experiments using isolated fresh potato tuber mitochondria respiring on succinate it was determined that all electrodes operated independently and no electrical cross-talk was observed, i.e. recordings made with individual electrodes looked the same as recordings made with combinations of electrodes. With a working system it was now possible to simultaneously measure three bioenergetic parameters in isolated mitochondria. Figure 3.9 shows that upon addition of ATP to potato mitochondria respiring on succinate the changes in vO2, Qr/Qt and  occur simultaneously13 . Setting up the 3-electrode system was successful and recordings were made with both potato and S. pombe mitochondria. It was however decided not to use it as the standard technique. Setting up the system is a delicate matter. Both the Q-electrode and the TPP+ - electrode signals need some time to stabilise at the beginning of the experiment. Then the TPP+ -electrode needs to be calibrated which is a time consuming process. 13 Given that the change in oxygen consumption rate is small it cannot be appreciated from the oxygen concentration curve without looking at the first derivative of this trace.
  • 87 Figure 3.9 Activation of SDH by ATP. Three parameters: oxygen concentration (black), Qr/Qt (red) and  (blue) measured simultaneously. Additions: succinate (5 mM) ATP (0.2 mM). Mitochondrial protein used was 0.7 mg. The Q-electrode can be volatile in its behaviour and has at times the tendency to show drift at the end of a trace when anoxia is reached. Because of this no values can be assigned to the Q recording as the Q-signal under anoxia is used as a reference value. Problems with the Q-electrode arose more often in the 3-electrode setup than with the combination of just the oxygen and Q-electrode. It was deemed more effective to do duplicate experiments with two set-ups (oxygen-Q and oxygen-TPP+ ) on the same day with the same mitochondrial preparation and combine the results. Determining vO2, Qr/Qt and  in parallel was the standard way of doing experiments throughout the remainder of this work. The results shown in figures 3.7-3.9 indicate that addition of adenine nucleotides to potato mitochondria respiring on succinate does indeed lead to an increase in . But this in itself does not prove that it is the change in  which stimulates SDH. Figure 3.10 shows a modular representation (see section 1.5) of the ETC of fresh potato mitochondria respiring on succinate. There is only one Q-reducing pathway (SDH in combination with the dicarboxylate carrier) and one Q-oxidising pathway (the cytochrome pathway). Arrows indicate the rates of Q-pool reduction (a) and Q-pool oxidation (b). In
  • 88 terms of this model three hypotheses can be formulated to explain the observations so far described: 1) Addition of ATP or ADP (in the presence of CAT) leads to an increase in  through interaction with a component which is outside the defined system (essentially Affourtit’s hypothesis, inhibition of PmitoKATP [21], see section 3.1.1). The increase in  stimulates SDH, this leads to an increase in rate a and a concomitant increase in reduction of the Q- pool, as an effect of this, rate b will also increase which leads to an increased vO2 (cf. Figure 3.5). 2) Addition of ATP or ADP (in the presence of CAT) leads to a direct stimulation of SDH, this leads to an increase in rate a and a concomitant increase in reduction of the Q-pool, as an effect of this rate b increases which leads to increased activity of the cytochrome pathway. The electron transfer rate through complexes III and IV increases and so does the rate of proton translocation.  is a passive follower of events and not an effector in this case. 3) Addition of ATP or ADP (in the presence of CAT) has a dual effect. Both SDH and  are affected, which leads to a simultaneous change in both reducing and oxidising pathway kinetics. Figure 3.10 Modular representation of the ETC in potato mitochondria respiring on succinate, see text. To test hypothesis 1 experiments were done in the presence of an uncoupler.
  • 89 3.2.4 Stimulation of SDH by adenine nucleotides in the presence of uncoupler—In the following experiment the effect of ATP on SDH activity was assessed in the presence of uncoupler, see Figure 3.11. In the absence of ATP addition of succinate (5 mM) generates a membrane potential of 210 mV. Addition of an uncoupler increases proton permeability of the IMM which leads to dissipation of p. Because of this the activity of the ETC increases in a futile attempt to restore p which is concomitant with rapid respiration [1]. It can be seen that upon addition of CCCP (1 M)  is dissipated but vO2 only increases 1.4 times in contrast to what is seen when ADP is added (cf. Figure 3.3) where vO2 is increased ~3.3 times. This suggests that SDH is partially deactivated. Subsequent addition of ATP (0.2 mM) approximately 2 minutes later results in an increased respiration rate (~2.8), but no change in  is seen. The same experiment but with ADP (0.2 mM) instead of ATP gives similar results (data not shown). Figure 3.11 Stimulatory effect of ATP on SDH in the presence of uncoupler. Representative  (red) and oxygen consumption (black) traces. Additions: succinate (5 mM), CCCP (1 M) and ATP (0.2 mM). Membrane potential values in mV, oxygen consumption rate values in nmol O2 / min / mg protein. Mitochondrial protein used was 0.5 mg. 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 0 80 160 240 320 400 480 0 2 4 6 8 10 TPP+ signal(V) oxygen(nmol) time (min) 210 82 115 324 succinate CCCP ATP
  • 90 Affourtit hypothesized that the activity of SDH by adenine nucleotides is modulated via the protonmotive force [21] but could not demonstrate this, concluding that: “the data presented in this paper remain inconclusive as to whether activation of SDH by adenine nucleotides is also wholly mediated by the protonmotive force” (taken from [21]). This question can now be answered conclusively, Figure 3.11 demonstrates that addition of ATP to uncoupled potato mitochondria respiring on succinate leads to activation of SDH but not to a change in . Based on these results it was concluded that adenine nucleotides under uncoupled conditions can stimulate SDH directly. Hypothesis 1 can be discarded. Hypothesis 2 can explain the results shown thus far, a stimulatory effect of adenine nucleotides on SDH leads to both an increase in Qr/Qt and , with the latter being a passive follower of events. Further investigation of the modulatory role of  on SDH stimulation will reveal that hypothesis 2 is also incorrect. 3.2.5 Stimulation of SDH by oligomycin—Affourtit et al. found an activating effect on SDH by oligomycin (an inhibitor of the ATP synthase, see section 1.2.9). Addition of oligomycin to fresh potato mitochondria respiring on succinate (no ATP incubated) results in a large increase in Qr/Qt (cf. Figure 3A in [21]) which is concomitant with a small but significant increase in vO2 [21]. This was interpreted as a stimulation of SDH, however, upon addition of uncoupler, (which dissipates p) the respiratory rate did not increase. This suggests that the oligomycin induced activation of SDH was reversed by uncoupler, or that SDH was not activated in the first place at all. A similar effect was seen with N,N’- dicyclohexylcarbodiimide (DCCD) another inhibitor of complex V [21], suggesting that oligomycin has no direct effect on SDH. The activating effect of oligomycin on SDH was not to the same extent as that by ATP, maximal activation by oligomycin was ~ 85% of the stimulation observed at saturating ATP concentrations [21]. Previous work done in this laboratory by Fricaud et al. has shown that addition of oligomycin (and other ATP synthase inhibitors such as efrapeptin and aurovertin) to potato mitochondria respiring on succinate, in the absence of ATP, results in an increase in magnitude of  (~ 20 mV) [110].
  • 91 Figure 3.12 Effect of oligomycin on potato mitochondria respiring on succinate. Representative  (blue) Qr/Qt (red) and oxygen concentration (black) traces, measured simultaneously using the 3-electrode system. Additions: succinate (5 mM), oligomycin (1 M). Amount of protein used was 0.7 mg. Figure 3.12 shows a representative experiment where oxygen consumption (black), Qr/Qt (red) and  (blue) were determined simultaneously (using the 3-electrode setup). Upon addition of succinate (5 mM) a  of 214 mV was established, with a concomitant vO2 of 66 nmol O2 / min / mg protein and a Qr/Qt of 24%. Addition of oligomycin (1 M) led to an increase in magnitude of  to 230 mV (agreeing well with results by Fricaud et al. [110]) with a concomitant increase in Qr/Qt to 80 % (agreeing well with results by Affourtit et al. [21]) vO2 however decreased slightly, to 60 nmol O2 / min / mg protein. The results in Figure 3.12 confirm both Affourtit’s and Fricaud’s earlier findings showing that addition of oligomycin leads to an increase in Qr/Qt and an increase in  respectively, in potato mitochondria respiring on succinate. The observed decrease in vO2 is at variance with Affourtit’s findings [21] but in agreement with results from Fricaud et al. [110, 111] who found that upon addition of oligomycin to potato mitochondria respiring on succinate, respiration was inhibited by 31% [111]. Interestingly enough it was found in the same study that addition of ATP (0.25 mM) to potato mitochondria respiring on succinate also led to inhibition of respiration (by 25%) [111].
  • 92 Inhibitory effects of ATP and oligomycin on respiration were also seen with NADH as a substrate [110] which indicates that these effects are not specific for succinate dependent respiration. This suggests that the inhibitory actions of ATP and oligomycin exert their effects at the cytochrome pathway side. The only apparent difference between the experiments of Affourtit and Fricaud is the time interval between the addition of succinate and the addition of either ATP or oligomycin. In Affourtit’s experiments ATP or oligomycin were added typically within 1-3 minutes after respiration was initiated by the addition of succinate, whereas in Fricaud’s experiments this interval was between 5-7 minutes. Potato mitochondria in this study were isolated using the exact same protocol as used by Affourtit14 (see section 2.1.3) and RCR values are in the same range (~3-4) as in Affourtit’s study [153]. The results shown so far are in good agreement with results obtained by Affourtit previously [21] and it is therefore assumed that the quality and the respiratory kinetics of the potato mitochondria in this study are essentially the same as those in [21]. The RCR values from the study of Fricaud are in the range of 3-4 as well. The protocol for isolating potato mitochondria used by Fricaud [38] is somewhat different to the one used by Affourtit and this author. There are some small differences in concentration of some of the chemicals in the grinding medium: Chemical Affourtit / this study Fricaud MOPS 40 mM 20 mM cysteine 10 mM 7 mM EDTA 2 mM 10 mM and in the reaction medium: Chemical Affourtit / this study Fricaud MOPS 20 mM 10 mM MgCl2 1 mM 5 mM KH2PO4 5 mM 10 mM 14 Dr Affourtit personally supervised the isolation of the potato mitochondria during the initial stage of this D. Phil project
  • 93 More importantly though, in Fricaud’s study [38] succinate (1 mM) was added to the washing medium to minimise SDH inactivation. Possibly the differences in respiratory kinetics between the studies of Affourtit and Fricaud were due to slight differences in the method of isolation. Since the potato mitochondria used in this study are essentially the same as those used by Affourtit (and are therefore expected to behave in a similar way) this hypothesis was tested by repeating some of the experiments done by Fricaud et al. 3.2.6 Adenine nucleotides and oligomycin inhibit succinate dependent respiration in potato mitochondria—It was shown that addition of ATP or oligomycin to potato mitochondria respiring on succinate leads to an inhibition of respiration [110]. It was already shown that addition of oligomycin leads to a decrease in vO2, see Figure 3.12. To investigate the effects of ATP on succinate dependent respiration, experiments were done using the oxygen electrode. In order to appreciate changes in oxygen consumption rate it is useful to take the first derivative of the oxygen concentration trace. Figure 3.13 shows the slow activation of SDH by succinate (SDH is a homotropic enzyme), the oxygen concentration (black) decreases slowly until anoxia is reached and the oxygen consumption rate (red) is seen to increase slowly over time until after ~9 minutes a steady state rate is reached. Since oxygen concentration decreases over time the derivative of this trace has only negative values.
  • 94 Figure 3.13 SDH activation by succinate in potato mitochondria. Representative oxygen consumption trace (black) and its first derivative (red) which represents the oxygen consumption rate. Succinate was added to initiate respiration (5 mM), mitochondrial protein used is 0.8 mg. To test the effect of ATP on vO2, experiments were done where ATP was added to potato mitochondria respiring on succinate either 2 or 6 minutes after respiration was started with succinate. Figure 3.14 shows the time dependent effects of ATP on succinate dependent respiration in potato mitochondria. Succinate (5 mM) was added to initiate respiration, ATP (0.2 mM) was added after 2 minutes (A) or 6 minutes (B). It can be seen in Figure 3.14A that upon addition of ATP vO2 stabilises (at ~50 nmol O2 / min / mg protein) whereas in 3.14B it is clearly inhibited (stabilising at ~45 nmol O2 / min / mg protein). Interestingly enough the final vO2 reached with succinate alone (~87 nmol O2 / min / mg protein) is higher than with ATP present (data not shown). 0 100 200 300 400 500 600 -100 -80 -60 -40 -20 0 0 2 4 6 8 10 12 oxygen(nmol) vO2 nmolO2 /min/mgprotein time (min) succinate anoxia
  • 95 Figure 3.14 Time dependent effects of ATP on succinate dependent respiration. Oxygen consumption (black) and its first derivative (red). Succinate (5 mM) was added to the medium to initiate respiration, ATP (0.2 mM) was added 2 minutes (A) or 6 minutes (B) after succinate addition, traces done with same mitochondrial preparation, mitochondrial protein used is 0.8 mg. 250 300 350 400 450 500 550 -60 -50 -40 -30 -20 -10 0 10 0 2 4 6 8 10 oxygen(nmol) vO2 nmolO2 /min/mgprotein time (min) succinate ATP A 100 200 300 400 500 600 -80 -70 -60 -50 -40 -30 -20 -10 0 2 4 6 8 10 12 oxygen(nmol) vO2 nmolO2 /min/mgprotein time (min) succinate ATP B
  • 96 Steady state vO2 values from a series of duplicate experiments are given in Table 3.1. Mitochondria were well coupled (RCR 4.6)15 and the respiratory rate under ADP limited conditions was relatively low. Table 3.1 Averaged steady state oxygen consumption rates of potato mitochondria respiring on succinate. Rates obtained from one mitochondrial isolation, averaged values from duplicate experiments. Concentrations: succinate (5 mM), ATP (0.2 mM). Mitochondrial protein used was 0.8 mg. From Table 3.1 it can be seen that addition of ATP has an inhibitory effect on succinate dependent respiration in potato mitochondria as the highest oxygen consumption rate is reached when only succinate is present. As described in section 3.1.3, SDH is a homotropic enzyme, i.e. succinate is not only substrate but also activator. Activation of SDH by succinate is a slow process and can take up to 15-20 minutes [124]. It can be seen in Figure 3.13 that vO2 starts accelerating the moment succinate is added and after approximately 9 minutes a steady state rate is reached. In Figure 3.14A it can be seen that upon addition of ATP acceleration stops immediately and a steady state oxygen consumption rate is reached. Taking the average oxygen consumption rate prior to and after addition of ATP would lead one to conclude that addition of oxygen consumption is increased upon addition of ATP. When comparing Figure 3.14A with Figure 3.13 it becomes apparent that addition of ATP in fact limits succinate dependent respiration. This does not disprove however that ATP stimulates SDH activity in potato mitochondria, Figure 3.11 clearly shows that in the presence of uncoupler ATP activates SDH. It was also shown by comparison of reducing pathway kinetics [21] that SDH activity is higher in the presence than in the absence of ATP. 15 state 3 rate / state 4 rate Additions averaged steady state vO2 (nmol O2 /min /mg protein) succinate 87 succinate + ATP (added after 2 min) 50 succinate + ATP (added after 6 min) 45
  • 97 It was shown by Fricaud et al. that addition of ATP led to inhibition of succinate dependent respiration [110]. The effects of ATP addition on  however were not determined. Figure 3.7 showed that addition of ATP, within 3 minutes after addition of succinate, leads to an increase in . The averaged vO2 after addition of ATP is also increased slightly. An experiment was done where ATP (0.2 mM) was added ~6 minutes after initiation of respiration with succinate (5 mM), see Figure 3.15. It can be seen clearly that  increases upon addition of ATP, vO2 however decreases considerably. The same experiment was repeated, this time measuring vO2 and Qr/Qt simultaneously, see Figure 3.16. Again it can be seen that upon addition of ATP (~6 minutes after initiation of respiration with succinate) Qr/Qt increases (cf. Figure 3.5) vO2 however decreases considerably. It was reported by Fricaud et al. that ATP and oligomycin also had an inhibitory effect on NADH dependent respiration [110], suggesting that these effects are substrate independent. Experiments with the oxygen electrode revealed that in potato mitochondria respiring on NADH addition of either ATP or oligomycin led to a decrease in oxygen consumption rates (data not shown), comparable to [110]. Figure 3.15 Inhibition of succinate dependent respiration by ATP. Representative vO2 (black) and  (red) traces. Respiration was initiated with succinate (5 mM), ATP (0.2 mM) was added ~6 minutes later. Amount of mitochondrial protein used was 0.8 mg. -120 -100 -80 -60 -40 -20 0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 0 2 4 6 8 10 vO2 nmolO2 /min/mgprotein TPP+ signalV time (min) succinate ATP
  • 98 Figure 3.16 Inhibition of succinate dependent respiration by ATP. Representative vO2 (black) and Qr/Qt (red) traces. Respiration was initiated with succinate (5 mM), ATP (0.2 mM) was added ~6 minutes later. Amount of mitochondrial protein used was 0.5 mg. The results from figures 3.13-3.16 indicate that the differences in respiratory kinetics between the studies of Affourtit and Fricaud are not due to a difference in isolation of the mitochondria since Fricaud’s findings could be reproduced in potato mitochondria which are essentially the same as the mitochondria used in Affourtit’s study. Figures 3.14 to 3.16 indicate that with mitochondria from the same preparation one can get different responses upon addition of ATP. -100 -80 -60 -40 -20 0 20 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 time (min) vO2 nmolO2 /min/mgprotein Qr/Qt succinate ATP
  • 99 3.3 DISCUSSION In this study a modulatory role of the protonmotive force on SDH regulation in fresh potato tuber mitochondria was investigated. In a previous study done in this laboratory the activation of SDH by adenine nucleotides was quantified in terms of Q-redox poise and oxygen consumption rates [21]. It was believed that oligomycin also had an activating effect, which was unexpected. It was hypothesized that SDH was activated indirectly by adenine nucleotides using p as an intermediate. Evidence was given suggesting a possible role for the mitochondrial K+ ATP channel in this process [21]. The potato mitochondria isolated in this study are of good quality and the respiratory kinetics agree well with what has been found previously in this laboratory [21, 110] and by others [157], (figures 3.2-3.4). The stimulatory effect of adenine nucleotides on SDH (in terms of Qr/Qt and vO2) as found by Affourtit et al. [21] was reproduced (Figure 3.5). It was previously shown in this laboratory that incubation in the presence of ATP fully activates SDH in potato mitochondria [207]. It was shown that addition of ATP to potato mitochondria in the absence of substrate leads to a transient  which was generated by the ATP synthase via hydrolysis of ATP (Figure 3.6). The continuous presence of  was not a requirement to keep complex II activated until the moment of succinate addition. Possibly the transient  activated SDH which remained in an active configuration after  was dissipated. This is unlikely however as it was determined that deenergization of potato mitochondria respiring on succinate (in the absence of ATP) led to a deactivation of SDH [21]. It was hypothesized originally by Affourtit et al. [21] that addition of ATP would lead to an increase in . This was confirmed in an experiment where  was determined using a TPP+ -electrode [154] (Figure 3.7). It was determined that in the presence of CAT, addition of ADP to potato mitochondria respiring on succinate leads to stimulation of SDH in a way similar to stimulation by ATP [21]. Through inhibition of the adenylate kinase ADP was prevented from being converted into ATP and it was concluded that the stimulatory effect was due to ADP per se. Subsequent addition of ATP does not lead to any further significant stimulation of SDH (cf. Figure 2A in [21]), this suggests that both adenine nucleotides activate SDH via the same mechanism [21]. Addition of ADP to potato mitochondria respiring on succinate, incubated
  • 100 in the presence of CAT was found to increase  (Figure 3.8), again confirming the hypothesis by Affourtit that addition of adenine nucleotides16 can lead to an increase in . Using a 3-electrode set-up it was possible to determine vO2, Qr/Qt and  simultaneously. It was shown in potato mitochondria respiring on succinate that upon addition of ATP the parameters , Qr/Qt and vO2 all change simultaneously (Figure 3.9). The 3-electrode system performed adequately but proved in practice too volatile to use effectively for experimental work. It was decided to do duplicate experiments using two set-ups (oxygen-Q and oxygen-TPP+ ) using mitochondria that were isolated the same day. It was shown that in the presence of uncoupler (p dissipated) the addition of ATP or ADP could stimulate SDH (Figure 3.11). Therefore the presence of  is not required for adenine nucleotides to activate SDH. This does not exclude the possibility that under coupled conditions there could be an indirect effect of adenine nucleotides on SDH activation via a change in . It was found that a decrease in  by addition of uncoupler leads to deactivation of SDH (cf. Figure 3A in [21]) which suggests that an increase in  might activate SDH. Affourtit discusses several mechanisms via which the addition of adenine nucleotides could lead to an increase in magnitude of  in potato mitochondria [21]: A possible role for the uncoupling protein: ATP could have an inhibitory effect on the mitochondrial plant uncoupling protein (UCP, see section 1.2.10). Potato tuber mitochondria express an uncoupling protein [44, 180, 208]. Its activity leads to an increased proton conductivity of the IMM which could lead to a decrease in the magnitude of . It is known that mammalian UCP activity can be inhibited by purine nucleotides [1]. Borecký et al. expressed the Arabidopsis thaliana uncoupling protein in E. coli cells and found that both ATP and ADP could inhibit its activity [209]. The effects of ADP and ATP on the membrane potential in potato mitochondria could be explained in terms of UCP inhibition. After Affourtit’s study was published however it was found that in potato mitochondria, addition of ATP had no effect 16 mitochondria incubated in the presence of CAT when ADP is added.
  • 101 on fatty acid induced uncoupling [179], i.e. UCP activity in potato mitochondria is insensitive to the addition of ATP (it is inhibited by GTP [210]). Therefore the increase in  caused by ATP addition to potato mitochondria could not have occurred through the inhibition of UCP. A possible role for CAT-insensitive transport of adenine nucleotides: SDH in potato mitochondria respiring on succinate, incubated in the presence of CAT, is activated by either ATP or ADP, this suggests that the activation occurs at the cytosolic side of the IMM. This interpretation was not fully conclusive though as ATP might enter the matrix via a CAT-insensitive nucleotide carrier [21]. The mitochondrial ATP-Mg/Pi transporter was reported to catalyse the exchange of ATP-Mg for Pi [211]. The name ATP-Mg/Pi transporter was later found to be a misnomer as this protein can in fact exchange AMP and ADP as well [212]. The exchange of adenine nucleotides for phosphate is electroneutral and occurs in the presence of CAT [212]. An ortholog of this protein has been putatively identified in Arabidopsis thaliana [212]. Experiments done by Leach in a previous study in this laboratory [207] suggest however that CAT-insensitive nucleotide transport does not occur in potato mitochondria. In the absence of both Mg2+ and Pi and presence of CAT addition of ATP led to activation of SDH. This suggests strongly that ATP activates SDH on the cytosolic side and does not cross the IMM in a CAT- insensitive way. If ADP were to be transported by the ATP-Mg/Pi transporter in the presence of CAT this could activate the ATP-synthase (inducing a state 3) which would result in a decrease of Qr/Qt and , but this is not observed, see Figure 3.8 and compare figures 1B and 2A in [21]. A possible role for the K+ -ATP channel: The existence of a mitochondrial K+ ATP channel in plants (PmitoKATP) has been demonstrated [172]. This channel allows K+ ions to flow into the matrix thereby depolarising the existing  across the IMM. This channel can be inhibited by both ATP and ADP [172] which leads to an increase in . SDH is stimulated by ATP and ADP and
  • 102 is possibly deactivated by a decrease in . Given the parallels between both processes, suggesting a relationship between PmitoKATP inhibition and SDH activation is very reasonable. This channel was found to be selective for K+ and Cs+ which both could collapse  in respiring mitochondria, whereas Na+ and Li+ had no effect [172], therefore in order for this channel to be able to exert an effect on , potassium has to be present in the medium. It has been reported however that the flux through PmitoKATP is too small to decrease  significantly (only 1 to 2 mV) [213]. Affourtit claims that “when mitochondria are incubated in the presence of KCl, succinate alone is incapable of generating a ” referencing [172]. This however is not true. Work done previously in this laboratory and by others, demonstrates that potato mitochondria, incubated in the presence of KCl, generate a  upon addition of succinate [38, 110, 111, 139, 175, 181, 210]. In this study it was also found that potato mitochondria incubated in the presence of KCl generate a membrane potential upon addition of succinate, see figures 3.7-3.9, 3.11, 3.12 and 3.15. Upon reading [172] it also becomes apparent that Pastore et al. never claim that in mitochondria incubated in the presence of KCl succinate is incapable of generating a . Figure 2D in [172] clearly shows that durum wheat mitochondria incubated in the presence of various concentrations of KCl generate a  upon addition of succinate. Only at a high concentration of KCl (60 mM) is the generation of a  prevented. What the article does show is that in the presence of ATP, potassium induced depolarisation can be partially prevented (cf. Figure 2B in [172]). To investigate a possible role of PmitoKATP in the activation of SDH by adenine nucleotides the role of K+ in SDH activation was studied [21]. Figure 3.16 shows a figure taken from [21] (Figure 4A) in which an effect of K+ on SDH activation can be clearly seen. Reduction of the Q-pool in time was measured in potato mitochondria under various energetic conditions. At T = 0 succinate was added to the reaction medium. Experiments 1, 3 and 5 were performed in potato reaction medium (see section 2.1.3.2), which contains K+ ; experiments 2, 4 and 6 were performed in potato reaction medium, but with Na+ substituted for K+ . Trace 1 shows time resolved Q-reduction upon addition of succinate; no steady state is reached before anaerobiosis. In the absence of K+ a steady state is reached quickly, trace 2. When incubated with the uncoupler CCCP, SDH is not activated at all irrespective of the medium used, traces 3 and 4. When incubated with ATP SDH activation is maximal, traces
  • 103 5 and 6. A clear effect of K+ on SDH activation is demonstrated by the difference in traces 1 and 2. However, this effect is not related to an effect of the PmitoKATP as indicated by experiments 5 and 6. ATP is assumed to activate SDH indirectly via  which is assumed to increase due to inhibition of the PmitoKATP by ATP. In the absence of K+ in the medium PmitoKATP will be inactive and has no influence on . Inhibiting PmitoKATP in the absence of K+ therefore will have no effect on  and trace 5 should look similar to trace 1. SDH activation cannot not occur via inhibition of PmitoKATP. Figure 3.16 The effect of K+ on SDH activation. Figure 4A from [21], see text for details. Concluding, also under coupled conditions PmitoKATP is not involved in the process of SDH activation. K+ has been found to directly inhibit SDH [214], it was suggested that potassium induces a conformational change of the enzyme to an inactive form. Other salts such as NaCl, choline chloride and NaNo3 were shown to have a similar inhibitory effect. It was also found previously that anions activate SDH [170, 206, 215]. Perhaps these ion induced changes in SDH activation are due to direct electrostatic effects and Affourtit’s potassium experiments show a direct inhibitory effect of K+ on SDH. It has been found that anions favour dislodgement of OAA from SDH [124]. Possibly the presence of potassium favours binding of OAA to SDH.
  • 104 SDH activity has been shown to be dependent on the protonmotive force [21, 169, 216]. The experimental results presented in this chapter suggest that ATP and ADP can activate SDH in the absence of  which indicates that SDH activity is not modulated by  exclusively which leaves the possibility open that under coupled conditions Affourtit’s hypothesis could still be true. After investigating the mechanisms with which adenine nucleotides could affect  in potato mitochondria, taking into account recent work that was published after Affourtit’s publication the possibility that ADP and ATP can stimulate SDH indirectly via  seems unlikely. ADP and ATP most likely interact directly with SDH. It was shown that both nucleotides stimulate SDH cytosolically [21]. Based on its known structure (see figures 1.5 and 1.6) the only tentative binding sites for adenine nucleotides could be located on the small fragments of subunits C and D which extend into the IMS. A phosphorylation site on the SDH flavoprotein in potato mitochondria has been identified [217], this subunit however extends into the matrix and is therefore not involved in cytosolic stimulation of SDH. Recently it has become apparent that complex II in plant mitochondria is structurally different from its mammalian, protist and fungal counterparts. Complex II has been characterized for bacteria, protozoa, fungi and animals and nearly always is composed of four subunits [218] : SDH1 to SDH4 (see section 1.2.4). Recently however it was determined by Eubel and colleagues using 2D Blue-native/SDS PAGE that SDH in potato mitochondria contains seven subunits [219] of which three were positively identified as SDH1, SDH2 and SDH3. In follow-up research Millar et al. subsequently found eight complex II subunits in Arabidopsis thaliana, bean and potato mitochondria [218]. In line with existing nomenclature the new subunits were named SDH5 to SDH8, apparent molecular masses being: 65 kDa (SDH1), 29 kDa (SDH2), 18 kDa (SDH5), 15 kDa (SDH6), 12 kDa (SDH3), 7 kDa (SDH7), 6 kDa (SDH4) and 5 kDa (SDH8). As in other heterotrophic eukaryotes all subunits are nuclear encoded, i.e. also in plant mitochondria complex II is the only main ETC complex, which does not contain mitochondrial-encoded subunits. Sequence comparisons between subunits SDH5 to SDH8 with protein databases did not allow identification of similar proteins of known functions [218]. Possibly one or more of the newly identified subunits of the plant SDH face the IMS and contain(s) phosphorylation sites. The difference in structure may also explain differences in
  • 105 mammalian and plant SDH regulation. Affourtit found that SDH in rat liver mitochondria is fully activated by succinate alone and does not require addition of ATP (cf. Figure 6B in [21]). In another study it was found that ATP has no effect on SDH in rat liver mitochondria as the addition of ATP to rat liver respiring on succinate did not lead to an increase in , (cf. Figure 1B in [44]). Future research will have to clarify this. Time dependent effects of ATP addition: Fricaud et al. determined that potato mitochondria synthesize ATP under ADP limited conditions [110]. It was found that upon addition of succinate there was a significant increase in ATP concentration. Experiments showed that endogenous ADP present upon isolation was used for phosphorylation and the ATP produced was converted back to ADP by adenylate kinase. This explains the effects of ATP-synthase inhibitors under ADP limited conditions,  is increased and vO2 is decreased because the continuous proton influx through the ATP synthase is blocked, which results in decreased IMM conductivity. As discussed in section 1.2.9 ATP synthase activity is regulated by the natural inhibitor protein IF1. Upon increase of  this protein unbinds and ATP synthase activity increases. When ATP concentration increases (addition of ATP or after a state 3 to state 4 transition) IF1 is stimulated to bind to the ATP synthase and inhibits it. This was also found with NADH as a substrate, therefore the effects of added ATP or oligomycin are not specifically related to SDH activity [110]. Addition of these substances (ATP or oligomycin) has an inhibitory effect on cytochrome pathway activity, which leads to a reduction in rate b, see Figure 3.10. This will therefore lead to an increase in Qr/Qt. The addition of ATP early during the trace will result in stimulation of SDH but at the same time it works as a ‘brake’ on respiration by inhibiting the ATP synthase, as can be seen in Figure 3.14A where acceleration of vO2 is stopped immediately upon addition of ATP. This ‘brake’ does not occur in the situation where succinate is the only added substance since higher vO2 rates are attained in the absence of ATP. Addition of ATP leads to the establishment of a steady state which is reached instantaneously whereas upon addition of succinate a steady state is only reached after a relatively long time. When ATP is added after ~6 minutes to potato mitochondria respiring on succinate, SDH is almost completely activated and SDH has very
  • 106 little control on respiration. It is known that under ADP limited conditions in potato mitochondria the proton leak has strong control [133]. Inhibiting part of this leak through the ATP synthase (with either ATP or oligomycin) will have a strong effect. This effect may be masked early on during the experiment since SDH was not fully activated yet. As long as SDH is activating the oxygen consumption rate will increase, inhibition of proton leak during this period of activation may therefore not be apparent. When SDH is fully activated though; a decrease in oxygen consumption rate due to inhibition of proton leak can no longer be masked (cf. figures 3.14B, 3.15 and 3.16). Does uncoupling lead to decreased succinate transport? Succinate is transported into the matrix via the dicarboxylate carrier which catalyses the electroneutral exchange of dicarboxylates (e.g. malonate, malate and succinate), inorganic phosphate and inorganic sulfur containing compounds (e.g. sulfite) [220]. The carrier is inhibited by butylmalonate [221]. In combination with the phosphate carrier which catalyses the symport of inorganic phosphate and a proton [43] succinate uptake can be viewed as a net succinate/proton symport [222], see Figure 3.17. Figure 3.17 Succinate antiport coupled to phosphate proton symport, the dicarboxylate/phosphate translocase and the H+ :Pi- symport (equivalent to a OH- /Pi- antiport). succinate Pi Pi H+ H+
  • 107 Both transporters catalyse electroneutral processes so dissipation of  by uncouplers should not affect their activity. Uncouplers also dissipate any pH present, which would decrease the driving force for the ‘succinate/proton symport’ process. However, as mentioned in section 3.1.2 potato mitochondria contain a highly active K+ /H+ exchanger which keeps the pH very low. Even though the driving force is small, an effect of uncoupling on succinate transport in plant mitochondria has been reported [223]. In castor bean (Ricinus communis) mitochondria dicarboxylate accumulation in the matrix is inhibited by addition of uncouplers (CCCP and DNP17 ) and by inhibitors of the ETC (antimycin A and KCN). This was interpreted as an effect on phosphate uptake. Interestingly enough it was found that addition of ATP led to an increase in dicarboxylate accumulation into the matrix [223]. Which was interpreted as a direct effect on the dicarboxylate/phosphate translocase. This ATP induced increase in succinate transport into the matrix was not affected by oligomycin. These findings suggest that dissipation of the protonmotive force (due to uncoupling or inhibition of the ETC) does not affect SDH activity directly. Furthermore, a stimulatory effect of ATP on succinate dependent respiration could be due to an increase in the rate of succinate transport. Oestreicher et al. used the PMS-DCIP assay (see section 3.1.3) to demonstrate that in plant mitochondria (cauliflower and mung bean) addition of uncoupler does not deactivate SDH [171] which suggests that any effects of uncoupling on succinate dependent respiration may be indirect, e.g. by inhibition of succinate transport. Is SDH activity regulated by ? By inhibition of succinate transport the protonmotive force can affect SDH activity, but is there an additional effect of the protonmotive force in SDH directly? Affourtit reports that SDH, in the presence of ATP is more active than in the absence of ATP. Figure 3.18 was taken from [153] (Figure 6.6C). It is readily apparent that in the presence of ATP, at a certain value of Qr/Qt the respiratory activity is higher with ATP-activated SDH in comparison to inactive SDH (no ATP present). The ATP-activated data points are in fact from two data sets, state 3 data () and state 4 () data. The choice of pooling the ATP- 17 carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol respectively.
  • 108 activated SDH data points and fitting it with one curve is interesting. This would suggest that there are no differences in SDH activity depending on protonmotive force, which is the exact opposite of what Affourtit was trying to demonstrate. Figure 3.18 Reducing pathway kinetics in potato mitochondria, figure taken from [153], the same data was used in Figure 5B in [21]. Inactive SDH: reducing pathway kinetics from potato mitochondria respiring on succinate in the absence of ATP. ATP-activated SDH: reducing pathway kinetics of pooled state 3 () and state 4 () data. Data modelled according to [76]. The data points from Figure 3.18 were extracted from the plot (a very useful feature of the Kaleidagraph software package) and new fits were made. This time states 3 and 4 were treated separately and it can be seen in Figure 3.19 that there is no single curve which can fit both these data sets (this might explain the reason why the state 3 and state 4 data points were combined in [21]). In fact, under state 4 conditions, where the magnitude of the protonmotive force is higher than under state 3 conditions, SDH is less active, i.e. at a certain value of Qr/Qt the respiratory activity under state 4 conditions is lower than under state 3 conditions, which is the opposite of what Affourtit hypothesized. In the view of this author, Figure 5B in [21] if represented as done in Figure 3.19 would have made a very 0 150 300 0.0 0.5 1.0 SĘ 3Ę T,4Ę inactive fit activated fit V(nmolO2 min-1 mgprotein-1 ) Q-reduction (fraction) inactive SDH ATP-activated SDH C
  • 109 strong argument in favour of the hypothesis that SDH activity in potato mitochondria is indeed modulated by the protonmotive force. Figure 3.19 Data from Figure 3.18, reducing pathway kinetics under state 3 (red line) and state 4 (green line) separately modelled according to [76]. values (see section 2.4.5) for state 3 (-0.68) and for state 4 (-0.92). Linear correlation coefficients (R) for state 3 and state 4 were 0.95143 and 0.64871 respectively. Still, the data in Figure 3.19 can be explained in terms of an effect on phosphate transport as opposed to a direct effect of the protonmotive force on SDH. This begs the question whether or not there are any studies which show conclusively whether or not the protonmotive force can directly affect SDH activity. The only known example of regulation of SDH by  is in Bacillus subtilis [224]. Complex II in B. subtilis is structurally different from its eukaryotic counterparts as it has two heme groups [225] and it therefore does not belong to the same class of SQORs (see section 1.2.4) as the SDH complexes in plant, yeast or mammalian mitochondria [226]. Complex II in B. subtilis uses menaquinone (MK) as an electron acceptor. The electron transport from succinate (E° = +30 mV) to MK (E°  -80 mV) [224] is endergonic. It was found that in B. subtilis and other bacteria which use MK as the respiratory quinone that addition of uncoupler (CCCP) or valinomycin (which 0 150 300 0.0 0.5 1.0 SĘ 3Ę T,4Ę inactive fit activated fit V(nmolO2 min-1 mgprotein-1 ) Q-reduction (fraction) inactive SDH ATP-activated SDH state 4 state 3
  • 110 dissipates the membrane potential) specifically inhibited succinate dependent respiration and menaquinone oxidoreductase activity [224]. Upon oxidation of succinate electrons are transferred ultimately to a heme group which is close to the extra cellular side of the membrane, i.e. to the positive side. The electron transfer from the heme group close to the cytosolic side (negative) to the heme group close to the extra cellular side is driven by the protonmotive force. It was suggested by Schirawski et al. that it is the protonmotive force which provided the driving force for MK reduction [224]. Although this mechanism allows for direct stimulation of SDH by the protonmotive force it cannot be used as a means to modulate SDH activity in eukaryotic mitochondria for the simple reason that complex II in eukaryotic systems only has one heme group (see figures 1.5 and 1.6). Structural differences aside, in a later study by Azarkina et al. [227] it was determined that the stimulating effects of energization on electron transfer in the respiratory chain of B. subtilis is not associated uniquely with complex II activity, i.e. stimulating effects of energization were also observed with other substrates and it was hypothesized that electron transfer in the respiratory chain of B. subtilis might be controlled by membrane energization at the level of MK reduction by the dehydrogenases. In short, SDH activity in B. subtilis does not appear to be directly modulated by the protonmotive force. Concluding, given the current experimental evidence available it seems unlikely that SDH activity is modulated by the protonmotive force directly, whereas inhibition of succinate transport could explain the effects of membrane deenergization on succinate dependent respiration. Summary: ADP and ATP do not stimulate SDH via . It has recently become apparent that plant SDH is structurally different from mammalian and yeast SDH, containing at least 8 extra sub-units with unknown functionality. ADP and ATP probably stimulate SDH cytosolically, there is no evidence suggesting the presence of phosphorylation sites on subunits C and D, however phosphorylation sites might be present on one or more of the newly identified subunits, which still need to be characterised. Findings by Affourtit and Fricaud appeared to be at variance. It was shown that findings of both Affourtit and Fricaud could be reproduced in the same system. ATP and oligomycin affect respiratory rates in a
  • 111 substrate independent manner by inhibiting complex V which is continuously active. Inhibition of the ATP-synthase decreases both IMM conductivity and cytochrome pathway activity. Decreased cytochrome pathway activity, just like a titration with antimycin, will result in an increase in Qr/Qt concomitant with a decrease in vO2. When succinate is added to potato mitochondria SDH slowly becomes activated by succinate itself as it is a homotropic enzyme. Naturally addition of an activator such as ATP will not stimulate SDH any further when it is already fully activated, but a decrease in respiration rate would not be expected. ATP addition has a dual effect. It activates SDH (if it is not fully activated already by succinate) and simultaneously inhibits cytochrome pathway activity. At the beginning of an experiment, this inhibitory effect is not apparent because SDH is spontaneously activating and the net result of addition of ATP will be a net increase in vO2. Expressing SDH activation in terms of Qr/Qt was an innovative idea which allowed for a quantitative measure of SDH activation state, determining SDH activity in this way requires a tightly coupled environment, which turns out to be the Achilles heel of this approach. In a tightly coupled environment in which the Q-redox poise is determined by the interaction of Q-reducing and Q-oxidising pathways quantification of enzyme activity in terms of Qr/Qt is only possible if a change in Qr/Qt is caused only by the enzyme under investigation, which is not the case when ATP is used as an activator. It is the author’s view that at present the best way of determining SDH activity is by spectrophotometic determination of the rate of artificial electron acceptor reduction by SDH.
  • 112 Chapter 4 Respiratory characteristics of Schizosaccharomyces pombe mitochondria 4.1 INTRODUCTION The yeast Schizosaccharomyces pombe also referred to as ‘the other yeast’ [95] is increasingly the preferred model system to investigate a wide range of processes such as the cell cycle [96], DNA repair [97], microtubule formation, meiotic differentiation, cellular morphogenesis and stress response mechanisms [98] over the traditionally used Saccharomyces cerevisiae. S. pombe divides by fission [228] and is one of the few free- living eukaryotic species whose genome has been completely sequenced at the time of writing [99]. Although S. pombe has been extensively used to investigate the cell cycle and genome repair mechanisms, in comparison to S. cerevisiae, relatively little work has been done on S. pombe metabolism [103] and even less has been done on the respiratory characteristics of its mitochondria [104, 105]. S. pombe has been used in this laboratory to express the alternative oxidase (AOX) protein from the plant Sauromatum guttatum in order to investigate structure function relationships [59]. AOX is a non-protonmotive terminal oxidase which catalyses the oxidation of ubiquinol (QH2) to ubiquinone (Q) and the reduction of O2 to H2O [229]. The characteristics of AOX activity and its influence on overall respiration in S. pombe mitochondria expressing AOX are described in chapters five and six (see section 1.2.11 for a description of the alternative oxidase). To interpret the results obtained from work on S. pombe mitochondria expressing the alternative oxidase it is necessary to have an accurate and complete understanding of the respiratory characteristics of the ‘wild type’ S. pombe (sp.011 wt) mitochondria. In this study, for the first time, the membrane potential (∆) in S. pombe mitochondria was measured using the TPP+ electrode [154]. The presence of a ∆ across the inner mitochondrial membrane of S.
  • 113 pombe mitochondria was already inferred from transport studies done by Moore et al. [104]. The magnitude of the membrane potential in isolated yeast mitochondria under ADP limited conditions is in the range of –180 to –200 mV [160, 187], which is intermediate of what has been reported in mammalian and plant mitochondria (see section 3.1.2) although lower values have been reported [230]. Characterisation studies on sp.011 wt mitochondria have been performed previously [26, 104, 105]  however was not determined in these studies. Attempts to optimise the in-house isolation method led to a small improvement in the quality and yield of isolated mitochondria. Therefore a characterisation of the S. pombe mitochondria used in this study will be given, presenting values for the membrane potential, under different energetic conditions, for the first time. Under certain energetic conditions cytochrome pathway kinetics were obtained which appeared to be different from what has been seen previously in mitochondria from other sources. Cytochrome pathway kinetics in sp.011 wt mitochondria were investigated extensively and comparisons were made with cytochrome pathway kinetics from potato and Saccharomyces cerevisiae mitochondria. 4.2 RESULTS 4.2.1 General characterisation of Schizosaccharomyces pombe respiratory kinetics 4.2.1.1 Respiratory rates with different substrates—Table 4.1A shows averaged oxygen consumption rates (vO2) under different energetic conditions. Concentrations of added chemicals, the number of data points and the amount of mitochondrial isolations are given in the legend. NADH-dependent respiration: Under state 2 conditions the average vO2 was 91 nmol O2 / min / mg protein. Upon addition of a limited amount of ADP (0.2 mM) the averaged vO2 increased almost three times to 281 nmol O2 / min / mg protein (state 3), which demonstrates that electron transfer is coupled to
  • 114 phosphorylation. Upon depletion of ADP (state 4) the average vO2 decreased to 110 nmol O2 / min / mg protein. Upon addition of an uncoupler (CCCP, 2 M) the vO2 increased almost six times compared to the state 2 rate, which indicates that the mitochondria upon isolation are relatively intact. The IMM functions as an isolating membrane, which is a requirement for an energy transducing system (see postulate 4 on page 35). The state 3 rate / state 4 rate RCR is 2.6 and the uncoupled / state 2 RCR is 5.9 (Table 4.1B). Succinate-dependent respiration: Addition of succinate alone led to an averaged vO2 of 57 nmol O2 / min / mg protein which is considerably lower than the NADH state 2 rate. Comparable to plant species, SDH needs to be activated [21, 216]. Successive additions of ATP [123] and glutamate [104] (for removal of oxaloacetate) led to further increases in vO2. The mechanism by which ATP activates SDH is still poorly understood [21] but possibly ATP interacts directly with SDH cytosolically (see chapter 3). Unlike in potato mitochondria the effect of adding ATP to S. pombe mitochondria is modest, only small increases in Qr/Qt and  are seen (cf. figures 3.5 and 3.7 respectively) a possible explanation for this difference between plant and yeast mitochondria is that apart from stimulating complex II [123] ATP has been found to stimulate complex IV in yeast mitochondria [230]. Stimulation of an oxidising pathway will lead to a decrease in steady state Qr/Qt. Oxaloacetate is known to inhibit SDH when bound to it, addition of glutamate results in transamination of oxaloacetate to aspartate [231]. A decrease in protonmotive force (p) is known to lead to a decrease of SDH activity [21, 26]. Uncoupled rates (144 nmol O2 / min / mg protein) are lower than state 3 rates (156 nmol O2 / min / mg protein) which suggests that SDH is deactivated due to dissipation of p. Deactivation of SDH is also reflected in the succinate RCR values. For succinate the state 3 / state 4 RCR value is 1.6 and the uncoupled / state 2 RCR value is 1.4 (Table 4.1B). Deactivation of the dehydrogenases will be discussed in section 4.2.3.
  • 115 Glutamate-dependent respiration: Addition of glutamate itself induces a rate (32 nmol O2 / min / mg protein) which can be increased further by addition of ATP (43 nmol O2 / min / mg protein). This has been interpreted as SDH activation by Crichton [26]. He hypothesizes that addition of glutamate leads to generation of succinate inside the matrix. This hypothesis is supported by findings in this study. Addition of glutamate led to a slowly accelerating vO2, as seen when succinate is added as a substrate (cf. Figure 3.13), which suggests that SDH is slowly activating. Addition of ATP led to an increase in vO2 which can be interpreted as activation of SDH by ATP [21]. Addition of ADP or CCCP did not lead to an appreciable increase in rate, which can be interpreted as deactivation of SDH. Most convincingly though is the observation that upon addition of malonate, an inhibitor of complex II (see section 3.1.3) the glutamate-dependent respiration is completely inhibited (data not shown). The glutamate-dependent respiratory rate (in the presence of ATP) is lower than the succinate-dependent respiratory rate. This could be due to SDH not being fully activated when the concentration of succinate generated after glutamate addition is subsaturating. Possibly the addition of glutamate leads to release of a limited amount of oxaloacetate, which then (via the TCA cycle) is converted to succinate. If indeed succinate concentration would be subsaturating then the membrane potential with glutamate as a substrate should be lower than with succinate as a substrate. This would be the basis for an interesting experiment in future work. S. pombe mitochondria contain glutamate dehydrogenase (SPCC132.04c)18 which can couple the oxidation of glutamate to -oxoglutarate to the reduction of NAD+ thereby generating matrix NADH [43]. This could also lead to glutamate dependent respiration but given the results obtained with malonate this seems unlikely. Table 4C shows the ADP/O ratios obtained with NADH or succinate as a substrate being 1.36 and 1.22 respectively. The values are somewhat low. S. pombe mitochondria do not express complex I therefore only complexes III and IV translocate protons across the IMM. The expected ADP/O value for either NADH or succinate would be 1.5 [1]. Poor phosphorylative capacity may reflect the in vivo role of mitochondria in S. pombe. 18 http://www.genedb.org/genedb/Search?organism=pombe&name=SPCC132.04c&isid=true
  • 116 Table 4D shows the sensitivity of sp.011 wt mitochondria to inhibitors. KCN, antimycin A and myxothiazol completely inhibit NADH and succinate dependent respiration which indicates that no alternative oxidase is expressed in S. pombe mitochondria as reported previously [104]. Octyl gallate, a potent inhibitor of AOX [70] at a concentration of 16 µM slightly inhibits both NADH-and succinate-dependent respiration. SDH can be fully inhibited by malonate. Carboxyattractyloside (CAT) is a potent inhibitor of the adenine nucleotide translocator [126], addition of 5 L of a 1 mg/ml solution of CAT completely prevents generation of a state 3 upon addition of an aliquot of ADP. Table 4 E shows averaged membrane potentials obtained with NADH or succinate (ATP and glutamate incubated). The obtained ∆ values with either substrate are approximately the same, indicating that, with respect to the membrane potential there are no substrate dependent differences. Under state 4 conditions with either substrate the magnitude of ∆ is smaller than under state 2 conditions. The increased vO2 and decreased ∆ under state 4 conditions are suggestive of an increased conduction of the IMM. These observations may indicate the involvement of adenylate kinase (see section 1.3.1). This enzyme catalyses the reaction: ATP+AMP  2 ADP [110]. With each state 3 more ATP will be available for ADP recycling, leading to increased ATP-synthase activity. The presence of adenylate kinase in S. pombe mitochondria has been demonstrated [112]. Yeast AK is located in the IMS where it can move freely, unlike in plants where it is bound to the outer face of the IMM [232].
  • 117 A Respiratory Rate (nmol O2 min-1 mg-1 protein) substrate(s) state 2 state 3 state 4 uncoupled NADH 91 (13) 281 (39) 110 (22) 537 (68) succinate * 106 (15) 156 (18) 97 (7) 144 (25) glutamate 32 (4) - - - glutamate + ATP 43 (8) - - - B RCR substrate state 3 / state 4 uncoupled / state 2 NADH 2.6 5.9 succinate * 1.6 1.4 C ADP/O NADH 1.36 (0.13) [20,11] succinate * 1.22 (0.08) [8,4] D Sensitivity to inhibitors inhibitor percentage of oxygen consumption rate inhibition antimycin A (2.3 µM) 100% KCN (270 µM) 100% myxothiazol (2.5 L of 1 mg/ml) 100% malonate (9 mM) 100% octyl gallate (16 M) 10% E ∆ values (mV) substrate state 2 state 3 state 4 NADH 208 (6) [6,4] 188 (7) [4,2] 204 (11) [4,2] ** succinate * 200 (7) [4,4] 180 (10) [3,2] 195 (5) [3,2] ** Table 4.1 Respiratory characteristics of sp.011 wt mitochondria. Table A: respiration rate ( S.D.) (see Appendix 2A for full details regarding amount of mitochondrial isolations and experimental traces performed). Concentrations of chemicals added: NADH (1.8 mM), succinate (9 mM), ATP (0.2 mM), glutamate (9 mM). Uncoupled conditions are defined as respiratory
  • 118 activity in the presence of an uncoupler (2 µM CCCP). The concentration of added ADP to induce a state 3 to state 4 transition was 0.2 mM in all cases. State 3 conditions are defined as respiratory activity in the presence of 1 mM ADP. The RCR values (see section 2.4.4.3) in table B were calculated using the vO2 values from Table 4.1A. Table C: ADP/O value ( S.D.) [traces, preps]. Table D: percentage inhibition by inhibitors. Table E:  values ( S.D.) [traces, preps]. Nomenclature used is discussed in section 2.4.4.2. * SDH is activated stepwise by addition of ATP (0.2 mM) and glutamate (9 mM). vO2 with just succinate is 57 (7) addition of ATP subsequently leads to a vO2 of 72 (12). ** ∆ for the first state 4 only Figure 4.1 shows a representative trace of an experiment where ∆ and vO2 were measured simultaneously. Upon addition of NADH (1.8 mM) a membrane potential of 210 mV was generated with a concomitant vO2 of 120 nmol O2 / min / mg protein. Upon addition of ADP (0.2 mM) ∆ was lowered to 190 mV and the respiratory rate increased to 287 nmol O2 / min / mg protein, which indicates that electron transfer is coupled to phosphorylation. After depletion of ADP  stabilised at a value of 203 mV with a concomitant vO2 of 110 nmol O2 / min / mg protein. Upon addition of CCCP (2 M)  was dissipated and vO2 increased to 630 nmol O2 / min / mg protein. These findings are in agreement with what has been found previously in mitochondria of S. cerevisiae [160], plants [157] and mammals [1]. The protonmotive force (pmf) in mitochondria is composed of an electrical potential (∆) and a proton concentration gradient (∆pH) [1]. Nigericin is an ionophore which can bind both H+ or K+ , this molecule, when added to a solution of mitochondria, will catalyse the electroneutral exchange of H+ for K+ , which leads to dissipation of ∆pH [1]. After addition of nigericin the pH will be converted to . Addition of nigericin to S. pombe mitochondria respiring on either NADH or succinate did not lead to a change in TPP+ - electrode signal (data not shown), indicating that as in potato mitochondria [157] the ∆pH component of the pmf in S. pombe mitochondria is negligible. The absence of a detectable ∆pH suggests the presence of a K+ /H+ exchanger [188] in S. pombe mitochondria. Figure 4.2 shows representative oxygen consumption and  traces from sp.011 wt mitochondria respiring on succinate. Addition of succinate (9 mM) generated a membrane potential of 193 mV with a low concomitant vO2 of 39 nmol O2 / min / mg protein. Addition of ATP (0.2 mM) stimulated SDH as seen in the increase of vO2 to 64
  • 119 nmol O2 / min / mg protein,  however was seen to decrease slightly (using a fresh ATP stock), addition of glutamate (9 mM) led to further increases in both vO2 and , to 96 nmol O2 / min / mg protein and 198 mV respectively. Addition of ADP led to a decrease in  to 179 mV and an increase in vO2 to 138 nmol O2 / min / mg protein, which indicates that respiration is coupled to phosphorylation, but not to the same extent as with NADH. This can be interpreted as deactivation of SDH. After depletion of ADP vO2 decreased to 109 nmol O2 / min / mg protein and  increased to 197 mV (state 4). Addition of ADP (0.2 mM) resulted in a second state 3 to state 4 transition. Figure 4.1 State 3 to state 4 transition in NADH respiring sp.011 wt mitochondria. Simultaneous measurement of oxygen uptake (—) and membrane potential (). 1.8 mM NADH, 0.2 mM ADP, 2 M CCCP. The numbers adjacent to the respiratory traces represent specific O2-uptake rates (nmol O2 / min / mg protein, bold font) and ∆ values (mV, standard font). The traces shown are representative of data from repeated experiments. Amount of mitochondrial protein used was 0.5 mg. 0 100 200 300 400 500 600 0.85 0.9 0.95 1 1.05 1.1 1.15 0 1 2 3 4 5 6 oxygen(nmol) TPP+ signalV time (min) 120 287 110 630 210 203 184 NADH ADP CCCP anoxia
  • 120 Figure 4.2 sp.011 wt mitochondria respiring on succinate. Simultaneous measurement of oxygen uptake (—) and membrane potential (). 9 mM succinate, 0.2 mM ATP, 9 mM glutamate, 0.2 mM ADP, 0.2 mM ADP. The numbers adjacent to the respiratory traces represent specific O2-uptake rates (nmol O2 min-1 mg-1 ) and ∆ values (mV). The traces shown are representative of data from repeated experiments. Amount of mitochondrial protein used was 1 mg. Figure 4.3 shows a representative trace of an experiment where Qr/Qt and oxygen consumption were measured simultaneously in sp.011 wt mitochondria respiring on succinate19 . Addition of succinate (9 mM) resulted in an artifactual signal followed by slow SDH activation until a steady state Qr/Qt of 63% was reached with a concomitant vO2 of 56 nmol O2 / min / mg protein. Addition of ATP (0.2 mM) and glutamate (9 mM) led to further activation of SDH as reflected in increased Qr/Qt and vO2 values to 89% and 95 nmol O2 / min / mg protein respectively. Addition of ADP (0.2 mM) led to an increase in vO2 to 153 nmol O2 / min / mg protein and a decrease in Qr/Qt to 66% which indicates that respiration is coupled to phosphorylation. Upon depletion of ADP a state 4 was reached with Qr/Qt 92% and vO2 95 nmol O2 / min / mg protein. Another addition of ADP (0.2) led to a state 3 which coincided with anoxia where the Q signal stabilised at 100% Qr/Qt. The data shown in Figure 4.3 agree well with results previously obtained in this laboratory [26]. 19 An equivalent Qr/Qt trace with NADH cannot be produced because of the strong interference of NADH with the Q-electrode at high concentrations (1.8 mM). 1.2 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 0 100 200 300 400 500 600 0 5 10 15 20 TPP+ signal(V) oxygen(nmol) time (min) succinate ATP glutamate ADPADP anoxia 64 96 138 39 109 152 107 193 189 198 179 197 175 193
  • 121 Figure 4.3 Effects of ATP, glutamate and ADP on succinate-dependent respiratory activity. Simultaneous measurement of oxygen uptake () and redox poise of the mitochondrial Q-pool (…) in sp.011 wt mitochondria respiring on succinate. 9 mM succinate, 0.2 mM ATP and 9 mM glutamate, 50 M ADP and 0.1 mM ADP were added where indicated. The numbers adjacent to the respiratory traces represent specific O2-uptake rates (nmol O2 / min / mg protein) and Q-reduction levels (fraction QH2 in total pool). The traces shown are representative of data from repeated experiments. Amount of mitochondrial protein used was 0.7 mg. The sp.011 wt mitochondria isolated in this study were shown to be of good quality. With NADH as a substrate the RCR value (uncoupled / state 2) was ~5.9 which indicates that the IMM is relatively impermeable to protons (see postulate 4 on page 35). Respiration rates of sp.011 wt mitochondria respiring on succinate become limited under state 3 and uncoupled conditions which suggests deactivation of SDH upon a decrease in . The ability of these mitochondria to phosphorylate ADP is relatively poor with respect to what is seen in mammalian and plant mitochondria. The membrane potential generated and its response to changes in energetic conditions in sp.011 wt mitochondria is similar to what is seen in mitochondria isolated from S. cerevisiae [160], plants [157] (cf. Figure 3.2) and mammals [1]. 0 100 200 300 400 500 600 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 oxygen(nmol) Qr/Qt time (min) succinate ATP glutamate ADP ADP anoxia 0.63 0.64 0.89 0.66 0.92 0.66 56 67 95 153 95 150
  • 122 4.2.2 Schizosaccharomyces pombe - cytochrome pathway kinetics Cytochrome pathway kinetics (Qr/Qt vs. vO2) have been determined previously in both sp.011 wt mitochondria [104] and S. pombe mitochondria transformed with AOX [93], by titrating succinate-dependent respiration with malonate. Cytochrome pathway kinetics with NADH as a substrate were not determined in previous studies as NADH interacts with the Q-electrode. The NADH regenerating method (with Glucose-6-Phosphate dehydrogenase and Glucose-6-Phosphate incubated), in which subsaturating amounts of NADH are added to activate the external NADH dehydrogenase, can be used effectively, since additions of small amounts of NADH lead to only small effects on the Q-signal which can be corrected for (see section 2.4.4.4). Oxidising pathway kinetics of S. pombe mitochondria with NADH as a substrate have not been determined before. As part of a full characterisation of the sp.011 wt mitochondria it was decided to determine cytochrome pathway kinetics under various energetic conditions (states 2 to 4 and uncoupled) with NADH as a substrate measuring Qr/Qt, vO2 and ∆ simultaneously or in parallel. To obtain state 3 or uncoupled oxidising pathway kinetics; a saturating amount of ADP (1 mM) or a saturating amount of CCCP (2 M) was incubated. To obtain state 4 oxidising pathway kinetics a state 3 to state 4 transition was induced by adding an aliquot of ADP (~50 M). 4.2.2.1 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under ADP limited conditions with NADH as a substrate—Figure 4.4 shows the relationship between Qr/Qt and vO2 under state 2 and state 4 conditions. Under state 2 conditions no ADP is added (see section 2.4.4.2). State 4 conditions were achieved by titrating with subsaturating amounts of NADH until a low rate of vO2 was achieved followed by the addition of an aliquot of ADP (~50 µM) which led to a transient increase in vO2 and concomitant decrease in Qr/Qt, upon depletion of ADP the NADH titration was continued and only those data points were used to represent state 4 cytochrome pathway kinetics.
  • 123 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 0 10 20 30 40 50 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt A Figure 4.4 Relationship between Qr/Qt and vO2 under ADP limited conditions with NADH as a substrate. Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously under state 2 () and state 4 conditions (). State 4 conditions were achieved by titrating with subsaturating amounts of NADH (~ 3-5 M) until a low rate of vO2 was achieved followed by the addition of an aliquot of ADP (typically 50 µM) which led to a transient increase in vO2 and concomitant decrease in Qr/Qt, upon depletion of ADP the NADH titration was continued. State 2 and state 4 data points overlap and the relationship between Qr/Qt and vO2 is linear. For comparison the same relationship under state 4 conditions in fresh potato mitochondria, obtained with the NADH regenerating system is shown in inset A. This relationship is linear up until high values of Qr/Qt where its shape becomes slightly convex. The sp.011 state 2 and state 4 titration data were obtained from six and seven mitochondrial isolations respectively, data points were combined from nine and ten traces respectively. The state 4 potato titration data was obtained from one mitochondrial isolation combining data from two traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1 mg).
  • 124 The data points from both conditions overlap and the relationship between Qr/Qt vs. vO2 is linear. As a comparison, in the inset, the relationship between Qr/Qt and vO2 is shown in fresh potato mitochondria under state 4 conditions (state 4 conditions were achieved in the same way as for S. pombe mitochondria), which is primarily linear becoming slightly convex at high rates. Linear relationships (Qr/Qt vs. vO2) are typically seen under ADP limited conditions in mitochondria which do not have a branched respiratory chain [76, 86]. A linear relationship between Qr/Qt and vO2 under ADP limited conditions has been demonstrated previously in S. pombe mitochondria respiring on succinate (cf. Figure 2 in [104]), this suggests that there are no substrate dependent differences in cytochrome pathway kinetics under ADP limited conditions. These results agree well with data previously obtained in this laboratory and with those reported in the literature. Figure 4.5 shows the relationship between ∆ and vO2 under state 2 and state 4 conditions in sp.011 wt mitochondria. Data from both conditions overlap and it can be seen that under state 4 conditions higher respiratory rates are obtained, possibly reflecting the activity of adenylate kinase. Immediately apparent from the data is a non-ohmic relationship between ∆ and vO2 (see section 3.1.2). A non-ohmic p vs. proton conductance relationship under ADP limited conditions has been shown previously in S. cerevisiae mitochondria [233]. The results presented here agree well with the yeast literature. As a comparison the same relationship under state 4 conditions as determined in fresh potato mitochondria is given (data points taken from Figure 3.4). 4.2.2.2 The relationships of Qr/Qt vs. vO2 and ∆ vs. vO2 under state 3 conditions with NADH as a substrate—Figure 4.6 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011 wt mitochondria. It is apparent that the relationship is non- linear, showing a biphasic shape. This was surprising since in other tissues this relationship under state 3 conditions normally is found to be linear [37, 234], the inset shows the relationship in fresh potato mitochondria. At low Qr/Qt there is little respiratory activity, which increases considerably between 30 and 40 % Q-reduction in the sp.011 wt mitochondria.
  • 125 0 50 100 150 100 120 140 160 180 200 220 vO2 nmolO2 /min/mgprotein membrane potential (mV) Figure 4.5 The relationship between ∆ and vO2 under state 2 and state 4 conditions with NADH as a substrate. Using the NADH regenerating system both vO2 and ∆ were measured simultaneously under state 2 ( ) and state 4 () conditions. State 4 conditions were achieved by titrating with subsaturating amounts of NADH (~ 3-5 M) until a low rate of vO2 was achieved followed by the addition of an aliquot of ADP (50 M) which led to a transient increase in vO2, upon depletion of ADP the NADH titration was continued. The relationship between ∆ and vO2 under both state 2 and state 4 conditions is non-ohmic. For comparison the same relationship was determined in fresh potato mitochondria under state 4 conditions using the NADH regenerating system. The sp.011 state 2 and state 4 titration data were obtained from four and two mitochondrial isolations respectively, data points were combined from six and three traces respectively. The fresh potato titration data was taken from Figure 3.4. As already seen in Table 4.1A, the vO2 rates under state 4 conditions are higher than under state 2 conditions, possibly indicating adenylate kinase activity in S. pombe mitochondria. Mitochondrial protein used per experiment for S. pombe mitochondria (0.5-0.7 mg). 0 10 20 30 40 50 60 70 80 170 180 190 200 210 220 vO2 nmolO2 /min/mgprotein membrane potential mV
  • 126 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt A Figure 4.6 The relationship between Qr/Qt and vO2 under state 3 conditions with NADH as a substrate in sp.011 wt mitochondria. Using the NADH regenerating system both vO2 and Qr/Qt were measured simultaneously under state 3 conditions. Mitochondria were incubated with ADP (1 mM). The relationship between Qr/Qt and vO2 shows a biphasic pattern, a change in slope is observed at around 30 to 40 % Q- reduction. For comparison, the same relationship in fresh potato mitochondria under state 3 conditions (ADP 1 mM), obtained using the NADH regenerating system, is shown in inset A. In potato mitochondria a slight convex shape at high Qr/Qt is observed reminiscent of the pattern seen under state 4 conditions (see Figure 4.5 inset A).The sp.011 wt titration data were obtained from five mitochondrial preparations, combining data from eleven traces. The potato titration data were obtained from one mitochondrial isolation, combining data from two traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1 mg).
  • 127 Figure 4.7 shows the relationship between ∆ and vO2 under state 3 conditions in sp.011 wt mitochondria. A biphasic pattern is again seen in this relationship, and is different from what is seen in other tissues, where this relationship normally is linear [157], see inset A where the same relationship is given for fresh potato mitochondria. A representative trace is given in inset B clearly showing the biphasic nature of the kinetics in sp.011 wt mitochondria under state 3 conditions. By combined plotting of ∆, Qr/Qt and vO2 it became clear that the vO2 rate ( 50 nmol O2 / min / mg protein) at which the relationships of both Qr/Qt vs. vO2 and ∆ vs. vO2 become biphasic is the same, indicating that the change in both relationships occur simultaneously (data not shown). 4.2.2.3 The relationship between Qr/Qt vs. vO2 under uncoupled conditions with NADH as a substrate—Since ∆ is dissipated by CCCP only the relationship between Qr/Qt and vO2 was investigated. Figure 4.8 shows the relationship between Qr/Qt vs. vO2 under uncoupled conditions in sp.011 wt mitochondria. The relationship becomes biphasic around 40% Q-reduction, for comparison, the inset shows this relationship in fresh potato mitochondria. The vO2 rate at which the relationship becomes biphasic is the same as under state 3 conditions (around 50 nmol O2 / min / mg protein) also, the biphasic pattern is present whilst ∆ is dissipated which suggests that ∆ probably is not instrumental in causing the biphasic pattern. Are the biphasic cytochrome pathway kinetics NADH dependent? In previous work, using succinate as a substrate, the relationships between Qr/Qt and vO2 under state 3 and uncoupled conditions were determined in transformed S. pombe mitochondria in which AOX expression was repressed, i.e. mitochondria which only use the cytochrome pathway to oxidise the Q-pool [93]. The biphasic patterns as seen in figures 4.6 and 4.8 were not observed in that study and this raised the question whether or not the biphasic cytochrome pathway kinetics were NADH-dependent. Therefore cytochrome pathway kinetics under various energetic conditions (states 2 to 4 and uncoupled) were determined by titrating succinate-dependent respiration in the presence of malonate.
  • 128 0 50 100 150 200 250 300 350 400 120 130 140 150 160 170 180 vO2 nmolO2 /min/mgprotein membrane potential mV 0 20 40 60 80 100 162 164 166 168 170 172 174 vO2 nmolO2 /min/mgprotein membrane potential mV A 0 20 40 60 80 100 120 140 160 130 140 150 160 170 180 vO2 nmolO2 /min/mgprotein membrane potential mV B Figure 4.7 The relationship between ∆ and vO2 under state 3 conditions in sp.011 wt mitochondria respiring on NADH. Using the NADH regenerating system, ∆ and vO2 were determined simultaneously under state 3 conditions. ADP was incubated at a concentration of 1 mM. A biphasic pattern similar to the one seen in the relationship between Qr/Qt and vO2 (see Figure 4.6) is seen. For comparison the same relationship under state 3 conditions (1 mM ADP pre-incubated) obtained with the NADH regenerating system was determined in fresh potato mitochondria (inset A). Due to variability in the sp.011 data the presence of a biphasic pattern may not be apparent, therefore a representative individual trace is shown in inset B. It is clear that the relationship between ∆ and vO2 is linear in potato mitochondria whereas it is biphasic in S. pombe mitochondria. The sp.011 titration data were obtained from three mitochondrial isolations, combined data from five traces. The potato titration data was from one mitochondrial isolation, data from two traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1 mg).
  • 129 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 0 20 40 60 80 100 120 140 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt A Figure 4.8 The relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 wt mitochondria respiring on NADH. Using the NADH regenerating system both vO2 and ∆ were measured simultaneously under uncoupled conditions. Mitochondria were incubated with CCCP (2 M). The relationship between Qr/Qt and vO2 shows a biphasic pattern, a clear change in slope is observed at around 40 % Q-reduction. For comparison the same relationship in fresh potato mitochondria under uncoupled conditions, obtained by using the NADH regenerating system, is shown in the inset. A slight convex shape is observed in potato mitochondria. The sp.011 titration data were obtained from five mitochondrial preparations, combining data from five traces. The potato titration data were obtained from one mitochondrial isolation, combining data from three traces. Mitochondrial protein used per experiment for S. pombe (0.5-0.7 mg) and for potato (0.5-1 mg).
  • 130 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 4.2.2.4 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 and ∆ vs. vO2 relationships under ADP limited conditions—Figure 4.9 shows combined succinate and NADH Qr/Qt vs. vO2 data which clearly show that under ADP limited conditions there are no substrate-dependent differences in cytochrome pathway kinetics. Figure 4.9 Combined state 2 and state 4 cytochrome pathway kinetics of sp.011 wt mitochondria respiring on either NADH or succinate, Qr/Qt vs. vO2. NADH state 2 () and state 4 () data were taken from Figure 4.4. Succinate state 2 () and state 4 (∆) data were obtained with the malonate titration method. With succinate as a substrate a state 4 was induced by adding an aliquot of ADP (50M) after the mitochondria were energised with succinate (9 mM) + ATP (0.1 mM) + glutamate (9 mM), upon depletion of ADP respiratory activity was titrated with malonate (up to ~ 4 mM). State 2 and state 4 succinate titration data were obtained from one and two mitochondrial isolations respectively. Data points for succinate state 2 and state 4 were taken from one trace and nine traces respectively. Mitochondrial protein used per experiment was 0.5-0.7 mg. Figure 4.10 shows combined succinate and NADH  vs. vO2 data which clearly shows that under ADP limited conditions there are no substrate dependent differences in kinetics. It is clear that the relationship between  and vO2 is non-ohmic (cf. figures 3.1 and 3.4).
  • 131 0 50 100 150 100 120 140 160 180 200 220 vO2 nmolO2 /min/mgprotein membrane potential mV Figure 4.10 Combined state 2 and state 4 cytochrome pathway kinetics of sp.011 wt mitochondria respiring on either NADH or succinate, ∆ vs. vO2. NADH state 2 () and state 4 (∆) data were taken from Figure 4.5. Succinate state 2 () were obtained with the malonate titration method (0 – 4 mM malonate). Succinate titration data were obtained from one mitochondrial isolation, data points were taken from one trace. Mitochondrial protein used per experiment was 0.5-0.7 mg. 4.2.2.5 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 relationships under state 3 conditions—Figure 4.11 shows combined succinate and NADH Qr/Qt vs. vO2 data which show that under state 3 conditions there are no substrate dependent differences in cytochrome pathway kinetics. The succinate data points overlap with the NADH data points up until the point where vO2 becomes limiting, this reflects the lower activity of the SDH in comparison to the external NADH dehydrogenase.
  • 132 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 4.11 Combined state 3 cytochrome pathway kinetics of sp.011 wt mitochondria respiring on either NADH or succinate, Qr/Qt vs. vO2. NADH data () were taken from Figure 4.6. Succinate data () were obtained with the malonate titration method (0 – 4 mM malonate). A state 3 was brought about by incubating with ADP (1 mM). Succinate titration data were obtained from two mitochondrial isolations, data points were taken from five traces. Mitochondrial protein used per experiment was 0.5-0.7 mg. 4.2.2.6 A comparison of NADH and succinate dependent Qr/Qt vs. vO2 relationships under uncoupled conditions—Figure 4.12 shows combined succinate and NADH Qr/Qt vs. vO2 data that clearly show that under uncoupled conditions there are no substrate dependent differences in cytochrome pathway kinetics. The succinate data points overlap with the NADH data points up until the point where vO2 becomes limiting which, as under state 3 conditions, reflects the lower activity of the SDH in comparison to the external NADH dehydrogenase.
  • 133 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 4.12 Combined uncoupled cytochrome pathway kinetics of sp.011 wt mitochondria respiring on either NADH or succinate, Qr/Qt vs. vO2. NADH data () were taken from Figure 4.8. Succinate data () were obtained with the malonate titration method (0 – 4 mM malonate). Mitochondria were incubated with CCCP (2 M). Succinate titration data were obtained from five mitochondrial isolations, data points were taken from eleven traces. Mitochondrial protein used per experiment was 0.5-0.7 mg. From the data presented in figures 4.9 to 4.12 it was concluded that there were no substrate dependent differences in sp.011 wt cytochrome pathway kinetics. To further characterize the S. pombe respiratory kinetics, the reducing pathway kinetics with either NADH or succinate as a substrate were determined.
  • 134 4.2.3 Schizosaccharomyces pombe - reducing pathway kinetics Reducing pathway kinetics from S. pombe mitochondria expressing AOX have been determined previously under state 4 conditions both with either succinate or NADH as a substrate by titrating respiration with antimycin A [93]. Under ADP limited conditions  is relatively stable, i.e. a large decrease in respiration is concomitant with only a small decrease in  (see section 3.1.2). According to Affourtit an effect of the protonmotive force on reducing pathway kinetics in S. pombe mitochondria was not expected and reducing pathway kinetics were not determined under state 3 or uncoupled conditions [93]. It is however known from literature [21, 93] and from findings during this thesis work that SDH is deactivated upon dissipation of ∆p which led this author to expect that protonmotive force does have an effect on reducing pathway kinetics with succinate as a substrate in sp.011 wt mitochondria. 4.2.3.1 SDH reducing pathway kinetics in sp.011 wt mitochondria under state 2 and uncoupled conditions—Figure 4.13 shows combined SDH reducing pathway kinetics under state 2 and uncoupled conditions. A comparison between state 2 and uncoupled kinetic data should reveal clear differences if  were to have an effect on SDH activity. Deactivation of SDH is clearly reflected in the lower slope of the uncoupled data points. Under state 2 conditions, at every value of Qr/Qt the respiration rate is higher when compared to uncoupled conditions. This indicates that SDH in the presence of  is more active than in the absence of . The reducing pathway kinetics were determined by titrating respiration with antimycin A. It has been reported previously that S. cerevisiae mitochondria display linear relationships between bc1 complex inhibition and respiratory rate [27]. It was suggested that yeast mitochondria did not display pool behaviour. Figure 4.14 shows a representative trace of an antimycin A titration in sp.011 wt mitochondria respiring on succinate. A sigmoidal response can be seen which indicates that S. pombe mitochondria display pool behaviour [24] (see also section 1.2.5)
  • 135 0 50 100 150 200 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 4.13 Combined state 2 and uncoupled reducing pathway kinetics with succinate as a substrate, Qr/Qt vs. vO2. Reducing pathway kinetics under state 2 () and uncoupled conditions () were determined by titrating with antimycin A (0 – 40 nM), Qr/Qt and vO2 were measured simultaneously. SDH deactivation is reflected in the difference between state 2 and uncoupled reducing pathway kinetics. Additions: succinate 9 mM, ATP 0.2 mM, glutamate 9 mM and under uncoupled conditions CCCP 2 M. Mitochondrial protein used per experiment was 0.5-0.7 mg. Figure 4.14 Antimycin A titration of sp.011 wt mitochondria respiring on succinate, data taken from Figure 4.13. 0 40 80 120 160 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 136 0 10 20 30 40 50 60 70 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 4.2.3.2 External NADH dehydrogenase reducing pathway kinetics in sp.011 wt mitochondria under state 2 and uncoupled conditions—Figure 4.15 shows combined external NADH dehydrogenase reducing pathway kinetics under state 2 and uncoupled conditions. Respiration was initiated by titrating with sub-saturating amounts of NADH. Titration was stopped before the external NADH dehydrogenase was fully active in order to have a large range of Qr/Qt to titrate. When the external NADH dehydrogenase is fully active the Q-pool is highly reduced (cf. figures 4.4 and 4.8). Titration with antimycin A then would result in only a few data points from which it would be difficult to determine whether or not there were any differences in kinetics. It can be seen that the activity of the external NADH dehydrogenase in the presence of CCCP (2 M) is the same as the activity of the external NADH dehydrogenase in the absence of uncoupler as the data points of both energetic states overlap. Contrary to what was suggested previously [93] the SDH reducing pathway kinetics are dependent on energy status of the IMM. The external NADH dehydrogenase reducing pathway kinetics did not show any such dependency. Figure 4.15 Combined state 2 and uncoupled reducing pathway kinetics with NADH as a substrate, Qr/Qt vs. vO2. Reducing pathway kinetics under state 2 () and uncoupled conditions () were determined by titrating with antimycin A (0 – 40 nM), Qr/Qt and vO2 were measured simultaneously. The Q-pool was partially reduced with sub-saturating amounts of NADH (~14 M) using the NADH regenerating method. Under uncoupled conditions CCCP was pre-incubated at a concentration of 2 M. Mitochondrial protein used per experiment was 0.5-0.7 mg.
  • 137 4.2.4 Are the biphasic patterns due to cytochrome bc1 complex kinetics? Upon comparison of the cytochrome pathway kinetics obtained with either NADH or succinate as a substrate (see figures 4.9-4.12) it can be concluded that there are no substrate dependent differences in kinetics. An implication of this is that the biphasic patterns seen under state 3 (figures 4.6, 4.7 and 4.11) and under uncoupled conditions (figures 4.8 and 4.12) are not due to the activities of the dehydrogenases; i.e. it does not matter in which way the Q-pool is being reduced. Hence the underlying cause for the biphasic oxidising pathway kinetics must be somewhere downstream of the Q-pool. Since S. pombe mitochondria do not express an alternative oxidase (see Figure 1.14) these kinetics must then be a characteristic of cytochrome pathway activity. The first acceptor of reducing equivalents from the Q-pool is the cytochrome bc1 complex (see section 1.2.6) which is known to exist as a homodimer [235]. Interestingly enough there is no need for the cytochrome bc1 complex to be dimeric in order to accommodate for the protonmotive Q- cycle [1] as either monomer on its own can achieve this. Based on work done on isolated yeast (S. cerevisiae) cytochrome bc1 complexes Trumpower proposed a reaction mechanism in which only half of the monomers are active [33]. He hypothesized that the cytochrome bc1 complex could become fully active due to an effect of a change in protonmotive force or due to a high concentration of ubiquinol [33, 236]. The biphasic pattern observed in S. pombe cytochrome pathway kinetics could be a reflection of the regulatory characteristics of the yeast cytochrome bc1 complex. Activity of the cytochrome bc1 complex can be determined by following the reduction of ferricyanide spectrophotometrically [79, 237]. In mitochondrial isolations electrons can be transferred from cytochrome c to ferricyanide present in the reaction medium [237]. In order to avoid electron transfer through complex IV the mitochondria are incubated in the presence of KCN. Change in absorbance at 420 nm [237] was measured in solutions incubated with KCN with either potato or S. pombe mitochondria. The ferricyanide reduction rate was titrated with sub-saturating amounts of NADH using the NADH regenerating system. The aim of the experiments was to determine whether or not a biphasic pattern would be present in the relationship between substrate concentration and ferricyanide reduction rate in S. pombe mitochondria. Potato mitochondria were used for
  • 138 comparison. In this experimental system the only ‘electron sink’ available should be ferricyanide. Addition of KCN (1 mM) effectively blocks electron transfer to cytochrome c oxidase, this was verified using an oxygen electrode setup in a parallel experiment during the same day. In the presence of antimycin A (1.7 M) the cytochrome bc1 complex is completely inhibited, this was also verified. When the cytochrome bc1 complex is inhibited, no electrons can be transferred to cytochrome c and therefore no ferricyanide reduction can take place under these conditions. Control experiments were done to verify whether cytochrome c was the only electron donor present that was able to reduce the added ferricyanide. When ferricyanide was added to 1 ml of yeast reaction medium (see section 2.1.1.7) in the cuvette no change of absorbance at 420 nm was observed; the presence of any of the following substances: KCN (1 mM), NADH (1.8 mM), Glucose-6-phosphate dehydrogenase (10 units), glucose-6-phosphate (2.25 mM) and antimycin A (1.7 M) did not lead to any change in absorbance (results not shown). Ferricyanide is not reduced by the reaction medium or by any of the chemicals which are used during the experiments. Figure 4.16 shows a representative trace of an experiment using fresh potato mitochondria. With just mitochondria and ferricyanide in the reaction medium no change in absorption is seen. Upon addition of a saturating amount of NADH (1.8 mM) the reaction starts and within a few seconds a steady state change in absorption can be seen. Upon addition of antimycin A (60 ng) the reduction of ferricyanide was stopped completely. Similar results were obtained with the sp.011 wt mitochondria (results not shown). From these experiments it was concluded that in the presence of mitochondria and absence of substrate, there was no ferricyanide reduction. Addition of antimycin A completely stopped the reduction of ferricyanide, therefore the only pathway for electron transfer to ferricyanide is via cytochrome c. Figure 4.17 shows [NADH] vs. ferricyanide reduction rate in fresh potato mitochondria under uncoupled conditions; the relationship displays a convex shape. Figure 4.18 shows [NADH] vs. ferricyanide reduction rate in sp.011 wt mitochondria under uncoupled conditions, the relationship shows a biphasic pattern. These results suggest that the biphasic patterns observed under conditions of a lowered or dissipated ∆ are likely to be due to cytochrome bc1 complex kinetics.
  • 139 Figure 4.16 Ferricyanide reduction measured spectrophotometrically in potato mitochondria. Ferricyanide reduction was measured at 420 nm. The figure shows the change in absorption upon addition of NADH (2mM) and antimycin A (60 ng), the cuvette was incubated with 1 ml potato reaction medium (see section 2.1.3.2), 1 mM ferricyanide, 1 mM KCN and 0.2 mg of mitochondrial protein. Ferricyanide reduction rate increases upon addition of NADH and addition of AA completely stops ferricyanide reduction, indicating that there are no other pathways for electrons present but the cytochrome pathway. In order to add NADH and AA the cover of the spectrophotometer had to be opened upon which recording stops temporarily until the cover is closed again. The results in figures 4.17 and 4.18 demonstrate a clear difference in kinetics between potato and sp.011 wt mitochondria. Furthermore the sp.011 wt data suggests that upon increasing concentrations of NADH (which reflects an increase in Qr/Qt) a biphasic pattern occurs. These results corroborate the hypothesis that the biphasic pattern seen in sp.011 wt respiratory kinetics are possibly due to bc1 complex activity. Equally also, it demonstrates the presence of a biphasic pattern in the respiratory kinetics using a technique different from the Q-electrode and the TPP+ -electrode. The results shown in figures 4.17 and 4.18 show the relationship between NADH concentration and ferricyanide reduction rate. Increasing the concentration of NADH leads to an increase in Qr/Qt (cf. figures 4.4, 4.6 and 4.8). Originally, duplicate experiments were planned where in the presence of KCN and ferricyanide the relationship between NADH concentration and Qr/Qt could be determined. Unfortunately both KCN and ferricyanide react strongly with the Q-electrode and these compounds cannot be used in combination with this electrode. It is therefore not possible to establish quantitatively the Q-redox poise in the spectroscopy experiments. 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45 1.5 0 20 40 60 80 100 120 140 A420 time (s) NADH AA
  • 140 Figure 4.17 Ferricyanide reduction rates under state 2 and uncoupled conditions in potato mitochondria. Under both conditions the cuvette was incubated with 1 ml potato reaction medium, 1 mM KCN, 1 mM ferricyanide, 1 mM G6P and 10 units of G6PD, under uncoupled conditions 2 M CCCP was present. A steady state ferricyanide rate was induced by addition of a sub-saturating amount of NADH (0 – 100 M). One rate was determined per experiment. The data points are averaged rates from 2 mitochondrial isolations. Amount of mitochondrial protein used per experiment 0.1-02 mg. Figure 4.18 Ferricyanide reduction rates under uncoupled conditions in sp.011 wt mitochondria. the cuvette was incubated with 1 ml yeast reaction medium, 1 mM KCN, 1 mM Ferricyanide, 1 mM G6P, 10 units of G6PD and 2 M CCCP. A steady state ferricyanide rate was induced by addition of a subsaturating amount of NADH. One steady state rate was determined per experiment. The data points are averaged rates from 3 mitochondrial isolations. Amount of mitochondrial protein used per experiment 0.1 - 015 mg. 0 100 200 300 400 500 0 100 1 10-4 2 10-4 3 10-4 4 10-4 5 10-4 6 10-4 ferricyanidereductionrate (nmol/min/mgprotein) [NADH] M 0 100 200 300 400 500 600 700 0 100 5 10-5 1 10-4 1.5 10-4 2 10-4 2.5 10-4 3 10-4 ferricyanidereductionrate (nmol/min/mgprotein) [NADH] M
  • 141 The biphasic pattern in S. pombe mitochondrial respiratory kinetics may be explained in terms of yeast bc1 complex kinetics as reported by Trumpower et al. [33, 236]. The results obtained by Trumpower et al. were obtained from purified bc1 complexes isolated from S. cerevisiae mitochondria. If indeed the biphasic patterns are caused by yeast bc1 kinetics then it should be possible to reproduce the S. pombe findings in S. cerevisiae mitochondria. 4.2.5 Are biphasic respiratory kinetics a characteristic of yeast mitochondria? Figure 4.19 shows cytochrome pathway kinetics in mitochondria isolated from S. cerevisiae mitochondria. Oxygen consumption rate and Qr/Qt were measured simultaneously. Under state 2 conditions the relationship was linear (cf. Figure 4.4) whereas under uncoupled conditions a biphasic pattern is present (cf. Figure 4.8). These results are comparable to what was found in sp.011 wt mitochondria, which suggests that the biphasic respiratory kinetics are indeed a characteristic of yeast mitochondria. Figure 4.19 State 2 and uncoupled cytochrome pathway kinetics in S. cerevisiae mitochondria. State 2 () and uncoupled () cytochrome pathway kinetics were determined using the NADH regenerating system, titration range of NADH 0 – 55 M. Under uncoupled conditions CCCP was incubated at a concentration of 2 M. Amount of mitochondrial protein used 1.2 mg. The state 2 and uncoupled data were taken from 2 and 2 traces respectively, all data points are from one preparation. 0 10 20 30 40 50 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 142 4.3 Discussion 4.3.1 Respiratory characteristics of S. pombe mitochondria—In this study mitochondria isolated from S. pombe cells were characterised and the mitochondrial membrane potential was determined for the first time. Upon addition of NADH or succinate (with ATP and glutamate incubated) a ∆ is generated of about -200 mV (figures 4.1 and 4.2), addition of a limited amount of ADP leads to a state 3 state 4 transition and addition of CCCP leads to an increase in vO2 and dissipation of ∆. Additions of oligomycin or CAT prevent generation of a state 3 upon subsequent addition of ADP. These responses are essentially the same as is seen in plant [157] and mammalian [1] mitochondria. RCR values obtained with NADH as a substrate are different from the values obtained with succinate as a substrate. This is due to substrate dependent differences in reducing pathway kinetics (see section 4.2.3); i.e. respiratory rates under state 3 and uncoupled conditions with NADH as a substrate are considerably higher than with succinate as a substrate whereas the respiratory rates under ADP limited conditions with either succinate or NADH as a substrate are approximately the same. Upon addition of ADP or CCCP  is decreased and cytochrome pathway activity is stimulated. This is reflected in an approximately threefold increase in rate under state 3 conditions and an almost six times increase under uncoupled conditions with NADH as a substrate. A decrease in  not only stimulates the cytochrome pathway, it also deactivates SDH, whereas it has no effect on external NADH dehydrogenase activity (figures 4.13 and 4.15), hence the low RCR values obtained with succinate. Since S. pombe mitochondria do not express complex I there are only two coupling sites available to generate a pmf, which are both in the cytochrome pathway (complex III and IV) downstream from the Q-pool. Since SDH and the external NADH dehydrogenase feed electrons directly into the Q-pool a maximum ADP/O value of 1.5 would be expected [1]. The measured ADP/O values (NADH: 1.36, succinate: 1.22) are somewhat lower. A lowered ADP/O value can have a wide variety of causes such as transport of inorganic cations and various anionic metabolites, energy dissipation through uncoupling proteins, proton pump ‘slips’ which would alter the H+ /O ratio and the endogenous ion leak [238]. It could be that the current method of isolation yields mitochondria which have an IMM which is relatively permeable for protons. Alternatively it could be that a low ADP/O value
  • 143 is just a reflection of S. pombe physiology where mitochondria are not the primary source for ATP generation. This however would be in contrast with reported ADP/O values for S. pombe mitochondria by Jault et al. [105] (1.55-1.95) and Moore et al. [104] (~1.8) with NADH as a substrate. The main difference between the mitochondrial isolation methods used by those authors and the one used in this study lies within the enzyme digestion step (see section 2.1.1.5). Most yeast cell walls contain -linked glucan, which can be digested by gastric juice from the snail Helix pomatia [105]. S. pombe cell walls however also contain -linked glucan [239] which is resistant to -glucanases from snail gastric juice. Moore and Jault used the now no longer commercially available Novozyme 234 which has -glucanase activity. For this study a combination of enzymes was used: zymolyase 20/T which is only partially effective in producing spheroplasts in combination with lysing enzymes [26]. Incubation at 30 C with both enzyme mixtures present leads to 90% spheroplast production. It could be that the quality of the IMM also is affected by this treatment to a greater extent than with Novozyme 234. At some stage zymolyase became temporarily unavailable. As an alternative digestive enzyme Lyticase (Sigma) [240] was investigated. Unfortunately in order to be effective, very long incubation steps (several hours) and large quantities of the enzyme were needed, which made Lyticase unsuitable for the purpose of digesting the cell wall in S. pombe mitochondria. No other enzymes were investigated in this study. The hypothesis that digestion with zymolyase 20/T and lysing enzymes leads to decreased IMM quality provides the basis for future work in which the effectiveness of alternative enzymes can be investigated. Hopefully in the future a commercial product will become available which is as effective as Novozyme 234. Addition of either antimycin A, myxothiazol (both acting on complex III [1]) or KCN (acting on complex IV [1]) can completely inhibit respiration. This indicates that AOX is not expressed in S. pombe cells. This is a necessary requirement for these mitochondria to be used as a model system in which AOX is heterologously expressed. The presence of a cyanide-insensitive pathway in S. pombe mitochondria has been reported before but this has later been ascribed to a contamination of the isolated mitochondria [104], other studies on S. pombe mitochondria [26, 104, 105] did not show any cyanide- insensitive respiration.
  • 144 Membrane potential values obtained with either NADH or succinate20 were approximately the same (see Table 4.1E). The decrease in  upon addition of ADP was 20 mV which is comparable with mitochondria from other sources [157]. Addition of nigericin did not lead to a detectable change in TPP+ signal, this suggests that the pmf in S. pombe mitochondria essentially consists of a , which is, as in plant mitochondria [157], probably due to the presence of a K+ /H+ exchanger. It was postulated by Peter Mitchell that antiporters should be present in the IMM which electroneutrally exchange cations for protons in order to prevent osmotic swelling; the existence of the K+ /H+ exchanger21 was demonstrated in Keith Garlid’s laboratory [188]. At present there are no studies which show that S. pombe mitochondria express a K+ /H+ exchanger in the IMM. However, the presence of this exchanger in the IMM of S. cerevisiae mitochondria has been demonstrated [241] and its gene sequence is known (YOL027c). A BLAST search in which the YOL027c gene sequence was compared to the S. pombe genome revealed a homolog (SPAC23C11.17) which is annotated on http://www.genedb.org/ as a mitochondrial inner membrane protein involved in potassium ion transport. Its role was inferred from homology with YOL027c. This indicates, in all likelihood, that S. pombe expresses a K+ /H+ exchanger in the IMM of its mitochondria. These findings form the basis for future work. In a recent study by Froschauer et al. [241] the existence of the K+ /H+ exchanger in S. cerevisiae was demonstrated in an elegant set of experiments using submitochondrial particles (SMP’s) obtained from S. cerevisiae mitochondria. The SMP’s were loaded with either K+ -sensitive or H+ -sensitive fluorescent dyes. For determination of  an anionic dye which readily permeates depolarised membranes was used. K+ and H+ translocations across the membrane were inferred from changes in fluorescence. The experiments showed that K+ and H+ movement across the membrane as a reaction to imposed concentration gradients of either pH or K+ only occurred when K+ was exchanged for H+ , transport of either species did not occur independently. No change in membrane potential was detected during exchange of K+ for H+ demonstrating the electroneutrality of this process. Electrochemical equilibration between [K+ ] and [H+ ] brought about by nigericin was nearly identical to the equilibration 20 SDH fully activated with ATP and glutamate. 21 This exchanger is rather unselective and will in fact transport all alkali cations at similar rates, it is referred to as the K+ /H+ exchanger to distinguish it from the Na+ /H+ exchanger which is selective for Na+ [188].
  • 145 achieved by the endogenous system. Furthermore the process of K+ /H+ exchange was sensitive to either quinine, DCCD or Mg2+ , that are known inhibitors of this process. A mutant strain which lacks the gene for the K+ /H+ exchanger (yol027) was not able to exchange K+ for H+ . Expression of the human gene LETM1 (which is a homolog of YOL027c) restored the K+ /H+ exchange [241]. The experiments described above could readily be performed on S. pombe derived SMP’s. If, as expected, the presence of a K+ /H+ exchanger in S. pombe mitochondria can be demonstrated then subsequently S. pombe mitochondria could be used as a model system in medical research. It has been reported that a hemizygous deletion of the LETM1 gene in humans causes Wolf-Hirschhorn disease [241]. It is apparent from figures 4.1 and 4.2 that upon multiple additions of small aliquots of ADP successive state 4  values are lowered whereas the corresponding vO2 values are increased. This behaviour is reminiscent of potato mitochondria in which two molecules of ADP are generated (from ATP and AMP) via adenylate kinase activity in the intermembrane space [110]. It has been shown that under ADP limited conditions there is a continuous synthesis of ATP in liver and potato mitochondria [110, 186]. Addition of inhibitors of the ATP synthase or adenylate kinase under ADP limited conditions led to an increase in  and concomitant decrease in vO2 [111]. With each addition of ADP the total concentration of adenine nucleotides in the medium increases, therefore with each successive state 4 there is more ATP available for ADP generation via adenylate kinase. Since the presence of adenylate kinase has been shown in S. pombe mitochondria [112] it was an attractive notion to link the observed increased IMM conduction under state 4 conditions in S. pombe mitochondria to adenylate kinase activity. However, adenylate kinase activity could not be demonstrated conclusively. Under state 4 conditions the effect(s) of adding the following substances were studied: AMP, oligomycin, CAT and Ap5A (data not shown). Addition of oligomycin led to a decrease in vO2, however no change in  was seen. Addition of Ap5A (an inhibitor of the adenylate kinase [21]) did not appear to have any effect on either  or vO2. Addition of CAT led to a decrease in vO2 but no effect on  was seen. Addition of AMP had no effect. The decrease in vO2 under state 4 conditions observed when either oligomycin or CAT was added indicates that ATP was being synthesized at that time, this however should have been reflected also in a
  • 146 slight increase in . In potato mitochondria addition of AMP leads to the generation of a state 3 because ADP was generated via the adenylate kinase using AMP and ATP as substrates [21]. This was not seen in S. pombe. Addition of Ap5A had no effect. From these results it is unclear if adenylate kinase is active in S. pombe mitochondria. A BLAST search through the S. pombe genome (http://www.genedb.org/) does not reveal a mitochondrial adenylate kinase. Upon scrutinizing [112] it becomes apparent that no experimental evidence is given to support the original claim that S. pombe expresses a mitochondrial adenylate kinase. Based on experimental evidence obtained during this thesis work it is the authors opinion that S. pombe mitochondria do not express AK. What causes the increase in respiratory rates under state 4 conditions in S. pombe mitochondria remains unclear at present. 4.3.2 Are the biphasic patterns due to an experimental artefact?—The biphasic cytochrome pathway kinetics seen under state 3 conditions, Qr/Qt vs. vO2 and ∆ vs. vO2, (figures 4.6 and 4.7) and under uncoupled conditions, Qr/Qt vs. vO2, (Figure 4.8) have not been observed previously in mitochondria. It has been reported that in cyanide-sensitive mitochondria, electron flow through the cytochrome pathway is linearly dependent upon the redox state of the Q-pool [234]. To rule out whether or not the biphasic pattern is an artefact all possible non-physiological causes were investigated. When growing yeast cultures there is always the possibility of contamination (bacterial or the presence of another yeast). If a contamination was detected (cells being round instead of rod-like for instance) the yeast culture was discarded and all solutions and yeast plates were replaced. Yeast culture contamination occurred only rarely (two or three times during this study). To exclude the possibility that the biphasic respiratory kinetics were due to unnoticed22 contamination of the S. pombe cultures determination of cytochrome pathway kinetics was repeated many times. State 3 , Qr/Qt vs. vO2 data are from eleven traces from five different mitochondrial isolations. State 3, ∆ vs. vO2 data are from five traces from three mitochondrial isolations and the uncoupled, Qr/Qt vs. vO2 data are from five traces from five mitochondrial isolations. The mitochondrial preparations were isolated from yeast 22 Highly unlikely as the cultures were discarded if there was any doubt. Also, Dr M. Albury was always very helpful in giving a second opinion.
  • 147 cultures grown from two different yeast plates. A biphasic pattern is seen under state 3 or uncoupled conditions, but not under state 2 or state 4 conditions, this indicates that the cytochrome pathway kinetics show a dependency on energy status which is reflected in the biphasic pattern. If these biphasic kinetics were due to an experimental artefact why do the biphasic kinetics not appear under all energetic conditions? The biphasic pattern is seen in the relationships between Qr/Qt vs. vO2(state 3 and uncoupled) and  vs. vO2 (state 3), which means that the same pattern is seen in data sets from measurements done with different electrodes. The biphasic pattern cannot be attributed to the use of the same reference electrode since different ones were used in the Q-electrode set-up and the TPP+ electrode set-up. The biphasic pattern in S. pombe is also seen with succinate as a substrate thereby excluding the possibility that the biphasic pattern is due to the NADH titration method. The biphasic pattern is not seen in potato or arum mitochondria (see chapter 5) using the exact same experimental set-up which was used with the S. pombe system. A biphasic pattern was also seen using a completely different technique (spectrophotometry). In mitochondria expressing AOX the oxidising pathway kinetics under state 3 and uncoupled conditions were different (to be discussed in chapter 5). Addition of an extra oxidase led to a change in the biphasic pattern which indicates that it is a physiological process rather than an experimental artefact. In conclusion, the biphasic patterns as seen under state 3 and uncoupled conditions in the cytochrome pathway kinetics are a characteristic of S. pombe mitochondria. 4.3.3 Are the biphasic patterns due to cytochrome bc1 complex kinetics?—It was determined that the oxidising pathway kinetics in S. pombe mitochondria are the same with either NADH or succinate as a substrate (up until the point where vO2 rates become limiting with succinate dependent respiration); see figures 4.9 – 4.12. In other words, it does not matter in which way the Q-pool is reduced. That means that the underlying cause of the biphasic patterns in the oxidising pathway kinetics must be located downstream of the Q-pool. Since S. pombe mitochondria do not express AOX the biphasic kinetics must be a characteristic of the cytochrome pathway. The cytochrome pathway consists of the cytochrome bc1 complex, cytochrome c and cytochrome c oxidase with a stoichiometry of 3 : 14 : 7 [7]. On the basis of contemporary work done by Trumpower et al. on isolated yeast
  • 148 cytochrome bc1 complexes (see section 4.2.4) it was decided to investigate the possible role of the cytochrome bc1 complex in S. pombe mitochondria as a cause for the biphasic cytochrome pathway kinetics. For this purpose a different experimental approach was used. As opposed to simultaneously determine vO2 and either Qr/Qt or  (using the oxygen electrode in combination with either the Q-electrode or the TPP+ -electrode) a spectrophotometic technique was used. Ferricyanide (Fe(CN)6 3- ) is an artificial electron acceptor which can be reduced by cytochrome c. Mitochondria were incubated with ferricyanide, KCN (to inhibit cytochrome c oxidase), G-6-P and G-6-P-dehydrogenase (NADH regenerating system). It was determined that ferricyanide was the only pathway available to electrons to leave the respiratory chain (see Figure 4.16). Upon addition of subsaturating amounts of NADH the steady state change in absorption at 420 nm was determined. Increasing concentrations of NADH led to an increase in absorption change which reflects increased reduction of ferricyanide. This was done for both potato and S. pombe mitochondria under uncoupled conditions, see figures 4.17 and 4.18. It is apparent that the relationship of [NADH] vs. ferricyanide reduction rate in potato mitochondria has a convex shape whereas in S. pombe mitochondria there appears to be a biphasic pattern. These results indicate that, again, under uncoupled conditions there are kinetic differences in cytochrome pathway kinetics between potato and S. pombe mitochondria. Furthermore, the shape of the S. pombe kinetics appears to be biphasic, at low [NADH] there is low activity until a threshold value is reached upon which activity increases disproportionally. From the data available, this threshold NADH concentration value cannot be gauged, more experiments are needed in order to get a more continuous set of data points. It was concluded that it is probably cytochrome bc1 complex activity which causes the non-linear state 3 and uncoupled cytochrome pathway kinetics in S. pombe mitochondria. 4.3.4 Are biphasic respiratory kinetics a characteristic of yeast mitochondria?—The hypothesis that the biphasic respiratory kinetics in S. pombe mitochondria are due to bc1 complex kinetics was based on research done by Trumpower et al. on isolated bc1 complexes from S. cerevisiae mitochondria [33, 236]. If this hypothesis is correct then the biphasic respiratory kinetics should also be present in S. cerevisiae mitochondria. It was shown that under uncoupled conditions indeed the cytochrome pathway kinetics in S.
  • 149 cerevisiae mitochondria display a biphasic pattern, whereas under ADP limited conditions the relationship between Qr/Qt and vO2 is linear. These findings agree perfectly with the results obtained from S. pombe mitochondria and they corroborate the hypothesis that the observed biphasic patterns are caused by the bc1 complex. In order to understand how the kinetics of a single protein complex might determine mitochondrial respiratory kinetics Trumpower’s work on the S. cerevisiae bc1 complex will be reviewed in the general discussion chapter. Mechanisms proposed by Trumpower will be related to the biphasic respiratory kinetics found in S. pombe mitochondria. Having thoroughly characterised the respiratory kinetics of the S. pombe wild type mitochondria the next step was to incorporate the alternative oxidase protein in order to investigate its influence on respiratory kinetics.
  • 150 Chapter 5 Functional expression of the alternative oxidase in Schizosaccharomyces pombe mitochondria 5.1 INTRODUCTION Schizosaccharomyces pombe mitochondria heterologously expressing the alternative oxidase from the plant Sauromatum guttatum have been used in this laboratory to investigate structure-function relationships [59, 63, 94]. Many plant species as well as certain fungi23 and protists display cyanide-insensitive respiration [66]. The electron transfer chain in mitochondria of these species is branched at the level of the Q-pool, see Figure 1.4. Two pathways can accept reducing equivalents from the Q-pool; the conventional protonmotive cytochrome pathway and the non-protonmotive alternative pathway, which consists of the single AOX protein, see section 1.2.11. When the cytochrome pathway is inhibited (e.g. through incubation with antimycin A) several AOX expressing organisms can still generate a p through complex I [66]. S. pombe mitochondria however do not express complex I (see Figure 1.12) therefore S. pombe mitochondria expressing AOX, in the presence of an inhibitor of the cytochrome pathway cannot generate a p. It was found in plant mitochondria expressing AOX, when incubated in the presence of antimycin A, that succinate dependent respiration does not lead to the generation of p [176]. It was traditionally assumed that the alternative pathway functioned as an overflow pathway for reducing equivalents under conditions where cytochrome pathway activity was limited [37]. It has been shown however that both pathways can compete with each other for reducing equivalents [79]. Reducing equivalents can be diverted from the cytochrome pathway to the alternative pathway and vice versa [237]. Earlier work in this laboratory showed that under state 3 and state 4 conditions AOX 23 But not S. cerevisiae [242] or S. pombe [104].
  • 151 expression in S. pombe mitochondria led to a decrease in cytochrome pathway activity. In that study [93] oxygen consumption rate and Qr/Qt were measured simultaneously,  however was not determined. As shown in chapter 4, S. pombe mitochondria, respiring on either succinate or NADH can generate a  (see figures 4.1 and 4.2). The membrane potential in these mitochondria shows typical responses upon addition of ADP or uncoupler as seen in mitochondria from other yeasts [160] and from other species [1, 157]. It was concluded by Affourtit et al. that expression of AOX in S. pombe mitochondria leads to competition between the alternative and the cytochrome pathway for reducing equivalents, which leads to a lowered efficiency of energy transduction [93]. The same study showed a phenotypic effect of AOX expression in S. pombe, both growth rate and yield of S. pombe batch cultures were lowered when grown in the absence of thiamine (AOX expressed, see section 2.1.1). It was suggested that the change in phenotype was due to the effect of AOX expression on S. pombe respiratory kinetics [93]. It has been determined previously in this laboratory that S. guttatum AOX expressed in S. pombe mitochondria cannot be stimulated by pyruvate [59]. More recently it was found that S. guttatum AOX expressed in S. pombe mitochondria is constitutively active, as opposed to A. thaliana AOX expressed in S. pombe mitochondria, which can be stimulated by pyruvate [70]. It was hypothesized that during development of thermogenic species (like S. guttatum) high AOX engagement is ensured, at least in part, by expression of constitutively active isozymes [70]. In this chapter the respiratory kinetics of five types of mitochondria have been investigated, see Table 5.1. type source sp.011 wt non-transformed S. pombe ALU sp.011 AOX transformed S. pombe AOX expressed AU sp.011 AOX + T transformed S. pombe AOX repressed AUT sp.011 pREP transformed S. pombe empty vector AU A. maculatum isolated from fresh and cold stored spadices Table 5.1 types of mitochondria used and source material. A: adenine U: uracil L: leucine T: thiamine.
  • 152 Mitochondria isolated from S. pombe cells grown in the presence of adenine, leucine and uracil are referred to as sp.011 wt (wild type), see chapter 4. Mitochondria isolated from S. pombe cells which have been transformed with a plasmid coding for the S. guttatum AOX protein, grown in the presence of adenine and uracil, are referred to as sp.011 AOX. Mitochondria isolated from S. pombe cells which have been transformed with a plasmid coding for the S. guttatum AOX protein, grown in the presence of adenine, uracil and thiamine are referred to as sp.011 AOX + T (thiamine); AOX expression is repressed in these mitochondria due to the presence of the nmt1 promoter on the plasmid. Mitochondria isolated from S. pombe cells which have been transformed with the pREP plasmid (a vector lacking the gene coding for AOX), grown in the presence of adenine and uracil are referred to as sp.011 pREP. In previous work done in this laboratory the effects of a functionally expressed alternative oxidase in S. pombe mitochondria on respiratory kinetics were investigated [93]. Oxidising pathway kinetics were determined by titrating succinate dependent respiration with malonate under various energetic conditions. Limited data obtained with NADH as a substrate was found to overlap with the succinate data and it was concluded that there are no substrate dependent differences in oxidising pathway kinetics in S. pombe mitochondria expressing AOX. The amount of data obtained in that study was limited and the mitochondria used were of a lesser quality than the ones used in this study (see section 5.2.1). In this study the oxidising pathway kinetics were determined with NADH as a substrate using the NADH regenerating system (see section 2.4.4.4) in S. pombe mitochondria of good quality (see section 4.2.1 and 5.2.1). The oxidising pathway kinetics obtained with NADH as a substrate were different from the kinetics obtained by Affourtit et al. [93]. To investigate possible substrate dependent differences, oxidising pathway kinetics with succinate as a substrate were determined. Possible cause(s) for the differences between the results of this study and those of Affourtit were investigated. It was found that efficiency of energy transduction and cytochrome pathway activity was lowered in S. pombe mitochondria expressing AOX [93]. To investigate a possible decrease in protonmotive force due to AOX activity,  was determined in S. pombe mitochondria expressing AOX. For comparison with a system in which AOX is naturally expressed the respiratory kinetics of mitochondria isolated from A. maculatum were
  • 153 investigated. To investigate the effects of AOX expression on S. pombe respiratory kinetics results from experiments done on sp.011 AOX mitochondria (expressing AOX) were compared to results from the control group of sp.011 AOX+T mitochondria (AOX repressed). The act of transformation, possibly, could have altered respiratory kinetics of S. pombe mitochondria; this was investigated by comparing the results obtained with sp.011 AOX and AOX+T mitochondria to results obtained with sp.011 wt (the wild type, see chapter 4) mitochondria and with sp.011 pREP mitochondria (transformed S. pombe mitochondria with a vector lacking the AOX gene). 5.2 RESULTS 5.2.1 General characterisation of sp.011 AOX, AOX + T, pREP and wt respiratory kinetics Tables 5.2 and 5.3 show oxygen consumption rates and RCR values for sp.011 AOX, AOX + T, pREP and wt mitochondria. It was a priori expected that the respiratory characteristics of the sp.011 wt, sp.011 pREP and sp.011 AOX + T mitochondria would be similar, given that these mitochondria only have one oxidising pathway, the cytochrome pathway, as opposed to sp.011 AOX mitochondria which have both a cytochrome and an alternative pathway. sp.011 wt and pREP mitochondria have comparable oxygen consumption rates and RCR values. The sp.011 AOX + T oxygen consumption rates and RCR values are however different from the sp.011 wt and pREP values. Under ADP limited conditions the oxygen consumption rates of sp.011 wt, pREP and AOX+T are comparable. But under conditions where the protonmotive force is decreased (under state 3 and uncoupled conditions) the sp.011 AOX+T oxygen consumption rates are considerably lower than those of sp.011 wt and pREP. Also the sp.011 WT and pREP mitochondria appear to be better coupled than the sp.011 AOX+T mitochondria based on the RCR values. In order to compare sp.011 AOX cytochrome pathway activity with the other three types, oxygen consumption rates and RCR values were determined in the presence of octyl gallate (which potently inhibits AOX [59]). The obtained vO2 and RCR values are comparable to the sp.011 AOX+T values but are lower than the sp.011 wt and pREP values.
  • 154 sp.011 AOX Respiratory Rate (nmol O2 min-1 mg-1 protein) substrate(s) state 2 state 3 state 4 uncoupled NADH 199 (27) 328 (34) 224 (13) 531 (82) NADH + OG 92 (10) - - 396 (43) NADH + AA 81 (17) - - - succinate 142 (14) 198 (13) 136 (8) 176 (15) succinate + OG 83 (12) 123 (21) 72 (6) - succinate + AA 84 (13) - - - sp.011 AOX + T NADH 80 (13) 169 (29) 72 (9) 398 (55) NADH + OG 77 (12) - - 350 (48) succinate 83 (10) 136 (16) 87 (10) 124 (20) sp.011 pREP NADH 96 (13) 245 (25) - 543 (55) succinate 100 (13) 196 (24) 107 (23) - sp.011 wt NADH 91 (13) 281 (39) 110 (22) 537 (68) succinate 106 (15) 156 (18) 97 (7) 144 (25) Table 5.2 Respiratory rates in sp.011 AOX, AOX + T, wt and pREP mitochondria under various energetic conditions and various substrates. Format: rate ( S.D.). See appendix 2B for full details on amount of mitochondrial isolations and experimental traces. Concentrations of chemicals added: NADH (1.8 mM), succinate (9 mM), octyl gallate (14 M), antimycin A (~40 nM). Uncoupled conditions are defined as respiratory activity in the presence of an uncoupler (2 µM CCCP in all cases), which dissipates the protonmotive force leading to maximal activation of the cytochrome pathway. The concentration of added ADP to induce a state 3 to state 4 transition was 0.2 mM in all cases. State 3 conditions were induced by pre- incubation of ADP (1 mM). All succinate rates are from mitochondria incubated in the presence of ATP (0.2 mM) and glutamate (9 mM). The energy status had no effect on AOX activity, therefore the sp.011 AOX rates when incubated in the presence of antimycin A are the same under all energetic conditions.
  • 155 Based on similarities in vO2 rates and RCR values the various S. pombe mitochondria are divided into two groups. sp.011 wt and pREP mitochondria are well coupled (with NADH as a substrate the uncoupled / state 2 RCR is ~6) and display high uncoupled NADH vO2 rates ~ 540 nmol O2 / min / mg protein. sp.011 AOX (incubated in the presence of octyl gallate) and AOX+T mitochondria have somewhat lower couplings (with NADH as a substrate the uncoupled / state 2 RCR is 4-5) and uncoupled NADH vO2 rates are in the range of 350-400 nmol O2 / min / mg protein. sp.011 AOX mitochondria in the absence of octyl gallate (both pathways active) display higher vO2 rates under all energetic conditions when compared to sp.011 AOX+T mitochondria. Also the RCR with NADH as a substrate (uncoupled / state 2) is considerably lower (2.7) when compared to sp.011 AOX+T (5) indicating that AOX activity affects the efficiency of energy transduction in S. pombe mitochondria. Table 5.3 RCR values for sp.011 wt, pREP, AOX and AOX + T mitochondria with either NADH or succinate as substrate. NADH (1.8 mM) succinate (9 mM). The RCR values (see section 2.4.4.3) in Table B were calculated using the vO2 values from table 5.1. All succinate rates are from mitochondria incubated in the presence of ATP (0.2 mM) and glutamate (9 mM). sp.011 wt RCR's substrate S3 / S4 uncoupled / S2 NADH 2.6 5.9 succinate 1.6 1.4 sp.011 pREP NADH - 5.7 sp.011 AOX NADH 1.5 2.7 NADH + OG - 4.3 succinate 1.5 1.2 sp.011 AOX + T NADH 2.3 5 NADH + OG - 4.5 succinate 1.6 1.5
  • 156 It is not readily apparent why the cytochrome pathway respiratory characteristics of sp.011 AOX (incubated in the presence of OG) and sp.011 AOX+T mitochondria are different from sp.011 wt and sp.011 pREP cytochrome pathway kinetics. The act of transformation itself does not affect rates or coupling since the values obtained with sp.011 pREP compare well with sp.011 wt. The presence of thiamine in the yeast growth medium cannot be the cause for lowered respiratory rates and lowered coupling seen in sp.011 AOX+T (compared to sp.011 wt) since sp.011 AOX mitochondria (incubated in the presence of OG) show rates and couplings comparable to sp.011 AOX+T. The presence of leucine (in sp.011 wt) cannot be the causal factor for higher respiratory rates and coupling, compared to sp.011 AOX+T and sp.011 AOX (incubated in the presence of OG) mitochondria, since sp.011 pREP rates and coupling are comparable to sp.011 wt values. The only parameter which sp.011 AOX and sp.011 AOX+T mitochondria have in common is the fact that both types of mitochondria are isolated from S. pombe cells which have been transformed with a vector coding for AOX. How this in itself could affect cytochrome pathway activity is unclear. Importantly though, the cytochrome pathway characteristic of sp.011 AOX+T and sp.011 AOX mitochondria agree well with each other and the sp.011 AOX+T mitochondria constitute a proper control group for comparison with results obtained from sp.011 AOX mitochondria. To verify that AOX indeed was expressed (or repressed) in the transformed mitochondria, western blots were made (see section 2.2) using protein from isolated sp.011 AOX and AOX+T mitochondria. For comparison, protein from isolated A. maculatum mitochondria was used, see Figure 5.1. The A. maculatum protein bands (gel A lanes 6 and 7, gel B lanes 5 and 7) display both oxidised (~75 kDa) and reduced (~32 kDa) forms of AOX. It can be seen that AOX is expressed primarily in the reduced form (gel A, lanes 1-4 and gel B lanes 1-4) as indicated by the 32 kDa bands. The sp.011 AOX blots are marked by several bands of lower molecular weight. This was seen before in prior research (cf. Figure 5.1 in [26]) and it was attributed to protein degradation occurring during cold storage (-20°C). In order to prevent this, mitochondrial protein samples were incubated with a protease inhibitor cocktail (Roche Products Ltd) prior to cold storage, which clearly did not work. Importantly though, the AOX antibodies used, bound to a ~32 kDa protein band (which is the right size for an AOX monomer [243] ) and targeted both sp.011 AOX
  • 157 and A. maculatum mitochondria, but not sp.011 AOX+T in which AOX expression is repressed. Therefore sp.011 AOX mitochondria functionally express AOX. A B Figure 5.1 Immuno-detection of AOX in S. pombe and A. maculatum mitochondria. Lanes were loaded with 15 µg of protein and probed with antibodies raised against AOX (see section 2.2). Gel A was loaded with: sp.011 AOX mitochondria (1-4), sp.011 AOX + T mitochondria (5) and A. maculatum mitochondria (6-7). Gel B was loaded with sp.011 AOX mitochondria (1-4) and A. maculatum mitochondria (5 and 7), lane 6 is empty. Proteins were separated under non-reducing conditions.
  • 158 0 20 40 60 80 100 120 140 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 5.2.2 Oxidising pathway kinetics with NADH as substrate To obtain state 3 or uncoupled oxidising pathway kinetics a saturating amount of ADP (1 mM) or a saturating amount of CCCP (2 M) was incubated. To obtain state 4 oxidising pathway kinetics a state 3 to state 4 transition was induced by adding an aliquot of ADP (~50 M). 5.2.2.1 Comparing sp.011 pREP and wt cytochrome pathway kinetics with NADH as a substrate—The act of transforming S. pombe cells itself could possibly lead to changes in respiratory kinetics unrelated to the expression of AOX. To investigate an effect of transforming S. pombe cells with a vector on mitochondrial respiratory kinetics, cytochrome pathway kinetics of sp.011 wt and sp.011 pREP mitochondria were compared. Figure 5.2 shows the relationship between Qr/Qt and vO2 under state 2 and state 4 conditions. The sp.011 pREP vO2 rates at every value of Qr/Qt are slightly higher than the sp.011 wt rates. Both relationships are linear which agrees with results previously obtained in this laboratory [104] with succinate as a substrate. Figure 5.2 Combined state 2 and state 4 cytochrome pathway kinetics of sp.011 wt and sp.011 prep mitochondria. Using the NADH regenerating system (titration range 0 – 75 M) Qr/Qt and vO2 were measured simultaneously, sp.011 wt state 2 () and state 4 () data were taken from Figure 4.4. sp.011 pREP state 2 () data was obtained from three mitochondrial isolations, data points were combined from three traces. Mitochondrial protein used per experiment is 0.5-0.7 mg.
  • 159 0 50 100 150 100 120 140 160 180 200 220 vO2 nmolO2 /min/mgprotein membrane potential mV Figure 5.3 shows the relationship between ∆ and vO2 under state 2 and state 4 conditions in sp.011 pREP and sp.011 wt mitochondria respiring on NADH. The familiar non-ohmic relationship is seen (see section 3.1.2). The data sets from sp.011 wt and sp.011 pREP overlap, which suggests that the IMM conduction in both types of mitochondria is the same and that the act of transformation did not have an effect on proton permeability of the IMM. Figure 5.3 Combined state 2 and state 4 cytochrome pathway kinetics, ∆ vs. vO2, from sp.011 wt and sp.011 pREP mitochondria. Using the NADH regenerating system (titration range 0 – 75 M) ∆ and vO2 were measured simultaneously. sp.011 wt state 2 () and state 4 () data were taken from Figure 4.5. sp.011 pREP state 2 () data were obtained from two mitochondrial isolations, combining data points from four traces. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.4 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011 pREP and sp.011 wt mitochondria respiring on NADH. The sp.011 pREP vO2 rates at every value of Qr/Qt are slightly higher than the sp.011 wt rates . The same biphasic pattern as in sp.011 wt mitochondria can be seen.
  • 160 0 50 100 150 200 250 300 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 5.4 Combined state 3 cytochrome pathway kinetics, Qr/Qt vs. vO2, from sp.011 wt and sp.011 pREP mitochondria. Using the NADH regenerating system (titration range 0 – 75 M) Qr/Qt and vO2 were measured simultaneously. sp.011 wt state 3 () data were taken from Figure 4.6. sp.011 pREP state 3 () data were obtained from three mitochondrial isolations, combining data points from five traces. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.5 shows the relationship between ∆ and vO2 under state 3 conditions in sp.011 pREP and sp.011 wt mitochondria. The two data sets overlap but the typical biphasic pattern, as seen in sp.011 wt mitochondria is not seen in this sp0.11 pREP data set. The biphasic pattern was seen in sp.011 pREP data obtained with a TPP+ -electrode that was not properly calibrated, i.e. per tenfold change in [TPP+ ] ratio (see section 2.4.3) a value lower than 59 was obtained. This only affects the magnitude of the  values calculated and not the shape of the relationship between  and vO2 (see the inset in Figure 5.5). It can be concluded that under state 3 conditions the relationship between  and vO2 in sp.011 pREP is comparable to sp.011 wt.
  • 161 0 50 100 150 200 120 130 140 150 160 170 180 vO2 nmolO2 /min/mgprotein membrane potential mV 0 20 40 60 80 100 120 155 160 165 170 175 180vO2 nmolO2 /min/mgprotein membrane potential mV Figure 5.5 Combined state 3 cytochrome pathway kinetics, ∆ vs. vO2, from sp.011 wt and sp.011 pREP mitochondria. Using the NADH regenerating system (NADH titration range 0 – 75 M) Qr/Qt and vO2 were measured simultaneously. sp.011 wt state 3 () data were taken from Figure 4.7. sp.011 pREP state 3 () data (ADP 1 mM) were obtained from one mitochondrial isolation, combining data points from two traces. The inset shows the typical biphasic pattern which is also present in sp.011 pREP mitochondria, data from one trace. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.6 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 pREP and sp.011 wt mitochondria respiring on NADH. The sp.011 pREP vO2 rates at every value of Qr/Qt are slightly higher than the sp.011 wt rates. The sp.011 pREP data shows a biphasic pattern similar to the one seen in the sp.011 wt data. Under each energetic condition in sp.011 pREP mitochondria the vO2 is a bit higher at each value of Qr/Qt. One possible cause for this could be a difference in the quality of the IMM in both types of mitochondria. However, similar RCR values and overlapping state 2 ∆ vs. vO2 data suggest that both types of mitochondria have approximately the same IMM conductive characteristics. With the exception of somewhat higher respiratory rates in sp.011 pREP mitochondria it was concluded that the respiratory kinetics of sp0.11 wt and sp.011 pREP mitochondria are essentially the same.
  • 162 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 5.6 Combined uncoupled cytochrome pathway kinetics, Qr/Qt vs. vO2, from sp.011 wt and sp.011 pREP mitochondria. Using the NADH regenerating system (NADH titration range 0 – 75 M) Qr/Qt and vO2 were measured simultaneously. sp.011 wt uncoupled () data were taken from Figure 4.8. sp.011 pREP state 3 () data (ADP 1 mM) were obtained from three mitochondrial isolations, combining data points from six traces. Mitochondrial protein used per experiment is 0.5-0.7 mg. It was concluded that transformation of S. pombe cells does not lead to any significant changes in mitochondrial respiratory kinetics. It was already established that AOX is correctly targeted to the IMM in S. pombe mitochondria expressing AOX [59]. Affourtit found that the respiratory kinetics of sp.011 AOX mitochondria were different from those of sp.011 AOX+T mitochondria, it was assumed that these differences were due to AOX activity [93]. Having established that AOX is correctly targeted and that transformation does not change the S. pombe wild type respiratory kinetics; in a next series of experiments the oxidising pathway kinetics of sp.011 AOX and sp.011 AOX+T mitochondria respiring on NADH were investigated.
  • 163 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 5.2.2.2 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway kinetics with NADH as a substrate—To investigate any effects of AOX expression on respiratory kinetics, oxidising pathway kinetics of sp.011 AOX and sp.011 AOX+T mitochondria were compared. Figure 5.7 shows the relationship between Qr/Qt and vO2 under state 2 and state 4 conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on NADH. The vO2 rates at any value of Qr/Qt in sp.011 AOX mitochondria are considerably higher than in sp.011 AOX + T, i.e. the presence of AOX leads to an overall increase in vO2. Both relationships are linear as seen previously in sp.011 wt and sp.011 pREP (cf. Figure 5.2) Figure 5.7 Combined state 2 and state 4 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously (NADH titration range 0 – 75 M). sp.011 AOX state 2 () data were obtained from five mitochondrial isolations, combining data from eight traces. sp.011 AOX + T state 2 () and state 4 () data were obtained from four and one mitochondrial isolations respectively, combining data points from eight and two traces respectively. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.8 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011 AOX and sp.011 AOX+T mitochondria respiring on NADH. In sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T mitochondria. Interestingly, in the presence of AOX the biphasic pattern is no longer seen and the relationship between Qr/Qt and vO2 has become linear. The relationship between Qr/Qt and
  • 164 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt vO2 in the sp.011 AOX + T mitochondria is biphasic as seen in sp.011 wt and sp.011 pREP (cf. Figure 5.4). Figure 5.8 Combined state 3 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously (NADH titration range 0 – 75 M). sp.011 AOX state 3 () data were obtained from three mitochondrial isolations, combining data from six traces. sp.011 AOX + T state 3 () data were obtained from seven mitochondrial isolations, combining data points from twelve traces. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.9 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 AOX and sp.011 AOX+T mitochondria respiring on NADH. In sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T mitochondria. As seen under state 3 conditions (cf. Figure 5.8) the biphasic pattern appears to have become linear in sp.011 AOX mitochondria. However, when looking at individual experimental traces of sp.011 AOX mitochondria (see inset in Figure 5.9) it is evident that a biphasic pattern is still to be seen, indicating that the activity of AOX leads to a masking (but not disappearance) of the biphasic kinetics. The sp.011 AOX+T kinetics are biphasic as seen in sp.011 wt and sp.011 pREP, (cf. Figure 5.6).
  • 165 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 5.9 Combined uncoupled oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Using the NADH regenerating system Qr/Qt and vO2 were measured simultaneously (NADH titration range 0 – 75 M). sp.011 AOX uncoupled () data were obtained from five mitochondrial isolations, combining data from nine traces. sp.011 AOX + T state 3 () data were obtained from six mitochondrial isolations, combining data points from ten traces. The inset shows a representative trace of uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.10 shows the relationship between ∆ and vO2 under state 2 conditions in sp.011 AOX and sp.011 AOX+T mitochondria. Clearly the presence of AOX does not change the relationship. Data points from both sets overlap up until vO2 becomes limiting in sp.011 AOX+T mitochondria. The fact that the relationship between ∆ and vO2 does not change in the presence of AOX indicates that the functional expression of this protein does not lead 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 166 0 50 100 150 100 120 140 160 180 200 220 vO2 nmolO2 /min/mgprotein membrane potential mV to a disruption of the IMM. It is known that passive proton leak through the bulk phospholipid bilayer accounts for only a small proportion (between 5 to 25 %) of total mitochondrial proton leak [184, 244]. It has been hypothesized that the presence of protein complexes in the IMM can non-specifically increase the permeability of the IMM [245]. Incorporation of AOX into the IMM of S. pombe mitochondria could therefore lead to an increase in conductivity. Figure 5.10 however shows that the IMM conductivity of S. pombe mitochondria  AOX is the same. Therefore expression of AOX does not affect IMM conductivity and any differences in respiratory kinetics seen between mitochondria expressing AOX and mitochondria in which AOX is repressed are therefore likely to be due to AOX activity. Figure 5.10 Combined state 2 oxidising pathway kinetics, ∆ vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Using the NADH regenerating system ∆ and vO2 were measured simultaneously (NADH titration range 0 – 75 M). sp.011 AOX () data were obtained from two mitochondrial isolations, combining data from four traces. sp.011 AOX + T () data were obtained from two mitochondrial isolations, combining data points from three traces. Mitochondrial protein used per experiment is 0.5-0.7 mg. Figure 5.11 shows the relationship between ∆ and vO2 under state 3 conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on NADH. There does not appear to be any difference between both data sets. Interestingly the biphasic pattern is still present in sp.011 AOX mitochondria and does not look different from the pattern seen in the
  • 167 0 50 100 150 200 250 140 145 150 155 160 165 170 175 180 vO2 nmolO2 /min/mgprotein membrane potential mV mitochondria in which AOX is repressed (or in the wild type mitochondria, cf. Figure 4.7). As the ∆ is generated by activity of the cytochrome pathway, this indicates that the biphasic kinetics are a characteristic of cytochrome pathway activity, which corroborates the hypothesis that the biphasic kinetics are due to bc1 complex activity, see section 4.2.4. Due to experimental variation it cannot be concluded from the data in Figure 5.11 whether or not the presence of AOX has an effect on membrane potential. Figure 5.11 Combined state 3 oxidising pathway kinetics, ∆ vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Using the NADH regenerating system ∆ and vO2 were measured simultaneously (NADH titration range 0 – 75 M). sp.011 AOX () data were obtained from one mitochondrial isolation, combining data from three traces. sp.011 AOX + T () data were obtained from two mitochondrial isolations, combining data points from eight traces. Mitochondrial protein used per experiment was 0.5-0.7 mg. Addition of octyl gallate to sp.011 AOX mitochondria respiring under ADP limited conditions led to a decrease in vO2 but did not lead to a change in  (data not shown). This can easily be explained by the non-ohmic nature of the  vs. vO2 relationship under ADP limited conditions. Due to the increased conductivity of the IMM at high  (see section 3.1.2) a significant decrease of  will only be seen after a disproportional inhibition of respiration. Given the low capacity of the IMM it only requires the net transfer of 1 nmol of H+ per mg of protein across the membrane to establish a full  [1]. It was determined in S. pombe mitochondria under ADP limited conditions that a full  was
  • 168 established at a rate of ~50 nmol O2 / min / mg protein. If AOX activity under ADP limited conditions would compete with the cytochrome pathway to such an extent that the steady state vO2 generated by cytochrome pathway activity would decrease below 50 nmol O2 / min / mg protein then a decrease in  could occur. This however does not occur, the maximum rate attainable by AOX activity is approximately 80 nmol O2 / min / mg protein, see Table 5.1. NADH dependent respiration under ADP limited conditions in sp.011 AOX mitochondria is approximately 200 nmol O2 / min / mg protein and with succinate it is around 150 nmol O2 / min / mg protein. Therefore in both cases the minimum rate of 50 nmol O2 / min / mg protein (generated through cytochrome pathway activity) in order to establish a full  are easily attained and inhibition of AOX activity will not lead to a change in  under ADP limited conditions. Under state 3 conditions however the relationship between  vs. vO2 in S. pombe mitochondria becomes ohmic after the biphasic point, i.e. a change in cytochrome pathway activity leads to a proportional change in , see figures 4.7, 5.5 and 5.11. A linear relationship between  and vO2 under state 3 conditions has been demonstrated in potato mitochondria [133, 157]. As a control experiment for comparison with sp.011 wt respiratory kinetics the relationship between  vs. vO2 under state 3 conditions was determined in potato mitochondria and was found to be linear, in agreement with the literature, see inset A of Figure 4.7. Under state 3 conditions a decrease in cytochrome pathway activity would lead to a decrease in . If AOX and cytochrome pathway activity in sp.011 AOX mitochondria would be additive, i.e. cytochrome pathway activity is not affected by AOX activity, then inhibition of AOX should not lead to a decrease in . If AOX in sp.011 AOX mitochondria could actively compete with the cytochrome pathway for reducing equivalents then AOX activity would lead to a decreased cytochrome pathway activity (compared to the situation where AOX is absent) and  would therefore be proportionally lower. This follows directly from Ohm’s law: I = g*E I: current, g: conductance and E: electrical potential [246].
  • 169 Under state 3 conditions g is constant, indicated by the linear relationship between I (oxygen consumption rate) and E (). The relationship between I and E is proportional. When due to AOX activity the electron transfer rate through the cytochrome pathway would decrease (through competition for reducing equivalents) the oxygen consumption rate generated through cytochrome pathway activity would decrease and therefore also . To test this hypothesis the following experiment was done: a state 3 was induced in sp.011 AOX mitochondria respiring on either NADH or succinate by addition of a saturating amount of ADP (1 mM), when a steady state was reached a saturating amount of octyl gallate (14 M) was added to completely inhibit AOX. If AOX was actively competing with the cytochrome pathway then inhibition of this protein would lead to a diversion of electron flow from the alternative pathway to the cytochrome pathway. The increase in cytochrome pathway activity would then necessarily lead to an increase in . Figure 5.12 shows a representative experiment where the effect of AOX activity on  was investigated in sp.011 AOX mitochondria respiring on NADH. Figure 5.12 AOX activity has no effect on . Representative oxygen concentration and  traces of sp.011 AOX mitochondria respiring on NADH. Additions NADH (1.8 mM) ADP (1 mM) and octyl gallate (OG, 14 M). See text for details. 0 100 200 300 400 500 600 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 250 300 350 400 450 500 550 600 oxygen(nmol) TPP+ signalV time (s) TPP+ signal O2 NADH ADP OG 175 324 249
  • 170 It can be seen that upon addition of a saturating amount of octyl gallate the vO2 rate decreases significantly,  however remains constant. This suggests that in sp.011 AOX mitochondria the activities of the cytochrome and the alternative pathway are additive. Comparable results were obtained with succinate as a substrate (data not shown). Based on these results it was concluded that AOX activity does not have an effect on  in S. pombe mitochondria. The results shown in figures 5.7 to 5.9 show that under all energetic conditions, in the presence of AOX, at each value of Qr/Qt the vO2 rates are higher when compared to S. pombe mitochondria in which AOX is repressed. These results are different from what was found by Affourtit who found that under state 3 and state 4 conditions, in the presence of AOX vO2 rates were lower when compared to S. pombe mitochondria in which AOX is repressed [93]. Under uncoupled conditions no significant difference was found between S. pombe mitochondria in which AOX was either expressed or repressed [93]. The oxidising pathway kinetics determined by Affourtit were based on malonate titrations of S. pombe mitochondria respiring on succinate. In order to investigate the possibility of substrate dependent differences in oxidising pathway kinetics in S. pombe mitochondria expressing AOX it was decided to repeat these experiments. 5.2.3 Oxidising pathway kinetics with succinate as substrate 5.2.3.1 Comparing sp.011 AOX and sp.011 AOX + T oxidising pathway kinetics with succinate as a substrate—With the sp.011 AOX mitochondria it was difficult to obtain steady state respiratory rates with succinate after additions of malonate during the trace, this was due to a combination of an effect of malonate24 on the Q-electrode and the long time it takes for electron flow partition between the cytochrome and alternative pathway to stabilise after addition of malonate. In fact steady states were rarely reached. This problem did not occur with NADH as a substrate, presumably because the external NADH dehydrogenase has a much higher activity than SDH and a steady state electron flow to 24 Upon addition of malonate the Q-electrode signal deflects downwards and recovers slowly, this limits the amount of data points one can obtain during an experimental trace.
  • 171 both pathways is achieved relatively fast. In order to get around this problem sp.011 AOX succinate data points were obtained by using ‘standard titrations’ (see section 2.4.4.4). Steady state Qr/Qt and vO2 values were determined after additions of succinate, ATP and glutamate respectively (cf. Figure 4.3). This yields three data points which are relatively spaced apart. When incubated with malonate (thus avoiding effects of malonate addition on the Q-electrode signal during the trace) the range of these three data points can be shifted to the left. By combining data sets from several experiments nearly the whole range of Qr/Qt can be covered. Figure 5.13 shows the relationship between Qr/Qt and vO2 under state 2 and state 4 conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on succinate. In sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T mitochondria and both relationships are linear, comparable to what was seen in the NADH titration experiments, (cf. Figure 5.7). This result was the complete opposite of what was seen by Affourtit [93] who found that under ADP limited conditions, at each value of Qr/Qt, in the presence of AOX, vO2 values were lowered when compared to sp.011 AOX+T mitochondria. Figure 5.14 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011 AOX and sp.011 AOX + T mitochondria with succinate as a substrate. In sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T mitochondria, similar to what is seen with NADH as a substrate (cf. Figure 5.8). sp.011 AOX+T mitochondria display the familiar biphasic pattern (cf. figures 4.11, 5.4 and 5.8). The relationship between Qr/Qt vs. vO2 in sp.011 AOX mitochondria is linear (the biphasic point is masked), similar to what is seen with NADH as a substrate (cf. Figure 5.8). These results are different from what was found by Affourtit [93]. Under state 3 conditions he found that at Qr/Qt values higher than 40%, in the presence of AOX, the vO2 rate values were lower when compared to sp.011 AOX+T mitochondria. Figure 5.15 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 AOX and sp.011 AOX + T mitochondria respiring on succinate. In sp.011 AOX mitochondria the vO2 rates at each value of Qr/Qt are higher than in sp.011 AOX+T mitochondria, similar to what is seen with NADH as a substrate (cf. Figure 5.9). sp.011 AOX+T mitochondria display the familiar biphasic pattern (cf. figures 4.12, 5.6 and 5.9). The relationship between Qr/Qt vs. vO2 in sp.011 AOX mitochondria is linear (the
  • 172 biphasic point is masked), similar to what is seen with NADH as a substrate (cf. Figure 5.9). These results are different from what was found by Affourtit [93]. Under uncoupled conditions he found that the oxidising pathway kinetics of sp.011 AOX and sp.011 AOX+T mitochondria were virtually identical. Figure 5.13 Combined state 2 and state 4 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Succinate-dependent respiration was titrated with malonate with sp.011 AOX+T mitochondria. Standard titrations were used with both sp.011 AOX and sp.011 AOX+T mitochondria. Qr/Qt and vO2 were measured simultaneously. sp.011 AOX state 2 () and state 4 (∆) data were obtained from four and four mitochondrial isolations respectively, combining data points from six and five traces respectively. sp.011 AOX + T state 2 () and state 4 () malonate titration data were obtained from one and two mitochondrial isolations respectively, combining data points from two and five traces respectively. sp.011 AOX+T standard data points were obtained from five and four mitochondrial isolations respectively, combining data points from six and five traces respectively. Mitochondrial protein used per experiment is 0.5-0.7 mg. 0 50 100 150 200 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 173 Figure 5.14 Combined state 3 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Succinate-dependent respiration was titrated with malonate with sp.011 AOX+T mitochondria. Standard titrations were used with sp.011 AOX and sp.011 AOX+T mitochondria. Qr/Qt and vO2 were measured simultaneously. sp.011 AOX data () were obtained from six mitochondrial isolations, combining data points from eleven traces. sp.011 AOX + T data () were obtained from three mitochondrial isolations, combining data points from six traces (malonate titrations) and from five mitochondrial isolations, combining six traces (standard traces). Mitochondrial protein used per experiment is 0.5-0.7 mg. To investigate the cause(s) of the differences between the results of Affourtit [93] and the results obtained in this study several possibilities were considered. The method of isolation was different and it was known that the mitochondria used in Affourtit’s study were of different quality. The RCR value of sp.011 AOX+T mitochondria respiring on NADH (uncoupled / state 2) in this study is about 5 (Table 5.3). The equivalent RCR value in Affourtit’s study is estimated by this author to be around 2 (cf. Figure 2A in [93]). More importantly though, the reaction medium which was used in Affourtit’s study for the measurement of Qr/Qt and vO2 had a lower osmolarity from what is customarily used in yeast studies [104, 105, 160, 240, 247]. The reaction medium used in this study (see section 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 174 2.1.1.7) contains a mannitol concentration of 0.65 M whereas the medium used by Affourtit (potato reaction medium, see section 2.1.3.2) contains a mannitol concentration of 0.3 M. It has been reported that lowering of the osmolarity leads to a decrease in vO2 rates in S. pombe mitochondria [105]. To further investigate this, the relationship between Qr/Qt and vO2 under state 3 conditions was determined in sp.011 AOX mitochondria in potato reaction medium (0.3 M mannitol) and yeast reaction medium (0.65 M mannitol), see Figure 5.16. The oxidising pathway kinetics in the low osmotic medium level off at high values of Qr/Qt. These results suggest that a difference in osmotic strength of the reaction medium affects S. pombe respiratory kinetics. Figure 5.15 Combined uncoupled oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX and sp.011 AOX + T mitochondria. Succinate-dependent respiration was titrated with malonate with sp.011 AOX+T mitochondria. Standard titrations were used with sp.011 AOX mitochondria. Qr/Qt and vO2 were measured simultaneously. sp.011 AOX data () were obtained from three mitochondrial isolations, combining data points from seven traces . sp.011 AOX + T data () were obtained from two mitochondrial isolations, combining data from five traces (malonate titration data) and from three mitochondrial isolations, combining data from three traces (standard traces). Mitochondrial protein used per experiment is 0.5-0.7 mg. 0 50 100 150 200 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 175 Figure 5.16 Combined state 3 oxidising pathway kinetics, Qr/Qt vs. vO2, from sp.011 AOX mitochondria in reaction medium containing 0.3 M mannitol () or 0.65 M mannitol (). The oxidising pathway kinetics were obtained using the NADH regeneration method (NADH titration range 0 – 75 M). State 3 conditions were induced by pre-incubation with ADP (1 mM). The high osmolarity (0.65 M mannitol) data were obtained from two mitochondrial isolations, data points taken from three traces. The low osmolarity (0.3 M mannitol) data were obtained from two mitochondrial isolations, data points taken from four traces. Mitochondrial protein used per experiment is 0.5-0.7 mg. At the time when the research on S. pombe mitochondria was conducted by Affourtit it was unknown that S. pombe mitochondria display ethanol dependant respiration as was demonstrated later by Crichton [26]. Addition of small amounts of pure ethanol (as low as 1 l) will result in a significant oxygen consumption rate. Many of the substances added by Affourtit during his experiments were dissolved in ethanol, Q1 which was present in all experiments being one of them. The addition of ethanol to S. pombe mitochondria in solution will lead to ADH activity, (see section 1.3.1) which results in matrix NADH generation. Possibly the respiratory kinetics determined by Affourtit [93] with succinate as a substrate were a result of combined SDH and internal NADH dehydrogenase activities. Respiratory kinetics determined with NADH as a substrate were possibly a result of combined external and internal NADH dehydrogenase activities. 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 176 5.2.4 Oxidising pathway kinetics in sp.011 AOX mitochondria Results so far show that independent of substrate in S. pombe mitochondria expressing AOX, under all energetic conditions, at each value of Qr/Qt, the vO2 rates are higher when compared to sp.011 AOX+T mitochondria. Also, in the presence of AOX the biphasic kinetics under state 3 and uncoupled conditions are no longer seen and the relationship between Qr/Qt and vO2 becomes linear (the biphasic pattern is masked). Table 5.2 shows that steady state oxygen consumption rates with succinate as a substrate under all energetic conditions are lower than with NADH as a substrate. This could be due to SDH activity becoming limiting. For instance, in figures 4.11 and 4.12 the cytochrome pathway kinetics with either succinate or NADH as a substrate are clearly the same. With NADH as a substrate however the Q-pool can be reduced to a higher degree than with succinate, which results in higher maximum vO2 rates attainable with NADH as a substrate. sp.011 AOX oxidising pathway kinetics under various energetic conditions with either succinate or NADH as a substrate were plotted together to see if there were any substrate dependent differences in kinetics. 5.2.4.1 Comparing sp.011 AOX oxidising pathway kinetics with either NADH or succinate as a substrate—Figure 5.17 shows the relationship between Qr/Qt and vO2 under state 2 and state 4 conditions in sp.011 AOX mitochondria respiring on either NADH or succinate. It can be appreciated that at high Qr/Qt (~60%) and upwards the vO2 values attained with NADH as a substrate are higher than with succinate. Figure 5.18 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011 AOX mitochondria respiring on either NADH or succinate. It is clear that from Qr/Qt values around 40% and upwards with NADH as a substrate the vO2 rates at each value of Qr/Qt are higher than with succinate as a substrate. Figure 5.19 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 AOX mitochondria respiring on either NADH or succinate. The data suggests that from Qr/Qt values around 40% Qr/Qt with NADH as a substrate the vO2 rates at each value of Qr/Qt are higher than with succinate.
  • 177 Figure 5.17 Comparing state 2 and state 4 oxidising pathway kinetics in sp.011 AOX mitochondria respiring on either NADH or succinate. Succinate state 2 () and state 4 () were taken from Figure 5.13. NADH state 2 data () were taken from Figure 5.7. Figure 5.18 Comparing state 3 oxidising pathway kinetics in sp.011 AOX mitochondria respiring on either NADH or succinate. Succinate () data were taken from Figure 5.14. NADH () data were taken from Figure 5.8. 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 178 Figure 5.19 Comparing uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria respiring on either NADH or succinate. Succinate () data were taken from Figure 5.15. NADH () data were taken from Figure 5.9. The results shown in figures 5.17-5.19 indicate that there are substrate dependent differences under all energetic conditions in sp.011 AOX mitochondria respiring on either succinate or NADH. It was shown in chapter 4 (cf. figures 4.9-4.12) that wild type S. pombe mitochondria do not display any substrate dependent differences in cytochrome pathway kinetics. The results presented in section 5.2.2.1 (cf. figures 5.2-5.6) show that transformation of S. pombe mitochondria does not change its respiratory kinetics. However the sp.011 wt and sp.011 pREP respiratory kinetics are somewhat different from the sp.011 AOX+T and sp.011 AOX (incubated in the presence of OG) mitochondria as reflected in differences between vO2 rates and RCR values, see section 5.2.1. sp.011 wt and sp.011 pREP mitochondria may not be the proper control group. To verify whether or not the substrate dependent differences observed in sp.011 AOX mitochondria are due to altered cytochrome pathway kinetics induced by the transformation with a vector coding for AOX the cytochrome pathway kinetics with either succinate or NADH as a substrate from sp.011 AOX+T (AOX expression repressed) were compared. 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 179 5.2.5 Cytochrome pathway kinetics in sp.011 AOX+T mitochondria 5.2.5.1 Comparing sp.011 AOX + T cytochrome pathway kinetics with either NADH or succinate as a substrate—Figure 5.20 shows the relationship between Qr/Qt and vO2 under state 2 and state 4 conditions in sp.011 AOX + T mitochondria respiring on either succinate or NADH. Data points obtained with succinate overlap with those obtained with NADH. This suggests that under ADP limited conditions in sp.011 AOX+T mitochondria there are no substrate dependent differences in cytochrome pathway kinetics. Figure 5.20 Comparing state 2 and state 4 cytochrome pathway kinetics in sp.011 AOX + T respiring on either NADH or succinate. Succinate state 2 (, ) and state 4 (, ) data were taken from Figure 5.13. NADH state 2 () and state 4 (∆) data were taken from Figure 5.7. Figure 5.21 shows the relationship between Qr/Qt and vO2 under state 3 conditions in sp.011 AOX + T mitochondria respiring on either succinate or NADH. No apparent substrate differences can be seen in cytochrome pathway kinetics. Figure 5.22 shows the relationship between Qr/Qt and vO2 under uncoupled conditions in sp.011 AOX + T mitochondria respiring on either succinate or NADH. No apparent substrate dependent differences can be seen in the cytochrome pathway kinetics. 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 180 Figure 5.21 Comparing state 3 cytochrome pathway kinetics in sp.011 AOX + T respiring on either NADH or succinate. Succinate (, ) data were taken from Figure 5.14. NADH () data were taken from Figure 5.8. Figure 5.22 Comparing uncoupled cytochrome pathway kinetics in sp.011 AOX + T mitochondria respiring on either NADH or succinate. Succinate (, ) data were taken from Figure 5.15. NADH () data were taken from Figure 5.9. 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 181 It can be concluded from the results shown in figures 5.20-5.22 that there are no substrate dependent differences in sp.011 AOX+T cytochrome pathway kinetics. This suggests that the substrate dependent differences observed in sp.011 AOX mitochondria are not due to transformation of S. pombe cells with a vector containing the S. guttatum AOX gene. Equally also it can be concluded from figures 5.20-5.22 that inclusion of thiamine in the growth medium does not affect cytochrome pathway kinetics. Since the substrate dependent differences in oxidising pathway kinetics in sp.011 AOX mitochondria cannot be attributed to cytochrome pathway characteristics the most likely cause would be a substrate dependent difference in alternative pathway kinetics. 5.2.6 Alternative pathway kinetics in sp.011 AOX mitochondria 5.2.6.1 Comparing sp.011 AOX alternative pathway kinetics with either NADH or succinate as a substrate—To investigate substrate dependent differences in alternative pathway kinetics, sp.011 AOX mitochondria were incubated with antimycin A to inhibit the cytochrome pathway [1]. Using either the NADH regenerating system or the malonate titration method alternative pathway kinetics were determined with either NADH or succinate as a substrate. For this experiment four different mitochondrial isolations were used and per isolation several traces of both NADH and succinate dependent respiration were determined. Unfortunately there was quite some variation in vO2 rates between isolations and combining the data would give the impression that with succinate as a substrate higher vO2 rates are obtained at each value of Qr/Qt in comparison to NADH kinetics. However, when analysing the data per day it was obvious that there are no substrate dependent differences in alternative pathway kinetics. Figure 5.23 shows representative data from one of the experimental days. From these results it was concluded that the substrate dependent differences as seen in sp.011 AOX oxidising pathway kinetics are not due to underlying substrate dependent alternative pathway kinetics.
  • 182 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 5.23 Comparing alternative pathway activity in sp.011 AOX mitochondria respiring on either succinate or NADH. Qr/Qt and vO2 were determined simultaneously, antimycin A was incubated (2.5 µL of 1 mg/ml AA). Succinate data points () taken from two traces, NADH data points () taken from three traces. Mitochondrial protein used is 0.5 mg. The antimycin-insensitive respiration rates displayed by sp.011 AOX mitochondria respiring on either succinate or NADH are comparable (cf. Table 5.2). In many plants antimycin-insensitive respiration with succinate is relatively high when compared with NADH dependent respiration [74]; this is due to the fact that with exogenous NADH as substrate no pyruvate is generated which can activate AOX [79, 248]. NADH respiratory rates being comparable to succinate respiratory rates in sp.011 AOX mitochondria, incubated in the presence of antimycin A, indicate that AOX expressed in S. pombe mitochondria is constitutively active, as has been demonstrated previously [70]. The results so far show that S. pombe mitochondria heterologously expressing a plant AOX display substrate dependent oxidising pathway kinetics which cannot be explained in terms of cytochrome pathway or alternative pathway kinetics. The substrate dependent differences in oxidising pathway kinetics may be a particularity of the expression system used in this study. For comparison, oxidising pathway kinetics with either succinate or NADH as a substrate were determined in isolated mitochondria from Arum maculatum, a thermogenic plant species [249] which naturally expresses AOX [250].
  • 183 5.2.7 Oxidising pathway kinetics in Arum maculatum mitochondria 5.2.7.1 Investigating substrate dependent differences in oxidising pathway kinetics in Arum maculatum mitochondria—Arum maculatum is, just like Sauromatum guttatum [251] a thermogenic plant species. During the development of thermogenesis A. maculatum spadices pass through a recognizable sequence of developmental stages (termed -) which results in a rapid rise in respiration that is responsible for a period of heat production during pollination [88]. Heat production can lead to a temperature difference with the environment of about 15 C [251], the aim of generating heat being to volatilise aromatic compounds to attract pollinators [66]. In A. maculatum mitochondria isolated during the -stage the alternative pathway is barely active, whereas during the / stage alternative pathway activity is much more pronounced. During the -stage virtually all respiration is due to alternative pathway activity [88]. In this study arum spadices were collected in local Sussex woods during the / stage in order to investigate possible substrate dependent differences in oxidising pathway kinetics in an organism naturally expressing both the cytochrome and the alternative pathway. When arum spadices are cold stored (4 C) overnight, prior to mitochondrial isolation both cytochrome pathway and SDH activities are increased, whereas alternative pathway activity remains unaltered [88]. Arum mitochondria were isolated from freshly picked or cold stored spadices (see section 2.1.4 for isolation protocol). In S. pombe mitochondria expressing AOX the activity of the alternative pathway is considerable given the fact that under all energetic conditions, at each value of Qr/Qt the respiratory rates in mitochondria expressing AOX are higher than in mitochondria in which AOX is repressed, see figures 5.7-5.9 and 5.13-5.15. However, under uncoupled conditions most of the respiratory activity is due to cytochrome pathway activity since the maximum alternative pathway activity is limited to 80-100 nmol O2 / min / mg protein and the total respiration rate in uncoupled sp.011 AOX mitochondria is around 530 nmol O2 / min / mg protein (rate with NADH as a substrate). It appears that in fresh A. maculatum mitochondria in the / stage the alternative pathway has a larger contribution to overall respiration rate than in S. pombe mitochondria. By cold storing the spadices the cytochrome pathway in A. maculatum mitochondria should become more active, which would increase its contribution to overall respiration rate, a situation more comparable to the yeast expression system.
  • 184 0 200 400 600 800 1000 1200 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt In both types of arum mitochondria (‘fresh’ and ‘cold stored’) both pathways were active. When incubated with OG and CCCP a limited respiration rate (cytochrome pathway activity) was measured. In the absence of OG and presence of CCCP the respiration rate was 3 to 4 times higher, demonstrating that the alternative pathway was active. Figure 5.24 shows the relationships between Qr/Qt and vO2 under uncoupled conditions in A. maculatum mitochondria respiring either on succinate or NADH. Data obtained from mitochondria isolated from freshly picked or cold stored spadices are combined. Although the data shows considerable variation it is clear that there are no substrate dependent differences in oxidising pathway kinetics. Equally also it is apparent that there are no differences between oxidising pathway kinetics of mitochondria isolated from fresh spadices or cold stored ones. Figure 5.24 Comparing uncoupled oxidising pathway kinetics in Arum maculatum mitochondria respiring on either succinate or NADH. Qr/Qt and vO2 were determined simultaneously. Succinate fresh Arum data () and cold stored Arum data (▲) were obtained from three and two mitochondrial isolations respectively, combining data points from six and five traces respectively (succinate 9 mM, ATP 0.2 mM). NADH fresh arum data () and cold stored arum data (∆) were obtained from four and two mitochondrial isolations respectively, combining data points from eight and six traces respectively. Mitochondrial protein used per experiment 0.1 – 0.3 mg. NADH and succinate data points were obtained using the malonate titration and the NADH regenerating method. NADH titration range (0-50 M) malonate titration range (0-4 mM)
  • 185 DOES THE ALTERNATIVE PATHWAY IN sp.011 AOX MITOCHONDRIA SERVE AS AN OVERFLOW? It has been a long held belief that the alternative pathway in AOX expressing mitochondria served as an overflow pathway under conditions where the cytochrome pathway is either saturated or inhibited [79]. It was originally found by Bahr and Bonner [252, 253] that upon addition of hydroxamic acids (inhibitors of AOX) switching of electron flow from the alternative pathway to the cytochrome pathway did not occur. Later studies showed that AOX only becomes engaged at relatively high Qr/Qt values [75, 78, 86] corroborating the overflow hypothesis. Results by Wilson [237] and Hoefnagel et al. [79] successfully challenged the overflow hypothesis by demonstrating that diversion of electron flow from the alternative pathway to the cytochrome pathway can occur. Also, the overflow hypothesis requires the cytochrome pathway to be saturated before electrons can be diverted to the alternative pathway, it was however demonstrated by van den Bergen et al. that the cytochrome pathway never attains a saturated rate, even at high levels of Q- reduction [76]. Assessment of the contribution of AOX to overall respiration rate by inhibition of AOX can lead to an underestimation, since electron flow can be diverted from the alternative pathway to the cytochrome pathway, possibly giving the impression that AOX was not engaged prior to addition of inhibitor. The alternative and cytochrome pathway share the same substrate (QH2), inhibition of one pathway will perturb the Qr/Qt ratio which will affect the activity of the uninhibited pathway [148]. To give an example, a naive approach to estimate the contribution of AOX to total respiration would be to measure total respiration, inhibit the alternative pathway (with octyl gallate or SHAM for instance), measure the residual respiration and then subtract these two rates from each other. Inhibition of AOX could lead to an increase in Qr/Qt, this will lead to an increase in cytochrome pathway activity. The rate thus measured does not reflect cytochrome pathway activity prior to addition of AOX inhibitor and subtracting this rate from total respiration rate will give an underestimation of AOX contribution to the total respiration rate.
  • 186 It is now firmly established that in plant mitochondria AOX does not function simply as an overflow pathway. The situation in plants however cannot simply be extrapolated to our yeast expression system. The alternative oxidase is a heterologous protein in the electron transfer chain of our expression system and it might be the case that in our system there is a preference for the cytochrome pathway over the alternative pathway. Bahr and Bonner [252] introduced an inhibitor titration technique which is used to provide an estimation of the contribution of AOX to respiration in the absence of inhibitors. Oxygen consumption is measured during an inhibitor titration of AOX activity (with octyl gallate for instance) in the presence and absence of a cytochrome pathway inhibitor. Plotting of these data sets against each other yields so called  plots [252], which gives a measure of AOX engagement during respiration in the absence of AOX inhibitor. If the relationship between the two sets is linear it can be concluded that the electron flux through the cytochrome pathway is independent from the flux through the alternative pathway. A ‘break’ in this relationship indicates that electron flow is diverted from the alternative pathway to the cytochrome pathway. Figure 5.25 shows a  plot where NADH dependent respiration was titrated with OG in the presence and absence of antimycin A. Both datasets were plotted against each other and it can be seen that there is a breakpoint in the relationship. This indicates that during the initial additions of OG electron flow was diverted to the cytochrome pathway and total respiration is seen to be resistant against OG inhibition. When the cytochrome pathway is saturated further addition of OG leads to a decrease in total respiration as no more electrons can be diverted from the alternative pathway to the cytochrome pathway. Figure 5.26 shows a similar experiment where sp.011 AOX mitochondria were titrated with antimycin A in the presence and absence of octyl gallate. This experiment indicates that upon inhibition of the cytochrome pathway electron flow can be diverted to the alternative pathway, i.e. the alternative pathway is not saturated. Similar results were obtained with succinate as a substrate (data not shown).
  • 187 Figure 5.25 Alternative pathway  plot. Two octyl gallate titration experiments were performed on sp.011 AOX mitochondria in the presence and absence of antimycin A. NADH (1.8 mM) antimycin A (2 M) octyl gallate titration range (0 – 1 M). Data from one mitochondrial isolation. Amount of protein used: 0.5 mg. Figure 5.26 cytochrome pathway  plot. Two antimycin A titration experiments were performed on sp.011 AOX mitochondria in the presence and absence of octyl gallate. NADH (1.8 mM) octyl gallate (2.3 M) antimycin A titration range (0 – 40 nM). Data obtained from one mitochondrial isolation. Amount of protein used: 0.5 mg. 0 50 100 150 200 0 20 40 60 80 100 120 OGtitration nmolO2 /min/mgprotein OG titration with AA incubated nmol O2 / min / mg protein 0 50 100 150 200 0 20 40 60 80 100 AAtitration AA titration with OG incubated nmol O2 / min / mg protein
  • 188 The results in figures 5.25 and 5.26 indicate that under ADP limited conditions neither pathway is saturated and that electron flow can be diverted from one pathway to the other. These results are in agreement with work done on plants naturally expressing AOX [79]. The main reason why the overflow hypothesis was not successfully challenged for almost two decades is due to the fact that only relatively recently it became clear that AOX is partially inactive in isolated plant mitochondria. Upon addition of pyruvate AOX is seen to become active at lower values of Qr/Qt [79]. In soybean cotyledon mitochondria intramitochondrial pyruvate is generated during succinate oxidation, but at low concentrations. Under these conditions inhibitor titrations may fail to demonstrate the ability of AOX to compete with the cytochrome pathway. In S. pombe mitochondria expressing the S. guttatum AOX it was found that AOX was constitutively active [70], a situation comparable to pyruvate activated AOX in plant mitochondria. The results in figures 5.25 and 5.26 agree well the work done by Hoefnagel et al. on fully activated AOX in soy bean cotyledon mitochondria [79]. In S. pombe mitochondria with either NADH or succinate as a substrate, under all energetic conditions, the respiratory rate at each value of Qr/Qt is higher in the presence than in the absence of AOX. Cytochrome pathway activity with NADH is higher than with succinate under state 3 and uncoupled conditions. Therefore with succinate as a substrate, under state 3 and uncoupled conditions, the cytochrome pathway clearly is not saturated. Since AOX is engaged under all energetic conditions, that means that in S. pombe mitochondria, the cytochrome pathway does not need to be saturated in order for the alternative pathway to become engaged.
  • 189 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Does the expression of AOX lead to a change in Q-pool behaviour? Classical pool behaviour [23, 24] was found in S. pombe mitochondria (see section 4.2.3.1 and [26]). In sp.011 AOX mitochondria an extra protein complex is introduced into the ETC. The IMM of mitochondria has a very high protein content25 when compared to other membranes. It was already found that insertion of AOX had no disruptive effects and the IMM conductivity was unchanged (cf. Figure 5.10). Although membrane permeability was not affected, it is possible that introducing an extra respiratory complex to a membrane which already is relatively protein packed may affect the fluidity with the membrane phase becoming more viscous. Perhaps the substrate dependent differences in sp.011 AOX oxidising pathway kinetics are due to a change in ETC organisation with a concomitant departure of Q-pool behaviour. In order to investigate this reducing pathway kinetics were determined with succinate as a substrate under various energetic conditions, see Figure 5.27. Figure 5.27 sp.011 AOX reducing pathway kinetics with succinate as a substrate. Reducing pathway kinetics were determined under state 2 () and uncoupled () conditions. State 2 conditions: Succinate (9 mM) was added to sp.011 AOX mitochondria incubated in the presence of ATP (0.2 mM) and glutamate (9 mM). Uncoupled conditions: like state 2 but with CCCP (2 M) incubated. Reducing pathway kinetics were obtained by titrating with antimycin A (0 – 40 nM). State 2 and uncoupled data were obtained from one mitochondrial isolation combining data points from 3 and 4 experimental traces respectively. Amount of mitochondrial protein used: 0.7 mg. 25 The IMM typically consists of 50% integral protein, 25% peripheral protein and 25% lipid [1].
  • 190 It can be seen that like in the wild type mitochondria (cf. Figure 4.13) there is a clear difference in reducing pathway kinetics depending on energy state reflecting SDH deactivation. Homogenous pool behaviour can be demonstrated by showing a sigmoidal relationship between respiration and increasing antimycin A concentration (which is reflected in increasing Qr/Qt), a so called resistance to inhibition (cf. Figure 1.9). It is difficult to distinguish between a linear and a sigmoidal relationship between respiration rate and Qr/Qt from Figure 5.27. Investigation of individual traces however clearly shows a sigmoidal relationship, see Figure 5.28, which indicates that S. pombe mitochondria expressing AOX display pool behaviour. Figure 5.28 Antimycin A titration of sp.011 AOX mitochondria , representative state 2 trace taken from Figure 5.26. It was concluded that functional expression of the alternative oxidase in S. pombe mitochondria does not affect Q-pool behaviour and the dependency of the SDH reducing pathway kinetics on energy status of the IMM is comparable to the wild type mitochondria. 50 100 150 200 0.7 0.75 0.8 0.85 0.9 0.95 1 vO2 /nmolO2 /min/mgprotein Qr/Qt
  • 191 5.3 DISCUSSION 5.3.1 Differences between the various S. pombe mitochondria used—In this study the effects of a functionally expressed plant alternative oxidase in S. pombe mitochondria on respiratory kinetics were investigated. From the mitochondria used three types were isolated from transformed S. pombe cells. sp.011 AOX, mitochondria expressing AOX. sp.011 AOX+T, mitochondria isolated from S. pombe cells transformed with a plasmid containing the gene for AOX but grown in the presence of thiamine, which represses AOX expression. sp.011 pREP, mitochondria isolated from S. pombe cells transformed with a plasmid lacking the gene encoding AOX. It was a priori expected that the respiratory characteristics of sp.011 wt, sp.011 AOX+T and sp.011 pREP mitochondria would be comparable given that these mitochondria all lack the alternative pathway. It was found however that based on respiratory rates and RCR values (see Tables 5.2 and 5.3) that sp.011 wt and sp.011 pREP mitochondria are similar, with comparable respiratory rates and RCR values whereas the sp.011 AOX+T mitochondria displayed lower respiratory rates and RCR values. When incubated with OG alternative pathway activity in sp.011 AOX mitochondria is inhibited and the respiratory characteristics should be similar to S. pombe mitochondria lacking the alternative pathway. It was found that sp.011 AOX (incubated with OG) respiratory rates and RCR values were lower than those of sp.011 wt and sp.011 pREP mitochondria, but were comparable with values found in sp.011 AOX+T mitochondria. Based on this the S. pombe mitochondria used in this study were treated as two groups: sp.011 wt and sp.011 pREP mitochondria (which display relatively high respiratory rates and RCR values) and sp.011 AOX and sp.011 AOX+T mitochondria (displaying somewhat lower respiratory rates and RCR values). It can be seen in Table 5.1 that each type of mitochondria is isolated from yeast cultures with slightly different growth conditions. sp.011 wt cells are grown in the presence of leucine. This however cannot cause any specific differences in respiratory kinetics since the respiratory kinetics of sp.011 wt and sp.011 pREP (no leucine during growth) are comparable. The act of transformation itself cannot be the cause for a difference in kinetics since the respiratory kinetics of sp.011 wt (not transformed) mitochondria are comparable to those of sp.011 pREP mitochondria.
  • 192 The presence of thiamine cannot be the cause of differences in respiratory kinetics since the respiratory kinetics of sp.011 AOX+T mitochondria are comparable of those of sp.011 AOX (incubated with OG) mitochondria. The only thing that sp.011 AOX and sp.011 AOX+T mitochondria have in common in which they are different from sp.011 wt and sp.011 pREP is the fact that both types of mitochondria are isolated from S. pombe cells transformed with the plasmid containing the gene encoding for AOX. It is unclear in what way the presence of a plasmid containing the gene for S. guttatum AOX can affect the respiratory kinetics of S. pombe mitochondria. Although the differences between the two groups of S. pombe mitochondria cannot be satisfactorily explained it is clear that the sp.011 AOX+T mitochondria are a proper control group for comparison with sp.011 AOX mitochondria. When sp.011 AOX mitochondria are incubated with OG, inhibiting the alternative pathway, its respiratory kinetics are similar to those of the sp.011 AOX+T mitochondria. 5.3.2 Does transformation of S. pombe mitochondria lead to changes in respiratory kinetics?—It could be argued that transformation of yeast cells may cause a change in mitochondrial respiratory kinetics. Figures 5.2-5.6 indicate that the respiratory kinetics of transformed S. pombe mitochondria (sp.011 pREP) are not significantly different from those of the wild type mitochondria (sp.011 wt). Under ADP limited conditions the vO2 vs. Qr/Qt relationship is linear (Figure 5.2) and under state 3 and uncoupled conditions the vO2 vs. Qr/Qt relationships display a biphasic pattern, in agreement with what was seen in the wild type mitochondria. Also under ADP limited conditions the vO2 vs.  relationships of sp.011 pREP and sp.011 wt mitochondria overlap (Figure 5.3), which suggests that the IMM conductivity is not affected by the transformation. 5.3.3 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics with NADH as a substrate—In a previous study done in this laboratory oxidising pathway kinetics were determined in sp.011 AOX and sp.011 AOX+T mitochondria by titrating succinate dependent respiration with malonate [93]. Succinate was chosen as substrate since NADH at saturating concentrations interferes strongly with the Q-electrode. The amount of data obtained in that study was somewhat limited and the mitochondria used were of relatively
  • 193 poor quality. Hoefnagel et al. [146] demonstrated that NADH can be used in combination with the Q-electrode to determine oxidising pathway kinetics by using an NADH regenerating system that requires addition of sub-saturating amounts of NADH; which only has a limited effect on the Q-electrode signal, that can be corrected for, see section 2.4.4.4. This method allows for probing of the relationship between vO2 and Qr/Qt covering the whole range of Qr/Qt (which is more difficult when titrating succinate dependent respiration with malonate). It was decided to determine the oxidising pathway kinetics in sp.011 AOX and sp.011 AOX+T mitochondria under various energetic conditions using the NADH regenerating system, see figures 5.7-5.11. It was found that under all energetic conditions, in the presence of AOX, the vO2 rates, at each value of Qr/Qt are higher when compared to sp.011 AOX+T mitochondria with NADH as a substrate. These results were different from Affourtit’s study [93] who reported that under state 3 and state 4 conditions, in the presence of AOX, vO2 rates were lowered, whereas under uncoupled conditions expression of AOX did not seem to affect oxidising pathway kinetics (with succinate as a substrate). Under ADP limited conditions the dependency of respiration on Qr/Qt was proportional for both sp.011 AOX and sp.011 AOX+T mitochondria, similar to what is seen in sp.011 pREP and sp.011 wt mitochondria (see Figure 5.2). Under state 3 and uncoupled conditions the sp.011 AOX+T mitochondria display a clear biphasic relationship between vO2 and Qr/Qt (see figures 5.8 and 5.9), similar to what is seen in sp.011 pREP and sp.011 wt mitochondria (see figures 5.4 and 5.6). Interestingly the relationship between Qr/Qt and vO2 under state 3 and uncoupled conditions in sp.011 AOX mitochondria is linear (see figures 5.8 and 5.9). When scrutinizing individual traces, it is clear that the biphasic pattern is still present under uncoupled conditions although the biphasic point is shifted to the left. Apparently the introduction of an extra oxidising pathway leads to masking of the biphasic pattern under state 3 and uncoupled conditions. This result corroborates the hypothesis that the biphasic pattern seen in S. pombe mitochondria is a characteristic of the cytochrome pathway. In chapter 4 (see section 4.2.5.2) it was already indicated that addition of an extra terminal oxidase changed the oxidising pathway kinetics. If the biphasic pattern was a characteristic of the dehydrogenase kinetics then the introduction of AOX should not change the shape of the Qr/Qt vs. vO2 relationship.
  • 194 Incorporation of the alternative oxidase into the IMM of S. pombe mitochondria might have a disruptive effect, uncoupling the mitochondria. The increase in vO2 observed in sp.011 AOX mitochondria could be due to uncoupling. However, it was shown that under ADP limited conditions the relationship between  vs. vO2 in sp.011 AOX and sp.011 AOX+T mitochondria is the same (see Figure 5.10) which suggests that the conductivity of the IMM in both types of mitochondria is the same and that incorporation of an extra protein complex into the IMM did not lead to a change in proton permeability. Under state 3 conditions, in both sp.011 AOX and sp.011 AOX+T the  vs. vO2 relationship is biphasic (Figure 5.11), similar to what is seen in sp.011 pREP and sp.011 wt (see Figure 5.5). Since AOX is non-protonmotive, this indicates that the biphasic pattern is a characteristic of the cytochrome pathway. 5.3.4 Does AOX activity affect  in S. pombe mitochondria?—Due to the non-ohmic relationship between  vs. vO2 under ADP limited conditions activity of AOX will not have an effect on , see section 5.2.2.2. Under state 3 conditions however the relationship between  vs. vO2 in the region following the inflection point of the biphasic pattern is proportional, i.e. a change in cytochrome pathway activity leads directly to a change in . If both pathways were to compete with each other for reducing equivalents, then AOX activity could lead to a decreased electron flow though the cytochrome pathway. If under state 3 conditions in sp.011 AOX mitochondria the rate of electron transfer through the cytochrome pathway would be lower than in sp.011 AOX+T mitochondria, then because of the proportional relationship between vO2 and  the maximum attainable  in sp.011 AOX mitochondria should be lower than in sp.011 AOX+T mitochondria. Unfortunately, due to experimental variation this cannot be demonstrated with the available NADH titration data, see Figure 5.11. To investigate the possible effect of AOX expression on  under state 3 conditions a different approach was used. To sp.011 AOX mitochondria respiring on NADH a saturating amount of ADP was added, inducing a state 3, during this state a saturating amount of OG was added to inhibit AOX. If AOX had been competing with the cytochrome pathway then upon inhibition, electron flow would have been diverted from the alternative pathway to the cytochrome pathway. Because of the proportional relationship between  and vO2 under state 3 conditions and due to an increase of
  • 195 electron transfer rate through the cytochrome pathway  should increase. However, addition of OG during state 3 had no effect on  whatsoever, see Figure 5.12. This was seen with either NADH or succinate as a substrate. This indicates that under state 3 conditions the alternative and cytochrome pathway do not compete for reducing equivalents and there is no diversion of electron flow from the alternative pathway to the cytochrome pathway upon inhibition of AOX. It suggests that the activities of both pathways are additive. 5.3.5 Comparing sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics with succinate as a substrate—The oxidising pathway kinetics of sp.011 AOX mitochondria respiring on NADH (see section 5.3.3) were different from the kinetics obtained by Affourtit in a previous study [93]. The oxidising pathway kinetics in that study however were determined in sp.011 AOX and sp.011 AOX+T mitochondria respiring on succinate. The differences between the kinetics obtained in this study and Affourtit’s study could be substrate dependent. Therefore the oxidising pathway kinetics under various energetic conditions in sp.011 AOX and sp.011 AOX+T mitochondria were determined with succinate as a substrate. It was found, in the presence of AOX, under all energetic conditions, that at each value of Qr/Qt the vO2 rates were higher when compared to sp.011 AOX+T mitochondria, see figures 5.13-5.15. Similar to what was found with NADH in this study and opposite of what was found by Affourtit. Under ADP limited conditions, the Qr/Qt vs. vO2 relationship was linear, similar to what was seen in the wild type (see Figure 4.9). Under state 3 and uncoupled conditions in sp.011 AOX+T mitochondria the relationship between Qr/Qt vs. vO2 displayed the familiar biphasic pattern (see figures 5.14 and 5.15) as seen in the wild type (see Figure 4.11). Under state 3 and uncoupled conditions in sp.011 AOX mitochondria the relationship between Qr/Qt vs. vO2 is linear (see figures 5.14 and 5.15) similar to what was seen in sp.011 AOX mitochondria respiring on NADH (see figures 5.8 and 5.9). From these results it was concluded that in the presence of AOX, under all energetic conditions, at each value of Qr/Qt the vO2 rates are higher when compared to sp.011 AOX+T mitochondria. Furthermore the differences between kinetics obtained in this study and those of Affourtit’s study cannot be explained in terms of substrate dependent differences.
  • 196 5.3.6 Why are the oxidising pathway kinetics obtained in this study different from Affourtit’s study?—The RCR value of sp.011 AOX+T mitochondria respiring on NADH (uncoupled / state 2) in this study is about 5 (Table 5.3). The equivalent RCR value in Affourtit’s study is estimated by this author to be around 2 (cf. Figure 2A in [93]). Also the reaction medium used by Affourtit contains 0.3 M mannitol, whereas in yeast studies normally a concentration around 0.65 M is used. To investigate the effect of the osmotic strength of the reaction medium, state 3 oxidising pathway kinetics were determined for sp.011 AOX mitochondria respiring on NADH in reaction medium containing 0.3 M mannitol or reaction medium containing 0.65 M mannitol. Figure 5.16 shows that sp.011 AOX mitochondria in 0.3 M mannitol display lower vO2 rates than mitochondria respiring in 0.65 M mannitol. Finally, in all experiments done by Affourtit ethanol was present in the reaction medium (addition of Q1 dissolved in ethanol) which seriously affects oxidising pathway kinetics by generating internal NADH and activating the internal NADH dehydrogenase. Based on these differences it can be concluded that the kinetics reported in [93] are completely incomparable to the kinetics determined in this study. 5.3.7 Are there any substrate dependent differences in sp.011 AOX oxidising pathway kinetics?—It was found that under all energetic conditions with NADH as a substrate higher maximum respiration rates were attained than with succinate as a substrate. This could be due to dehydrogenase kinetics, with the external NADH dehydrogenase being more active than SDH and reducing the Q-pool to a higher degree. However it can be seen that under ADP limited conditions, from around 60% Qr/Qt at equivalent values of Qr/Qt with NADH as a substrate the vO2 rates are higher than with succinate (see Figure 5.17). Under state 3 and uncoupled conditions the same thing happens from around 40% Qr/Qt (see figures 5.18 and 5.19). The Qr/Qt values at which point the oxidising pathway kinetics with either NADH or succinate no longer overlap are estimated on the basis of the raw data points. A more quantitative approach would be to compare mathematical fits through the data; results from modelling the data, employing Q-pool kinetics (see section 2.4.5) will be presented in chapter 6. At this point however it can be concluded that sp.011 AOX mitochondria display substrate dependent differences in oxidising pathway kinetics.
  • 197 5.3.8 Are the substrate dependent differences a characteristic of the cytochrome pathway?—It was shown in chapter 4 that S. pombe mitochondria do not display any substrate dependent differences in cytochrome pathway kinetics (see figures 4.9-4.12). It can be argued however that transformation of S. pombe cells may affect mitochondrial respiratory kinetics thus introducing substrate dependent differences in cytochrome pathway kinetics which are not present in the wild type mitochondria. In order to investigate this the cytochrome pathway kinetics of sp.011 AOX+T mitochondria respiring on either succinate or NADH were plotted together, see figures 5.20-5.22. From these plots it was concluded that there are no substrate dependent differences in the cytochrome pathway kinetics of the transformed S. pombe mitochondria. 5.3.9 Are the substrate dependent differences a characteristic of the alternative pathway?—Substrate dependent differences in alternative pathway activity are well known [74, 254]. It is often found that alternative pathway respiratory rates with succinate are higher than with NADH [72] , this being due to pyruvate formation during succinate dependent respiration, pyruvate being an activator of plant AOX [79, 255]. When mitochondria, expressing AOX, respiring on NADH are incubated with pyruvate, the respiration rates attained are equivalent to what is seen with succinate. In our system however AOX is constitutively active [70] and a difference in AOX activity dependent on substrate is therefore not to be expected. This was investigated by determining alternative pathway activity in sp.011 AOX mitochondria incubated in the presence of antimycin A respiring on either NADH or succinate, see Figure 5.23. The reduction state of the matrix pyridine nucleotide pool has been reported to activate AOX by reduction of the regulatory sulfhydryl-disulfide system [256]. Inhibition of the cytochrome pathway can lead to a perturbation of the reduction state of the pyridine nucleotide pool, increases in the NAD(P)H / NAD(P)+ ratio can lead to rapid activation of AOX [71]. Inhibition of the cytochrome pathway therefore could lead to activation of AOX. However, as reported previously, S. guttatum AOX expressed in S. pombe mitochondria is constitutively active [70]. From the Western blots in Figure 5.1 it is also apparent that AOX is in its reduced state in S. pombe mitochondria. An increase in the reduction level of the matrix pyridine
  • 198 nucleotide pool could therefore not lead to change of AOX activation status. It was concluded that there are no substrate dependent difference in alternative pathway kinetics. 5.3.10 Are the substrate dependent differences a characteristic of the expression system used?—In order to investigate whether or not the substrate dependent differences in sp.011 AOX oxidising pathway kinetics are a characteristic of our expression system a comparison was made with oxidising pathway kinetics in a plant species naturally expressing AOX, A. maculatum. It was demonstrated that mitochondria isolated from A. maculatum do not display substrate dependent differences in oxidising pathway kinetics, see Figure 5.24. 5.3.11 Can the alternative pathway compete with the cytochrome pathway in S. pombe mitochondria expressing AOX?—Inhibitor titrations according to the method of Bahr and Bonner [252] were employed to generate -plots, see figures 5.25 and 5.26. From these results it is clear that electron flow can be diverted from the cytochrome pathway to the alternative pathway and vice versa. This indicates that neither pathway is saturated and that the results in this study agree well with what is found in plant mitochondria with fully activated AOX. 5.3.12 Does expression of the alternative oxidase lead to a change in Q-pool behaviour?— Incorporation of an extra protein into the relatively packed IMM could lead to a change in Q-pool behaviour. Figures 5.27 and 5.28 show that sp.011 AOX mitochondria display Q- pool behaviour. Equally also, SDH activity in sp.011 AOX mitochondria is dependent upon energy status of the IMM comparable to sp.011 wt mitochondria (cf. Figure 4.13).
  • 199 5.4 CONCLUSION The sp.011 AOX and sp.011 AOX+T oxidising pathway kinetics found in this study are different from what has been reported previously [93, 153]. The expression of an extra terminal oxidase results in overall higher respiratory rates, which is what one would expect [29]. Given the differences in experimental conditions it can be concluded that the results from this study are incomparable to the results obtained by Affourtit. The substrate dependent differences in sp.011 AOX mitochondria are not caused by cytochrome pathway kinetics, nor by alternative pathway kinetics. The substrate dependent differences are only seen when both pathways are active simultaneously. In order to be able to quantitatively determine at which value or Qr/Qt the substrate dependent oxidising pathway kinetics start to diverge it is necessary to apply mathematical curve fitting to the data. Results of mathematical modelling of the data will be presented in the next chapter. In order to gain more insight into the nature of the substrate dependent differences experiments will be presented where both NADH and succinate are used with the same mitochondria.
  • 200 Chapter 6 Modelling of oxidising pathway kinetics in Schizosaccharomyces pombe mitochondria expressing the alternative oxidase 6.1 INTRODUCTION Substrate dependent differences in oxidising pathway kinetics in sp.011 AOX mitochondria were described in the previous chapter. It was determined that substrate dependent differences were not a characteristic of either the cytochrome or the alternative pathway. Substrate dependent differences in kinetics were only seen when both pathways were active at the same time. The biphasic pattern in state 3 and uncoupling oxidising pathway kinetics (Qr/Qt vs. vO2) is masked with either NADH or succinate as substrate in sp.011 AOX mitochondria. A biphasic pattern can still clearly be seen under state 3 conditions in the  vs. vO2 relationship. In order to more accurately determine at which value of Qr/Qt the oxidising pathway kinetics start to diverge the raw data was fitted using reversible Michaelis-Menten kinetics according to [76] (see section 2.4.5 for a detailed description). With NADH as a substrate the combined activities of cytochrome pathway and AOX appear to be additive, whereas with succinate the total oxidizing pathway activity appears to be less than the sum of cytochrome and AOX pathway activities combined, see Table 5.2. These findings are suggestive of a possible limiting effect of succinate on total oxidizing pathway activity. To investigate whether or not succinate indeed had such an inhibitory effect experiments were done in which oxidising pathway kinetics were determined using both NADH and succinate to reduce the Q-pool.
  • 201 6.1 RESULTS 6.1.1 Modelling of sp.011 AOX oxidising pathway kinetics—In section 5.2.4.1 data was shown which suggest that the oxidising pathway kinetics in sp.011 AOX mitochondria respiring on either succinate or NADH start to diverge at low values of Qr/Qt, see figures 5.17-5.19. It was estimated for ADP limited conditions that the NADH and succinate oxidising pathway kinetics start to diverge at around 60% Qr/Qt, whereas under state 3 and uncoupled conditions this occurs at around 40% Qr/Qt. In order to arrive at a more accurate determination of the point where NADH and succinate kinetics start to diverge, the data was modelled using a reversible Michaelis-Menten equation according to [76]. Figure 6.1 shows the result of curve fitting the sp.011 AOX oxidising pathway data under ADP limited conditions with either NADH or succinate as a substrate. Figure 6.1 sp.011 AOX oxidising pathway kinetics under ADP limited conditions with either NADH (black circles) or succinate (red circles) as substrate. Data taken from Figure 5.17. Curve fitting according to [76]. As opposed to a Qr/Qt value of 60%, as was estimated from Figure 5.17 (see section 5.2.4.1), after curve fitting, it can be seen that NADH and succinate kinetics start to diverge at around 30% Qr/Qt which was not readily apparent from the raw data. 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 202 A more pronounced difference between succinate and NADH oxidising pathway kinetics under state 3 conditions in sp.011 AOX mitochondria was apparent from the data (cf. Figure 5.18). This is also reflected in the fitted curves through these data, see Figure 6.2. Figure 6.2 sp.011 AOX oxidising pathway kinetics under state 3 conditions with either NADH (black circles) or succinate (red circles) as substrate. Data taken from Figure 5.18. Curve fitting according to [76]. Curve fitting reveals that succinate and NADH oxidising pathway kinetics start to diverge at a much lower Q-redox poise than the previously estimated 40%. Based on the kinetic fits it can be concluded that under state 3 conditions, at each value of Qr/Qt, with NADH as a substrate the oxygen consumption rates are higher than with succinate as a substrate. Under uncoupled conditions it appears that at low values of Qr/Qt, the oxygen consumption rates with succinate as a substrate are slightly higher than with NADH, but at approximately 20% Qr/Qt the kinetics change considerably with oxygen consumption rates being higher at each value of Qr/Qt with NADH as a substrate, see Figure 6.3. 0 50 100 150 200 250 300 350 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 203 Figure 6.3 sp.011 AOX oxidising pathway kinetics under uncoupled conditions with either NADH (black circles) or succinate (red circles) as substrate. Data taken from Figure 5.19. Curve fitting according to [76]. The data in figures 5.17-5.19 suggested the presence of substrate dependent differences in oxidising pathway kinetics in sp.011 AOX mitochondria under various energetic conditions. Modelling the data using reversible Michaelis-Menten kinetics (a method which has been used successfully with isolated mitochondria in the past [21, 70, 76, 153, 216]) corroborates this. When the kinetic fits from figures 6.1-6.3 are combined in one plot, a dependency on energy status is seen which was not readily apparent from the individual figures, see Figure 6.4. The differences between succinate and NADH kinetics become more pronounced when going from coupled to uncoupled conditions. Differences in oxidising pathway kinetics should be independent from reducing pathway kinetics (see section 1.5). Oxidising pathway kinetics reflect the relationship between steady state respiration rate and steady state Qr/Qt. Differences in activity between dehydrogenases are cancelled out when Q-pool kinetics [216] are assumed. In other words, the means with which the Q-pool is reduced should not affect the oxidising pathway relationships. Still, it was found that energy status affects SDH activity (cf. figures 3.19, 4.13 and 5.27) but not that of the external NADH dehydrogenase (cf. Figure 4.15). 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 204 Figure 6.4 Combined kinetic fits of sp.011 AOX oxidising pathway kinetics with NADH or succinate as substrate. Each energetic state is indicated by colour (state 2 red, state 3 blue and uncoupled black). Of each colour, the lower curve represents the succinate kinetics. Data taken from figures 6.1-6.3. N = NADH, S = succinate. Both SDH activity and the extent of substrate dependent differences in oxidising pathway kinetics are dependent on energy status. Possibly these two effects are related, which could challenge the model of Q-pool kinetics as it suggests interaction between reducing and oxidising pathway activities in a non Q-dependent manner. It can be concluded that, overall, oxidising pathway activity with succinate appears to be lower than with NADH as a substrate. Possibly the total respiratory rate is lower than the sum of the individual pathway activities with succinate as a substrate. 6.1.2 Are the oxidising pathway activities additive?—It was established that sp.011 AOX mitochondria show distinct differences between oxidising pathway kinetics dependent on substrate. Under all energetic conditions, over nearly the whole range of Qr/Qt, the oxygen consumption rates with succinate are lower than with NADH as a substrate. Substrate dependent differences are not seen in the cytochrome pathway kinetics (see section 5.3.7) nor in the alternative pathway kinetics (see section 5.3.8). 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt state 2 state 3 uncoupled N N N S S S
  • 205 Substrate dependent differences are only apparent when the two pathways are active simultaneously. Possibly with succinate as a substrate either one or both oxidising pathways are engaged to a lower extent than with NADH as a substrate. Table 6.1 shows the sp.011 AOX state 2 respiratory rates with either succinate or NADH as a substrate, data taken from Table 5.2. sp.011 AOX Respiratory Rate (nmol O2 min-1 mg-1 protein) substrate(s) state 2 NADH 199 (27) NADH + OG 92 (10) NADH + AA 81 (17) succinate 142 (14) succinate + OG 83 (12) succinate + AA 84 (13) Table 6.1 sp.011 AOX state 2 rates under various energetic conditions. Data taken from Table 5.2. Assessing the engagement of oxidising pathways based on respiratory rates only, cannot be used to quantify the contribution of each pathway to total respiration [148]. Respiration rate data however can be used to give qualitative answers. The respiration rates given in Table 6.1 were obtained through addition of saturating amounts of substrate (NADH 1.8 mM or succinate 9 mM). Therefore the level of Q-reduction is only limited by the activity of the dehydrogenases. The steady state respiration under state 2 conditions with NADH is 199 nmol O2 / min /mg protein whereas with succinate it is 142 nmol O2 / min / mg protein. The difference in rates could be due to a difference in reducing power of the dehydrogenases, where the Q-pool is more reduced with NADH. It can be seen in Figure 6.1 that the Q-pool with NADH is indeed more reduced (~10%). However, based on the curve fits through the data, an increase in Qr/Qt to the same level as attained with NADH would increase the
  • 206 respiration rate only marginally and cannot account for the discrepancy between oxygen consumption rates in sp.011 AOX mitochondria respiring on either succinate or NADH (saturating concentrations). The data in Table 6.1 were obtained from respiratory measurements using the oxygen electrode only and therefore represent a dataset different from the titration data presented in Figure 5.17. Respiratory rates obtained in both sets of experiments are comparable which corroborates the hypothesis that there are substrate dependent differences in sp.011 AOX oxidising pathway kinetics. The activities of both pathways separately have also been determined, by incubating with either octyl gallate or antimycin A. The cytochrome pathway respiratory rates for NADH and succinate are 92 and 83 nmol O2 / min / mg protein respectively. The alternative pathway respiratory rates for NADH and succinate are 81 and 84 nmol O2 / min / mg protein respectively26 . It appears that under ADP limited conditions, with NADH as a substrate the activities of the cytochrome pathway and the alternative pathway are additive, whereas with succinate as a substrate total respiration appears to be less than the sum of the individual pathway activities. Under ADP limited conditions when only the cytochrome pathway is active the Q-pool is reduced to the same extent with either succinate or NADH as a substrate (cf. Figures 4.9 and 5.20) and respiratory rates are comparable. Figure 5.23 suggests that with NADH as a substrate the Q-pool is more reduced than with succinate as a substrate in sp.011 AOX mitochondria incubated in the presence of antimycin A (only the alternative pathway active). With succinate as a substrate the Q-pool was never reduced further than 80%. With NADH as a substrate in most cases the Q-pool was not further reduced than 80%, except during two experimental days (out of five) where the Q-pool was reduced somewhat more. It is tempting to conclude that there is a substrate dependent difference in alternative pathway kinetics as it would offer a simple explanation for the substrate dependent differences observed in total oxidising pathway kinetics, without having to challenge Q-pool kinetics. However, the data do not allow for such a generalisation since in the majority of traces, no substrate dependent differences in alternative pathway kinetics were seen. If with NADH the Q-pool would be more reduced than with succinate this would still only lead to an increase in AOX dependent respiration 26 Energy status has no effect on AOX activity and the respiratory rates are the same under all energetic conditions.
  • 207 of approximately 20 nmol O2 / min / mg protein (cf. Figure 5.23), which cannot account for any of the discrepancies in oxygen consumption rates at specific Qr/Qt values with either succinate or NADH. Any differences between NADH and succinate dependent alternative pathway respiration cannot be due to limitation of SDH since under ADP limited conditions both substrates can reduce the Q-pool to the same extent with only the cytochrome pathway active. Under ADP limited conditions with both pathways active, it appears that with NADH both pathways are fully active, whereas with succinate, even though the Q-pool is reduced to almost the same extent, a much lower respiratory rate is reached, which suggests that activity of either one or both pathways is/are limited in the presence of succinate. To investigate whether or not the presence of succinate has a limiting effect on oxidising pathway kinetics in sp.011 AOX mitochondria experiments were done where the Q-pool was reduced with a mixture of substrates. 6.1.3 sp.011 AOX mixed titration studies—To establish a full reduction of the Q-pool and maximum respiratory activity normally a combination of substrates are added to mitochondria [74, 243, 257]. The results discussed in section 6.1.2 suggest that the presence of succinate may have a limiting effect on sp.011 AOX respiratory kinetics when both pathways are active. If this is the case then such an effect should be apparent when oxidising pathway kinetics are determined using mixed substrate titrations of NADH and succinate. The Q-pool can be partially reduced by titration with sub-saturating amounts of NADH, in the presence of the NADH regenerating system, subsequently it can be further reduced by addition of succinate and either ATP, glutamate or both (to fully activate SDH). If succinate does not have any limiting effects it can be expected that the oxidising pathway kinetics determined by partially reducing the Q-pool with NADH and by subsequently reducing it further with succinate are comparable to oxidising pathway kinetics with just NADH as a substrate. If succinate indeed has a limiting effect then the oxidising pathway kinetics should be different from those obtained with NADH, in fact, at comparable levels of Qr/Qt lower oxygen consumption rates should be obtained. Apart from investigating a possible limiting effect of succinate on oxidising pathway activities, the mixed substrate titrations serve as a control experiment to confirm
  • 208 that there are indeed substrate dependent differences in sp.011 AOX oxidising pathway kinetics. The data points in figures 6.1-6.3 suggest that succinate oxidising pathway kinetics are different from NADH kinetics. Modelling of the data corroborates this. However as can be seen in Figure 6.3, under uncoupled conditions SDH activity is limiting as there are no succinate data points at Qr/Qt values of 50% and higher. Combining substrates allows for reducing the Q-pool to higher Qr/Qt values and investigation of succinate dependent kinetics at Q-reduction levels higher than 50%. Substrate dependent differences between sp.011 AOX mitochondria are most pronounced under uncoupled conditions (cf. Figure 6.4). Incubated in the presence of CCCP (2 µM) sp.011 AOX oxidising pathway kinetics were determined using a mixture of substrates. Figure 6.5 shows a representative trace of a 'mixed titrations' experiment. In the presence of the NADH regenerating system sp.011 AOX mitochondria are titrated with sub-saturating amounts of NADH in order to partially reduce the Q-pool, subsequently succinate is added followed by ATP and/or glutamate. It can be seen that upon addition of G-6-P both Qr/Qt and vO2 increase (endogenous NAD+ is converted to NADH). Subsequent addition of NADH (~2.5 M) leads to a proportional increase in activity. Further addition of NADH (~5 M) leads to a non-proportional increase in activity associated with the biphasic point (the biphasic pattern is masked but still present in sp.011 AOX mitochondria, cf. Figure 5.9). The inset shows what the oxidising pathway kinetics would look like if the NADH titration was continued (NADH titration done on the same day with the same mitochondrial preparation), after the biphasic point, an increase of ~40% Q-reduction yields a concomitant increase in vO2 of about 400 nmol O2 / min / mg protein with NADH. However, when succinate (9 mM) and ATP (0.2 mM) are added to further reduce the Q-pool (after partial reduction with NADH) the increase in vO2 is only about 40 nmol O2 / min / mg protein, whereas Qr/Qt is increased by about 30%. Activity is effectively limited by the addition of succinate and further activation of SDH. Figure 6.6 shows a similar titration where the Q-pool was further reduced (~50%) initially with sub-saturating amounts of NADH before succinate was added. Beyond the biphasic point the relationship between Qr/Qt and vO2 becomes very steep, addition of a third aliquot of NADH results in an increase in Qr/Qt of ~15% with a concomitant increase in vO2 of about 140 nmol O2 / min / mg protein. Subsequent addition of succinate (9 mM)
  • 209 leads to a ~25% increase in Qr/Qt whereas vO2 hardly changes, which indicates that with succinate as a substrate the combined oxidising pathway activities become limited. Figure 6.5 A representative trace of a mixed substrate titration under uncoupled conditions in sp.011 AOX mitochondria. Glucose-6-phosphate dehydrogenase is incubated. Titration is started with addition of glucose- 6-phosphate (G6P) followed by two additions of subsaturating amounts of NADH (~7 M) leading to a partially reduced Q-pool (40%). Subsequently succinate (9 mM) and ATP (0.2 mM) are added which lead to a further reduction of the Q-pool. The inset shows a representative trace of an NADH titration (0 – 75 M) under uncoupled conditions in sp.011 AOX mitochondria. Mitochondrial protein used per experiment is 0.5- 0.7 mg. 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt G6P NADHNADH succinate ATP 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 210 Figure 6.6 A representative trace of a mixed substrate titration under uncoupled conditions in sp.011 AOX mitochondria. Glucose-6-phosphate dehydrogenase is incubated. Titration is started with addition of glucose- 6-phosphate (G6P) followed by three additions of subsaturating amounts of NADH (~27 M) leading to a partially reduced Q-pool (~50%). Subsequently succinate (9 mM) is added which leads to a further reduction of the Q-pool (~75%). Mitochondrial protein used 0.5 mg. Several things can be learned from figures 6.5 and 6.6. If there were no substrate dependent differences in sp.011 AOX respiratory kinetics, then it should not matter in which way the Q-pool is reduced. Clearly that is not the case, the kinetics displayed in the inset of Figure 6.5 with just NADH as a substrate look very different from the kinetics obtained when both succinate and NADH are present. This experiment corroborates the hypothesis that oxidising pathway kinetics in sp.011 AOX mitochondria are substrate dependent. Addition of succinate (and subsequent addition of activators of SDH) limit NADH dependent respiration. Upon addition of succinate (in the presence of a sub-saturating concentration of NADH) the Q-pool is significantly reduced , the concomitant increase in vO2 however is negligible when compared to NADH kinetics. 0 50 100 150 200 250 300 350 400 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt G6P NADHNADH succinateNADH
  • 211 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 NADH mixed titrations succinate vO2 nmolO2 /min/mgprotein Qr/Qt Figure 6.7 shows combined uncoupled sp.011 AOX oxidising pathway kinetics with NADH, succinate and mixed substrates. From the mixed substrate data, the initial NADH points are omitted, i.e. only the succinate and subsequent ATP and/or glutamate data points obtained (by addition of these chemicals after the Q-pool was partially reduced with sub- saturating amounts of NADH) are shown. The mixed substrate kinetics look different from the NADH kinetics. Figure 6.8 shows the omitted NADH data from the mixed titrations plotted in combination with uncoupled NADH oxidising pathway data. These data sets overlap and confirm that the sp.011 AOX mitochondria used for the mixed titration studies displayed regular NADH oxidising pathway kinetics. Figure 6.7 Combined oxidising pathway kinetics in sp.011 AOX mitochondria respiring either on NADH, succinate or both. NADH ( ) data points are taken from Figure 5.9. Succinate () data points are taken from Figure 5.15. The mixed titration () data were obtained from four mitochondrial isolations, combining data points from eight traces. The G6P and NADH data points from the mixed titration data are omitted in the figure. Mitochondrial protein used per experiment is 0.5-0.7 mg.
  • 212 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 6.8 Uncoupled sp.011 AOX NADH oxidising pathway data points () combined with the omitted NADH () data points from the mixed substrate titrations. Mitochondrial protein used per experiment is 0.5- 0.7 mg It is clear that the mixed titration points look different from both the succinate and the NADH data. To investigate whether the mixed substrate kinetics are similar to either NADH or succinate oxidising pathway kinetics the data were modelled according to [76]. 6.1.4 Applying Q-pool kinetics to fit sp.011 AOX mixed substrate oxidising pathway data— Figure 6.9 shows kinetic fits of the uncoupled sp.011 AOX NADH and mixed titration data. It can be seen that the oxidising pathway kinetics start to diverge at around 40% Qr/Qt. Also the shape of both types of kinetics is different, whereas the NADH kinetics are slightly concave at high Qr/Qt, the mixed substrate kinetic are clearly convex. At low values of Qr/Qt, respiration with succinate appears to be higher than with NADH. These characteristics are reminiscent of the uncoupled succinate kinetics (cf. Figure 6.3).
  • 213 0 100 200 300 400 500 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt Figure 6.9 Combined uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria respiring either on NADH or mixed substrates. NADH () data points are taken from Figure 5.9. The mixed titration () data was taken from Figure 6.6. Using Q-pool kinetics the data were fitted. It is clear that the modelled kinetic curves for both sets of data are different from one another. Curve fitting according to [76]. Figure 6.10 shows kinetic fits of the uncoupled sp.011 succinate and mixed titration data. The oxidising pathway kinetics start to diverge at around 15% Qr/Qt. The shape of both curves are clearly convex. The mixed substrate oxidising pathway kinetics cannot be fitted exclusively by either NADH or succinate kinetic fits. This suggests that both dehydrogenases are active. The similarity in shape of the succinate and the mixed substrate oxidising pathway kinetics suggest that SDH has a stronger control on oxidising pathway activity than the external NADH dehydrogenase. This will be discussed in terms of reducing pathway kinetics in the next section.
  • 214 Figure 6.10 Combined uncoupled oxidising pathway kinetics in sp.011 AOX mitochondria respiring either on succinate or mixed substrates. Succinate () data points are taken from Figure 5.15. The mixed titration () data was taken from Figure 6.6 Using Q-pool kinetics the data were fitted. It is clear that the modelled kinetic curves for both sets of data are different from one another. Curve fitting according to [76]. 0 50 100 150 200 250 300 350 400 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt
  • 215 6.3 DISCUSSION In this study substrate dependent differences in sp.011 AOX oxidising pathway kinetics were further explored. Oxidising pathway data were modelled according to [76] (figures 6.1-6.3) and it was determined that succinate and NADH dependent oxidising pathway kinetics under ADP limited conditions start to diverge at a Q-reduction level of ~30%. Under state 3 conditions, over the whole range of Qr/Qt respiratory activity with NADH is higher than with succinate. Under uncoupled conditions the succinate and NADH dependent oxidising pathway kinetics start to diverge at a Q-reduction level of ~20%. These determinations would not have been possible on the basis of looking at the raw data alone (cf. figures 5.17-5.19). Another characteristic of the substrate dependent differences in sp.011 AOX oxidising pathway kinetics becomes apparent when kinetic curves obtained under various energetic conditions are plotted together (Figure 6.4). The substrate dependent differences in kinetics become more pronounced upon deenergization of the IMM. Experiments done with the oxygen electrode showed that under ADP limited conditions the respiratory rates obtained in the presence of NADH or succinate are different (Table 6.1). This could be explained in terms of the ability of the dehydrogenases to reduce the Q-pool with the external NADH dehydrogenase having more capacity than SDH. The Q-pool is slightly more reduced with NADH than with succinate (cf. 6.1), but this argument would only be relevant if both succinate and NADH data were on the same kinetic curve, which they are not. Suppose there was in fact only one kinetic curve, either the succinate or the NADH one in Figure 6.1, a ~10% increase in Qr/Qt would not lead to an increase in vO2 that could account for the differences in steady state respiratory rates with either succinate or NADH as a substrate. The combined activities of the cytochrome pathway (determined in the presence of OG) and the alternative pathway (determined in the presence of antimycin A) with NADH as a substrate appear to be additive. With succinate as a substrate the total respiratory activity appears to be less than the sum of the activities of the two pathways. This suggests that succinate has a limiting effect on oxidising pathway kinetics. It was already established that succinate has no limiting effects on either the cytochrome or
  • 216 the alternative pathway as no substrate dependent differences are seen in either cytochrome pathway kinetics (cf. figures 4.9,4.11,4.12 and 5.20-5.22) or alternative pathway kinetics (cf. Figure 5.23). Also, it was determined in previous work done in this laboratory that addition of succinate to sp.011 AOX mitochondria respiring on NADH does not have any effect on respiration (cf. Table 6.1 in [26]), i.e. succinate does not have an inhibitory effect on AOX per se. Therefore, when both pathways are active, one or both of them are limited by succinate dependent respiration. A series of experiments were conducted where the Q- pool was reduced with a mixture of NADH and succinate. This approach was taken to address three interlinked questions: 1) Experiments using the oxygen electrode and titration studies using a combination of the Q-electrode and the oxygen electrode revealed substrate dependent differences in oxidising pathway kinetics, this was further corroborated by modelling the data. To confirm the presence of these substrate dependent differences oxidising pathway kinetics in the presence of both NADH and succinate were determined. 2) To investigate any limiting effects of succinate on oxidising pathway kinetics succinate (and SDH activators) were added to sp.011 AOX mitochondria respiring on NADH. 3) According to Q-pool kinetics the oxidising pathway kinetics are only dependent on the Q redox poise, regardless as to how the Q-pool is reduced. Reducing the Q-pool with both succinate and NADH will reveal whether in sp.011 AOX mitochondria Q-pool kinetics apply. Addition of succinate (and SDH activators) to sp.011 AOX mitochondria respiring on sub- saturating amounts of NADH (in the presence of the NADH regenerating system) leads to a large increase in Qr/Qt with a concomitant low increase in vO2 leading to oxidising pathway kinetics which are unlike the kinetics seen with just NADH (figures 6.5 and 6.6). These findings suggest that succinate indeed has a limiting effect on oxidising pathways activity and it confirms that there are substrate dependent differences in sp.011 AOX oxidising pathway kinetics. Furthermore, these results suggest that Q-pool kinetics may not apply (discussed later). At Qr/Qt levels of 20% and higher the respiratory rates in the presence of NADH and succinate are higher than with succinate alone (Figure 6.7). This suggests that both dehydrogenases are active and that activity of SDH does not inhibit the
  • 217 0 50 100 150 200 250 300 0 0.2 0.4 0.6 0.8 1 vO2 nmolO2 /min/mgprotein Qr/Qt external NADH dehydrogenase. Curve fitting shows that the mixed substrate oxidising pathway kinetics resemble succinate kinetics more closely than NADH kinetics (figures 6.9 and 6.10). These results suggest that in the presence of both NADH and succinate SDH has more control on respiration than the external NADH dehydrogenase. In order to investigate why the mixed substrate kinetics are more ‘succinate-like’ than ‘NADH-like’ it is necessary to compare the reducing pathway kinetics. Figure 6.11 shows reducing pathway kinetics in S. pombe mitochondria for both SDH and the external NADH dehydrogenase. It can be seen that the relationship between Q-redox poise and oxygen consumption rate with the external NADH dehydrogenase in S. pombe mitochondria is very steep, i.e. a small change in Qr/Qt will result in a large change in vO2. These kinetics agree well with previously published results on external NADH dehydrogenase kinetics in potato mitochondria (cf. Figure 5 in [76]). The SDH kinetics display a considerably lower slope than the external NADH dehydrogenase kinetics, i.e. a small change in Qr/Qt will result in a relatively small change in vO2. Figure 6.11 Reducing pathway kinetics in S. pombe mitochondria. SDH data points () are taken from Figure 4.13. The external NADH dehydrogenase data points () are taken from Figure 4.3B in [26]. The comparison is justified as the S .pombe mitochondria isolated in that study are essentially the same as in this study and the results were obtained during the same time when results for this study were obtained, i.e. both researchers were working on the same system in the same period. Concentrations: succinate 9 mM (SDH activated with ATP (0.2mM) and glutamate (9 mM)), NADH 1.8 mM. Kinetics are modelled according to [76].
  • 218 The difference in slopes and shapes of the kinetic curves in Figure 6.11 reflect a difference in affinity for ubiquinone (the substrate for the dehydrogenases), see Figure 6.12. The reversible Michaelis-Menten equations used to model Q-pool kinetics can be condensed into a simple equation: (equation 2.6, section 2.4.5) The parameter  represents the affinity for ubiquinone and determines the shape (and therefore the slope) of the kinetic curves. A low value for  (e.g. –0.99) represents a high affinity for ubiquinone, whereas a high value represents a low affinity, i.e. the shape of the kinetic fits reveal at a glance any differences in substrate affinity. It is clear from Figure 6.11 that over the whole range of Qr/Qt SDH has a higher affinity for ubiquinone, which means that at increasing levels of Q-reduction (decreasing concentration of ubiquinone) SDH could ‘out-compete’ the external NADH dehydrogenase. The oxidising pathway kinetics of sp.011 AOX mitochondria respiring on either NADH or succinate look distinctly different. Mixed titration oxidising pathway kinetics do not overlap with either type of kinetics, but look more similar to succinate kinetics than NADH kinetics. A difference in affinity for ubiquinone may explain why SDH appears to have more control over oxidising pathway activity. However a difference in dehydrogenase kinetics cannot explain the occurrence of substrate dependent differences in oxidising pathways since S. pombe mitochondria expressing only one pathway do not display any substrate dependent differences. v    s     s  1
  • 219 Figure 6.12 Reducing pathway kinetics modelling different affinities for ubiquinone. Curve 1 represents modelled reducing pathway kinetics with a low value of , this represents a typical SDH kinetic curve. Curve 2 represents modelled reducing pathway kinetics with a high value of , this represents a typical external NADH dehydrogenase curve. Respiratory rates in arbitrary units, figure adapted from Figure 3 in [216]. In order for dehydrogenases to have an effect on oxidising pathway kinetics a deviation from Q-pool kinetics has to be assumed. There are several not mutually exclusive possibilities: 1) The Q-pool in S. pombe mitochondria expressing both the cytochrome and the alternative pathways is not homogeneous. 2) Either one or both of the dehydrogenases can interact with the oxidising pathways in a non Q-dependent manner. 0 5 10 15 20 0 0.2 0.4 0.6 0.8 1 respirationrateAU Qr/Qt 1 2
  • 220 It has been shown that the Q-pool in S. pombe mitochondria displays pool behaviour (cf. figures 4.13, 4.14, 5.27 and 5.28 and [26]). That would mean that with the introduction of an extra oxidising pathway the Q-pool should become non homogenous. The introduction of AOX itself does not lead to a deviation from pool behaviour (cf. Figure 5.27). Also in the presence of antimycin A (only the alternative pathway active) no substrate dependent differences are seen (cf. Figure 5.23). Therefore a deviation from pool behaviour would occur when both pathways are active. Maybe, by introducing an extra oxidase the demand for reducing equivalents exceeds the ability of SDH to keep the Q-pool homogeneously reduced, i.e. there would be regional differences in QH2 concentration so that the bc1 complex and AOX would face different concentrations of substrate. The strain on SDH to keep the Q-pool homogeneously reduced would increase if either the demand for reducing equivalents by the oxidising pathways increased or if SDH itself would become deactivated. Both happen during deenergization. Under state 3 or uncoupled conditions the protonmotive force decreases and the cytochrome pathway is no longer restrained by it, Figure 6.4 shows that (with either succinate or NADH as a substrate) upon going from coupled (ADP limited) conditions to uncoupled (presence of ADP or CCCP) conditions the oxidising pathway activity increases. Therefore upon increased deenergization the demand for reducing equivalents from the Q-pool increases. It was shown that SDH activity was dependent on energy status (cf. figures 3.19, 4.13 and 5.27). Therefore upon increased deenergization the ability of SDH to supply reducing equivalents to the Q-pool decreases and the Q-pool becomes functionally compartmentalized. It is clear from Figure 6.4 that upon increased deenergization the substrate differences become more pronounced. The external NADH dehydrogenase is not affected by energy status and has more reducing capacity than the SDH therefore it is expected that the external NADH dehydrogenase is able to keep the Q-pool homogenously reduced in the presence of two oxidases. Perhaps the deviation from Q-pool behaviour is due to a physical limitation of the expression system where SDH is not able to keep the Q- pool homogenously reduced. Deviation in Q-pool kinetics was not seen in a system naturally expressing AOX (cf. Figure 5.24) suggesting that under natural conditions SDH is able to keep the Q-pool homogenously reduced. The extra activity induced by functionally expressing AOX in S. pombe mitochondria is not trivial, under ADP limited conditions
  • 221 with NADH as a substrate the oxygen consumption rate increases by about 100 nmol O2 / min / mg protein, double the rate with only the cytochrome pathway active, incorporation of AOX leads to a considerable increase in demand for reducing equivalents from the Q- pool. Perhaps the ETC in S. pombe mitochondria, as there never was any evolutionary pressure to accommodate an extra oxidase, has no means of upregulating SDH expression in response to an increased demand for reducing equivalents. Another possibility could be that reducing pathway activities influence oxidising pathway activities in a non Q-dependent manner. To explain an interplay found between cytochrome and alternative pathway activity in mitochondria isolated from the amoeba Acanthamoeba castellanii Jarmuszkiewicz et al. [258] postulate an effect of matrix pH on AOX activity. An effect of pH on cyanide-resistant respiration has been found in mitochondria isolated from A. castellanii [82] and from several plant sources such as vita bean (Vigna unguiculata L.) [259], Arum italicum [260] and (important to this study) S. guttatum [83]. Due to cytochrome pathway proton pump activity the matrix pH can be changed. In the presence of antimycin A, matrix pH would not change and no substrate dependent differences would be seen in alternative pathway activity (cf. Figure 5.23). In the presence of octyl gallate, or in S. pombe mitochondria not expressing AOX a difference in matrix pH can be expected as cytochrome pathway activity with succinate is lower than with NADH as succinate dependent respiration becomes limiting under state 3 and uncoupled conditions, see figures 4.11 and 4.12. So possibly, when both pathways are active, with NADH, a higher rate of proton pumping is achieved than with succinate, leading to different matricial pH values depending on substrate, which affects AOX activity. In section 4.2.2.1 it was discussed that nigericin (which abolishes pH [1]) had no effect on  which suggests that no pH was present (due to K+ /H+ ) activity. A pH effect on AOX in mitochondria lacking an apparent pH would therefore not be expected, however it was shown in V. unguiculata mitochondria that AOX was affected by pH even when nigericin was present in the medium [259]. A mechanism like this would be compatible with the results presented in this study. When the cytochrome pathway is active matrix pH values could be different depending on substrate. Cytochrome pathway activity is not affected by pH (or not to the same extent as AOX) and no substrate dependent differences would be
  • 222 seen in cytochrome pathway kinetics. In the presence of antimycin A the cytochrome pathway would be inactive and the pH would be the same with either succinate or NADH and no substrate dependent differences would be seen in alternative pathway kinetics. When both pathways are active, with the cytochrome pathway more active with NADH as a substrate than with succinate there could be a substrate dependent difference in matrix pH which specifically affects AOX activity, which would only be apparent when both pathways are active. 6.4 CONCLUSION The substrate dependent differences in sp.011 AOX oxidising pathway kinetics are only seen when both oxidising pathways are active. In this study an alternative oxidase from a thermogenic species (S. guttatum) was functionally expressed in S. pombe. Substrate dependent differences were not found in another thermogenic species (A. maculatum), naturally expressing AOX. One way to interpret this would be to assume that the substrate dependent differences are a specific characteristic of our expression system. Introduction of an extra oxidase to the ETC of S. pombe mitochondria might put a strain on the system inducing deviation from Q-pool kinetics. In our laboratory S. pombe has also been used to express the Arabidopsis thaliana alternative oxidase [70]. It would be of interest to determine whether or not this system also displays substrate dependent differences. Other possible avenues to explore would be to inhibit AOX27 in sp.011 AOX mitochondria and determine if the substrate dependent differences disappear. In the event of the substrate dependent differences being a characteristic of the expression system and not a reflection of an in vivo situation our expression system is still very useful to address different questions on a more fundamental level concerning Q-pool kinetics, the interplay between pathways and to determine the limitations of dehydrogenases to keep the Q-pool homogeneously reduced. 27 Difficult as the Q-electrode reacts strongly with octyl gallate, requiring long incubation times with OG for the Q-signal to stabilise.
  • 223 Chapter 7 General Discussion In this study the yeast Schizosaccharomyces pombe has been used to functionally express the plant Sauromatum guttatum alternative oxidase in order to investigate the influence of AOX on respiratory kinetics. The alternative oxidase is non-protonmotive [261], i.e. the free energy released upon oxidation of ubiquinol is not stored in an electrochemical gradient of protons across the IMM. The activity of AOX leads to the dissipation of free energy. Another means for many mitochondria to lower the efficiency of energy transduction is via the uncoupling protein [262] which catalyses the transport of protons into the matrix thereby decreasing the magnitude of the protonmotive force. It is unclear whether or not AOX activity can lead to a decrease in the magnitude of p. It was determined previously that uncoupling had no effect on AOX activity [176]. In this study AOX activity was also found to be independent of energy status. S. pombe mitochondria do not express complex I [13] therefore p is generated through the combined proton pumping activities of complexes III and IV (see Figure 1.14). It has been found that in the presence of pyruvate the alternative pathway can compete with the cytochrome pathway for reducing equivalents [79]. Addition of pyruvate to S. pombe mitochondria expressing S. guttatum AOX does not lead to an increase in activity, it was concluded that the alternative oxidase in our expression system is constitutively active [70]. Since S. guttatum AOX in S. pombe mitochondria is fully activated it can be hypothesized that the alternative pathway is able to compete with the cytochrome pathway for reducing equivalents in a way similar to the case in soybean mitochondria [79]. If indeed reducing equivalents were diverted from the cytochrome pathway to the alternative pathway this might affect the membrane potential. In order to investigate this a TPP+ -electrode [154] was employed to determine the membrane potential. The respiratory characteristics of the wild type S. pombe mitochondria had been characterised to a certain extent previously [26, 104, 105]. The membrane potential however (to the best of our knowledge) had not been determined previously. Prior to
  • 224 investigating any effects of AOX activity on  in S. pombe it was necessary to get an idea of the bioenergetics in the wild type mitochondria. Also, due to several small alterations to the isolation method (omitting the final PercollTM purification step, using a bench top centrifuge for slow spins instead of a floor centrifuge) led to a higher yield and a small increase in quality. For these reasons it was decided to recharacterise the S. pombe mitochondria in this study which yielded some very unexpected respiratory kinetics. 7.1 Characterisation of the wild type S. pombe mitochondria The sp.011 wt mitochondria displayed respiratory characteristics similar to mitochondria from other sources (see figures 4.1 to 4.3 and Table 4.1). Membrane potential values were similar to what was seen in other yeast mitochondria. Under ADP limited conditions the familiar non-ohmic relationship between  and respiratory rate was observed (see Figure 4.5 and cf. Figure 3.4). The relationship between Qr/Qt and oxygen consumption rate under ADP limited conditions when the cytochrome pathway kinetics were determined using an NADH regenerating system [146] was linear, similar to what is seen in mitochondria from other sources (see Figure 4.4). However, under state 3 and uncoupled conditions, the cytochrome pathway kinetics (Qr/Qt vs. vO2) showed a distinct biphasic pattern (see figures 4.6 and 4.8). Especially interesting were the results obtained when determining the  vs. vO2 cytochrome pathway kinetics under state 3 conditions (see Figure 4.7). A biphasic pattern can also clearly be seen in the relationship between membrane potential and oxygen consumption rate (see inset B of Figure 4.7). This observation provided the first indication of the fact that the cause of the biphasic kinetics was located downstream from the Q-pool (the membrane potential in S. pombe mitochondria is generated by the cytochrome pathway). Equally also,  and Qr/Qt are measured with different electrodes, which argues against the possibility that the biphasic patterns were artifactual (the possibility that the biphasic patterns are due to experimental artefacts is thoroughly explored in section 4.3.2). The cytochrome pathway kinetics were determined using the NADH regenerating system as it has the advantage over the malonate titration method (with succinate as substrate) of obtaining more data points during one experimental trace. To rule out the possibility that the biphasic patterns were NADH dependent, the
  • 225 cytochrome pathway kinetics were again determined, this time with succinate as a substrate (see figures 4.9-4.12). No substrate dependent differences were seen which corroborated the notion that the cause for the biphasic patterns was not a characteristic of the dehydrogenases. The most conclusive observation came with the determination of oxidising pathway kinetics in S. pombe mitochondria expressing the alternative oxidase, see figures 5.8 and 5.9. If an extra terminal oxidase is present the biphasic pattern under state 3 and uncoupled conditions (Qr/Qt vs. vO2) becomes masked. Under state 3 conditions in the  vs. vO2 oxidising pathway kinetics in sp.011 AOX mitochondria the biphasic pattern can still be clearly seen and looks no different from S. pombe mitochondria not expressing AOX (see figure 5.11), i.e. the biphasic kinetics are still there and are a characteristic of the cytochrome pathway. Contemporary work by Trumpower et al. [33] suggested that the yeast bc1 complex displays kinetics which could explain our findings. In order to investigate this possibility we employed a spectrophotometric technique where ferricyanide was used as an artificial electron acceptor (see figures 4.16 to 4.18). The results showed that respiratory kinetics of S. pombe mitochondria are different from potato mitochondria. Also a biphasic pattern appears to be present in the S. pombe data and absent in the potato data. Our data suggests that it is indeed bc1 complex kinetics underlying the biphasic patterns observed under state 3 and uncoupled conditions. Trumpower and colleagues worked on bc1 complexes isolated from S. cerevisiae mitochondria. If indeed the biphasic pattern can be explained in terms of yeast bc1 complex kinetics then oxidising pathway kinetics of S. cerevisiae mitochondria should display a biphasic pattern; which is what we found, see Figure 4.19. 7.1.1 How does cytochrome bc1 complex activity lead to biphasic cytochrome pathway kinetics in S. pombe mitochondria?—The cytochrome bc1 complex in eukaryotic organisms is a homodimeric multi-subunit inner mitochondrial membrane protein complex [263] which catalyses the protonmotive Q-cycle [31] (see also section 1.2.6). The Q-cycle mechanism does not require the cytochrome bc1 complex to be dimeric and the functional relevance of its dimeric structure is unclear [33]. Bernard Trumpower over the years has postulated several mechanisms of cytochrome bc1 complex activity which could provide a ‘raison d'être for the dimeric structure of the enzyme’. Publications by Trumpower and co-
  • 226 workers on the functional significance of the dimeric structure of the cytochrome bc1 complex have been appearing as far back as 1990 [264] and the most recent one came out in June 2005 [235]. The first mechanism that was proposed (1990) has over the years evolved considerably, one can discern two stages where this mechanism was changed. The first two mechanisms appear highly relevant with respect to this study, the most recent mechanism is difficult to reconcile with my results. The early work In 1990 a publication came out in which half-of-the-sites reactivity of the cytochrome bc1 complex in S. cerevisiae was reported [264]. In this study the nuclear gene encoding for subunit 6 was deleted. This subunit is believed to be involved in binding cytochrome c in cooperation with cytochrome c1. This deletion strain could be grown on non-fermentable media indicating that the cells had a (partially) functional mitochondrial respiratory chain. Isolated bc1 complexes from this strain showed a 50% reduction in cytochrome c reductase activity when the salt concentration in the assay was similar to the expected intracellular ionic strength. This reduced activity was not seen when the ionic strength in the assay was lowered. The wild type bc1 complex activity did not show any such dependency on ionic strength. It was further found in the deletion strain that under conditions where half of the bc1 complexes are inactive all of the complexes retain the ability to bind inhibitors (myxothiazol in this case). It was proposed that the genetic deletion of subunit 6 mimics a reversible conformational change or dissociation of this complex thereby inactivating one of the monomers in the dimeric complex. It was hypothesized that a physiological effector which could interact with subunit 6 is the protonmotive force. Conformation of subunit 6 might change upon a change in membrane potential or a change in local pH. Under ADP limited conditions subunit 6 would be in a conformation such that only one of the monomers in the bc1 complex is active. If in S. pombe mitochondria the cytochrome bc1 complex behaves in a similar way as in S. cerevisiae then a membrane potential induced conformational change in subunit 6 could explain why the cytochrome pathway kinetics are biphasic under state 3 and uncoupled conditions but not under ADP limited conditions.
  • 227 Under state 2 and state 4 conditions, due to a high  subunit 6 would be locked in a conformation in which only one of the monomers per dimer is active. Upon decrease of  a conformational change occurs upon which both monomers now can become active simultaneously. However, the fact that both monomers now can be active does not mean that they will or that they would be equally active. This change in conformation does not in itself explain the occurrence of a biphasic pattern unless the monomers within the dimer become active sequentially. This is exactly what Trumpower et al. found next. The intermediate work After a twelve year hiatus28 several publications on the yeast bc1 complex reaction mechanism came out [33, 34, 236]. It was found that several inhibitory analogs of ubiquinol binding at center P could inhibit the wild-type S. cerevisiae enzyme with a stoichiometry of 0.5 per bc1 complex. One molecule of inhibitor could fully inhibit the dimeric enzyme. From this it was concluded that only one monomer per dimer was active at a time, displaying the so called half-of-the-sites reaction mechanism. This was found with stigmatellin and MOA-stilbene29 , but interestingly enough not with myxothiazol. This is peculiar because both myxothiazol and MOA-stilbene are structurally similar and have the same mechanism of inhibition (both inhibitors prevent electron flow from ubiquinol to the iron-sulfur protein), it was suggested that very subtle differences in ligand-protein interaction could account for this. Stigmatellin allows reduction of the iron- sulfur protein but traps its position proximal to cytochrome b thereby preventing oxidation by cytochrome c1. Inhibition with antimycin (which prevents reduction of ubiquinone or ubisemiquinone at the N site) led to a stoichiometry of 1 per bc1 complex. Based on these observations it was suggested that the yeast cytochrome bc1 complex oxidises ubiquinol by a half-of-the-sites mechanism. Further inhibitor studies using stigmatellin or MOA – stilbene showed that the yeast bc1 complex binds these inhibitors in an anti-cooperative way. A second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor is already bound. Trumpower et al. propose that ubiquinol binding is also anti- cooperative and that binding of ubiquinol to one monomer raises the Kd for ubiquinol 28 At the EBEC 2004 meeting in Pisa professor Trumpower, during a presentation, mentioned the fact that the “half-of-the-sites reaction mechanism in the yeast bc1 complex” research line had been shelved right after the 1990 publication. 29 MOA-stilbene – methoxyacrylate - stilbene
  • 228 binding in the other monomer. Based on these findings the regulation of the bc1 complex in yeast is hypothesized to occur in either of two ways: 1: Due to a conformational change the bc1 complex can switch from half-of-the sites reactivity to full activation. 2: The bc1 complex functions with two affinities for substrate and the second half of the dimer is only active at high ubiquinol concentration. In the early work it was suggested that under ADP limited conditions subunit 6 is in a conformation in which one of the monomers is inactivated. A physiological effector, such as the protonmotive force, could reversibly change this conformation, leading to full activation of the bc1 complex. Interestingly enough Trumpower does not link his earlier suggestion to hypothesis 1. It was concluded that: ‘one can only speculate as to what types of signals might bring about such a switch in the enzyme’. In S. pombe mitochondria the biphasic pattern is seen only under state 3 or uncoupled conditions, i.e. a change in  with respect to ADP limited conditions. So possibly this change in  leads to a conformational change after which both monomers become active. This however does not explain the occurrence of the biphasic pattern itself. Hypothesis 2 could accommodate for this. The relationship between Qr/Qt vs. vO2 becomes biphasic at around 40% Q-reduction under state 3 or uncoupled conditions in S. pombe mitochondria. So perhaps at 40% Qr/Qt half of the high affinity monomers are bound and upon a further increase in Q reduction, ubiquinol starts binding to the low affinity monomers. However if the biphasic oxidising pathway kinetics are simply a reflection of Qr/Qt reaching a threshold value why is there no biphasic pattern under state 2 or state 4 conditions? Under ADP limited conditions the Q-pool is further reduced than under state 3 or uncoupled conditions. Hypotheses 1 and 2 are not mutually exclusive. In order to explain the biphasic kinetics in S. pombe mitochondria a mechanism which requires two conformational changes is described next. Under ADP limited conditions only one monomer is active, upon a change in  a conformational change occurs and the second monomer can now become active as well. Under state 2 or state 4 conditions this
  • 229 conformational change never occurs and hence only one monomer per dimer is accessible to ubiquinol. When one monomer in a dimer is bound the binding affinity in the other monomer is increased. So even if both monomers become active upon a change in  first all high affinity monomers must be activated by ubiquinol before ubiquinol can bind to the low affinity monomers. This could explain the occurrence of the biphasic pattern and why it only occurs under conditions where  is decreased. The recent work  Between 2004 and 2005 several papers came out [235, 265] which focus on electron transfer between monomers in the bc1 complex via the bL cytochromes. The distance between the bL heme in one monomer to the other bL heme in the second monomer is 10-11 Å, this distance is bridged by three pairs of aromatic residues via which electron transfer could take place30 . Interestingly enough Trumpower states that ‘Some center P inhibitors have been shown to completely block bc1 complex activity upon binding to only half of the dimeric complex, suggesting anti-cooperative interaction between the ubiquinol oxidation sites in the dimer’ [265]. It is not readily apparent to this author how half-of-the sites reactivity suggests anti-cooperative interaction. In the early work it was established that under specific conditions the bc1 complex displayed half-of-the sites reactivity, this was not related to anti-cooperative binding of ubiquinol. Only during the intermediate work, from inhibitor studies it became apparent that the affinity for certain center P inhibitors on the second monomer was decreased if another certain center P inhibitor was already bound to the first monomer. This might explain the half-of-the sites reactivity, not the other way around. It is suggested in the recent work that one active P site can reduce two bH hemes (via intermonomer electron transfer). It is not clear how this mechanism relates to anti-cooperative binding of ubiquinol. Concluding, the latest work by Trumpower et al. on the regulation of the yeast bc1 complex does not seem to be a further development of the previous mechanisms described in the early and intermediate work but seems to be going on a tangent, no longer directly related to previous work nor related to the results described in this thesis. 30 Theoretically it could also be possible for electron transfer to occur without participation of the aromatic residues via electrontunneling [5].
  • 230 7.1.2 Future work suggestions pertaining to the biphasic patterns in S. pombe cytochrome pathway kinetics—The S. pombe spectroscopy data is somewhat limited. It can be seen in Figure 4.18 that the data points are not equally distributed over the NADH concentration range. It would be good if some extra experiments were done in which the whole concentration range was covered. It was hypothesized that the membrane potential was instrumental in the generation of the biphasic pattern in the oxidising pathway kinetics. It would be interesting to see if in the presence of sub-saturating concentrations of uncoupler, during the transition from coupled to uncoupled conditions a biphasic pattern gradually becomes more apparent. The S. cerevisiae data convincing as they may appear are from one experimental day only. This experiment should be repeated. Our data suggest that the biphasic patterns in oxidising pathway kinetics are a general characteristic of yeast bc1 complex kinetics. It would be very interesting to see if isolated S. pombe bc1 complexes display the same kinetics as their S. cerevisiae counterparts.
  • 231 7.2 Functional expression of AOX in S. pombe mitochondria yields substrate dependent differences in oxidising pathway kinetics. Having characterised the wild type S. pombe mitochondria we subsequently started to investigate the effects of heterologously expressed AOX in S. pombe mitochondria on respiratory kinetics and the membrane potential. Previous work had shown that under various energetic conditions the functional expression of the S. guttatum AOX in S. pombe mitochondria led to a decreased oxygen consumption rate at a certain value of Qr/Qt when compared to transformed S. pombe mitochondria in which AOX expression was repressed [93]. This result is counter intuitive. One would expect that the introduction of an extra terminal oxidase would lead to an overall increased oxygen consumption rate. This is exactly what we found in this study, see figures 5.7 to 5.9. Using the NADH regenerating system it was determined that under all energetic conditions (ADP limited, state 3 and uncoupled) in the presence of AOX at a certain value of Qr/Qt the oxygen consumption rate was higher when compared to S. pombe mitochondria in which AOX expression was repressed. Possible causes for the differences between the results between this study and the previous one done in this laboratory [93] were explored (see sections 5.2.3 and 5.3.6) and it was concluded that it was a combination of differences in RCR, reaction medium used and the fact that some of the substances added during Affourtit’s experiments were dissolved in ethanol. Apart from increased oxygen consumption rates it also became apparent that in the relationship between Qr/Qt vs. vO2 under state 3 and uncoupled conditions the biphasic pattern became masked (cf. figures 5.8 and 5.9). Whereas in transformed mitochondria in which AOX expression was repressed the biphasic patterns were still to be seen (cf. figures 5.8 and 5.9). The biphasic pattern was still clearly present in the  vs. vO2 relationship under state 3 conditions in the sp.011 AOX mitochondria (which again corroborates the notion that the biphasic pattern is a characteristic of the cytochrome pathway) and does not look different from the same relationship in sp.011 AOX+T mitochondria. Importantly also was the observation that under ADP limited conditions the familiar non-ohmic relationship was observed between  and vO2 in sp.011 AOX mitochondria, see Figure 5.10.
  • 232 It can be seen that the relationship between  vs. vO2 in sp.011 AOX mitochondria overlaps with the sp.011 AOX+T data. This indicates that the introduction of the alternative oxidase in the S. pombe IMM did not change its conductivity. It has been previously hypothesized that the presence of protein complexes in the IMM leads to non-specific proton leak [245]. The introduction of AOX to the S. pombe IMM might have affected its conductive properties which would make comparisons between the respiratory kinetics of mitochondria expressing AOX and mitochondria in which AOX is repressed problematic. However, it is clear from Figure 5.10 that the IMM conductivities of both sp.011 AOX and sp.011 AOX+T mitochondria are the same and any differences in respiratory kinetics between the two types of mitochondria can be attributed to the activity of AOX. 7.2.1 Does AOX activity affect  in S. pombe mitochondria ?—Under state 3 conditions the relationship between  and vO2 in the region after the inflection point of the biphasic pattern is proportional, similar to what is seen in potato mitochondria (cf. inset A of Figure 4.7). This means that a decreased activity of the cytochrome pathway will lead to a proportional decrease in . If in S. pombe mitochondria the alternative oxidase can compete with the cytochrome pathway to such an extent that the cytochrome pathway receives less reducing equivalents per unit time, then this should be reflected in a lowered . It can be seen in Figure 5.11 that the titration data obtained with the NADH regenerating system shows variation to such an extent that a possible effect of AOX activity on  cannot be discriminated. In a different approach to address this problem (see Figure 5.12) it became apparent that AOX activity does not affect the value of . This finding suggested that AOX in S. pombe does not compete with the cytochrome pathway under state 3 conditions. 7.2.2 Substrate dependent differences in oxidising pathway kinetics of S. pombe mitochondria expressing AOX—The oxidising pathway kinetic data in S. pombe mitochondria (described thus far) was obtained using the NADH regenerating system. The results were different from a previous study [93] where oxidising pathway kinetics were obtained using succinate as a substrate. To be certain, these experiments were repeated, see figures 5.13 to 5.15. Again, it was observed that the introduction of an extra terminal
  • 233 oxidase led to an increased oxygen consumption at a certain value of Qr/Qt when compared to mitochondria in which AOX was repressed. Also, under state 3 and uncoupled conditions the biphasic pattern in the relationship between Qr/Qt vs. vO2 is masked. The oxidising pathway kinetics obtained with succinate as a substrate were different from the results obtained in the previous study [93] and were in agreement with the NADH data, i.e. with either succinate or NADH the biphasic pattern is masked and the oxygen consumption rate at a certain value of Qr/Qt is higher in the presence of AOX than in the absence of AOX. Interestingly enough, when plotting the NADH and succinate data together it became apparent that there were substrate dependent differences in oxidising pathway kinetics, see figures 5.17 to 5.19. At a certain value of Qr/Qt the oxygen consumption rates with NADH appear to be higher than with succinate. Under ADP limited conditions (Figure 5.17) the oxidising pathway kinetics seem to diverge at a Qr/Qt value of ~60% whereas under state 3 and uncoupled conditions the kinetics seem to diverge at ~40%. These values were estimated on the basis of the raw data. In order to get more accurate values the data were fitted using a reversible Michaelis-Menten equation according to [76], see figures 6.1 to 6.3. Based on the kinetic fits it was estimated that under ADP limited condition the kinetics start to diverge at a Qr/Qt value of ~30%. Under state 3 conditions oxygen consumption rates with NADH as a substrate appear to be higher over the whole range of Qr/Qt and under uncoupled conditions the kinetics start to diverge at a Qr/Qt value of about 20%. 7.2.3 What causes the substrate dependent differences in oxidising pathway kinetics in S. pombe mitochondria expressing AOX?—Substrate dependent differences in total oxidising pathway kinetics could be attributable to either of the individual pathways, the cytochrome pathway or the alternative pathway. It was already shown in the wild type mitochondria that the cytochrome pathway kinetics did not show any substrate dependent differences, see figures 4.9 to 4.12. Possibly the act of transformation altered cytochrome pathway kinetics leading to substrate dependent differences, this was also ruled out, see figures 5.20 to 5.22. There were no substrate dependent differences in the cytochrome pathway kinetics of sp.011 AOX+T mitochondria.
  • 234 If not the cytochrome pathway, could there be substrate dependent differences in the alternative pathway kinetics? This possibility was also ruled out, see Figure 5.23. No substrate dependent differences were seen in sp.011 AOX mitochondria incubated in the presence of AA. The substrate dependent differences are only seen when both pathways are active simultaneously. As a comparison to a system in which AOX is naturally expressed and where both pathways are active simultaneously we determined oxidising pathway kinetics with either NADH or succinate as a substrate in mitochondria isolated from Arum maculatum spadices, see Figure 5.24. No substrate dependent differences were seen. This could be interpreted as such that the substrate dependent differences are a particularity of our expression system. 7.2.4 Are the substrate dependent differences in oxidising pathway kinetics in S. pombe mitochondria expressing AOX due to dehydrogenase characteristics?—According to the Q-pool model (see section 1.5) the dehydrogenases do not interact directly with the oxidising pathways, i.e. differences in reducing pathway activities are compensated for by the Q-pool. The ubiquinol oxidases (bc1 complex and alternative oxidase) can only ‘sense’ the Q-redox poise. How this redox poise is brought about (through SDH or the external NADH dehydrogenase) should not matter. However, upon combining the kinetic fits from figures 6.1 to 6.3 in a composite figure (see Figure 6.4) it became apparent that the substrate dependent differences in oxidising pathway kinetics became more pronounced depending on the degree of deenergization of the IMM. It has been shown in both potato and S. pombe mitochondria that SDH activity is dependent on energetic status31 , see figures 3.19, 4.13 and 5.27, whereas external NADH dehydrogenase activity is not dependent on energy status, see Figure 4.15. Possibly, in our expression system, with both pathways active, dehydrogenase activity could determine oxidising pathway kinetics. 31 Which is probably not a direct effect on SDH but on succinate transport, see section 3.3.
  • 235 It was shown that succinate appears to have a stronger control on oxidising pathway kinetics, see figures 6.5-6.7, 6.9 and 6.10. When the Q-pool is partially reduced with sub- saturating amounts of NADH, subsequent further reduction by addition of succinate (and SDH activators) leads to kinetic curves which are more similar to the oxidising pathway kinetics obtained with succinate than with NADH. In order to explain the substrate dependent differences in terms of non Q-pool behaviour three observations are relevant: 1) SDH has a higher affinity for ubiquinone than the external NADH dehydrogenase (see Figure 6.12). 2) SDH is physically located closer to the alternative oxidase (as both are facing the matrix) than the external NADH dehydrogenase. Equally also the external NADH dehydrogenase is physically located closer to the bc1 complex than SDH. 3) It has been shown that the Km of the alternative oxidase for duroquinol (3.3 mM) is considerably higher than the Km for duroquinol of the cytochrome pathway [266], i.e. the cytochrome pathway will always operate faster than the alternative pathway. The observations in our expression system might be explained in terms of ‘tunnelling’. In the situation where the two pathways are active simultaneously, the ‘slow’ dehydrogenase (SDH) has a preference for the ‘slow’ oxidising pathway (alternative pathway), whereas the ‘fast’ dehydrogenase (external NADH dehydrogenase) has a preference for the ‘fast’ oxidising pathway (cytochrome pathway). This would mean that the Q-pool would be partitioned into functionally separate pools of ubiquinone. Perhaps in the presence of two oxidases SDH in S. pombe mitochondria cannot keep the Q-pool homogeneously reduced, a situation which would be exacerbated by deactivation of SDH as occurs during deenergization of the IMM. It can however compete effectively with the external NADH dehydrogenase for ubiquinone molecules, hence the strong control of SDH on oxidising pathway kinetics as seen in figures 6.5 and 6.6. Another explanation would be a non Q-dependent interaction between reducing and oxidising pathways. It is known that AOX activity in Acanthamoeba castellanii mitochondria is pH dependent [82, 258]. Importantly for this study, S. guttatum AOX also shows such a dependency [83]. Possibly, in S. pombe mitochondria expressing AOX there
  • 236 is a substrate dependent difference in matrix pH which could affect AOX activity. This would only be apparent under the condition where both pathways are active at the same time and this mechanism could be compatible with our results. 7.2.5 Future work suggestions pertaining to the substrate dependent oxidising pathway kinetics in S. pombe mitochondria expressing AOX—In order to determine quantitatively the distribution of reducing equivalents between the cytochrome pathway and the alternative pathway with either succinate or NADH as a substrate there are at present two approaches available. Under state 3 conditions, using the ADP/O method [267] one can determine the distribution of reducing equivalents between the cytochrome and alternative pathway. The method rests on the assumption that AOX activity does not contribute to ATP formation and the ADP/O value in the presence of AOX is lowered as opposed to the situation where it is inhibited (with OG for example) and can only be applied under state 3 conditions. This method, applied to S. pombe mitochondria expressing AOX could under state 3 conditions quantitatively show the difference in engagement of the alternative and the cytochrome pathway dependent on substrate. Another method which has been used to quantify the distribution of reducing equivalents between the alternative and the cytochrome pathway is the oxygen isotope discrimination method [268]. The cytochrome and alternative pathway discriminate differently against the isotope 18 O. Employing mass spectrometry it is feasible to asses the partition of reducing equivalents between pathways in mitochondria. This technique however requires expensive equipment and is not easy to use. In our laboratory another alternative oxidase, from a non thermogenic species (Arabidopsis thaliana) has been expressed successfully in S. pombe [70]. This system provides the opportunity to answer the question whether or not substrate dependent differences are specific to the S. pombe expression system in future work.
  • 237 7.3 Conclusion SDH regulation in plants appears to be more complicated than previously assumed. The recent discovery of extra subunits make a direct comparison of results obtained in mammalian and yeast systems with plant SDH difficult. Although SDH regulation has been studied for over 50 years now it is clear that this subject is still as interesting as ever and worthy of investigation. Biphasic respiratory kinetics were an unexpected finding. Our results indicate that they may be a general characteristic of yeast mitochondria, these results open new avenues of research investigating the bc1 complex in mitochondria. The expression system used in this laboratory has been and still is being used for the investigation of structure-function relationships of the alternative oxidase. This study has revealed some interesting findings which may shed more light on the process of AOX regulation. The substrate dependent differences found could not be reproduced in a system naturally expressing AOX, which may indicate that it is a characteristic of the system itself. If that were the case, then our system is still very useful for the purpose of investigating bioenergetic questions of a more fundamental nature, such as investigating the limitations of the abilities of reducing pathways to keep the Q-pool homogenously reduced or mechanisms via which reducing pathways can interact with oxidising pathways in a non Q- dependent manner.
  • 238 Appendix 1 Modelling of Q-pool data—The fits used to describe the Q-pool kinetics presented throughout this thesis were modelled according to van den Bergen et al. [76]. In this model, each of the Q(H2)-interacting pathways is assumed to exhibit reversible Michaelis-Menten kinetics in accordance with the following scheme: where the forward reaction represents the oxidation of quinol substrate (S) to give quinone product (P) catalysed by enzyme (E). Described below is a derivation of a rate equation which differs from the general reversible Michaelis-Menten equation in one aspect, namely the sum of product and substrate is assumed to be constant (the ‘pool assumption’) as opposed to the conventionally used initial conditions assumption. The following definitions and assumptions apply:  [S] + [P] + [ES] = q  [E] + [ES] = e  [ES]  e << [S], [P] so that with [S] = s and [P] = p s + p = q  solution kinetics apply (rates are proportional to concentrations)  ES reaches a steady state The steady state equation is: [k1s + k4 p]  [e – [ES]] = [k2 + k3]  [ES] [1] PEESES 3 4 1 2  k k k k
  • 239 The rate (oxidation of ubiquinol) is defined as: [2] The net forward rate is: v = k1se – [k1s + k2]  [ES] = -k4pe + [k4p + k3]  [ES] [3] Eliminating ES yields: [4] This can be rearranged to: [5] Using common definitions for Ks, Kp, Vs and Vp: Vs = k3e 4 32 k kk Kp   Vp = k2e the rate equation becomes: dt Sd v ][  )()( 34 4 21 1 kpk pekv ksk sekv      ][][ ][ 4132 4231 pkskkk epkkskk v    1 32 k kk Ks  
  • 240 [6] Using the ‘pool assumption’ where s + p = q, p can be substituted for q – s which yields: [7] For fitting purposes it is convenient to use a simpler formula. By dividing both numerator and denominator by (1 + q / Kp) the following equation is obtained: [8] With:                Kp p Ks s Kp pVp Ks sVs v 1                                      pps p p p p s s K q s KK K qV s K V K V v 1 11 v    s     s  1   Vp Kp 1   Kp Ks         Vs  Vp Kp 1   Kp Ks         1 Kp 1
  • 241 Appendix 2A Table 4.1A including data on number of mitochondrial isolations and amount of experimental traces. Format: respiratory rate ( S.D.) [experimental traces, preps]. * SDH is activated stepwise by addition of ATP (0.2 mM) and glutamate (9 mM). vO2 with just succinate is 57 (7) [14,11] addition of ATP subsequently leads to a vO2 of 72 (12) [14,9] A Respiratory Rate (nmol O2 min-1 mg-1 protein) substrate(s) state 2 state 3 state 4 uncoupled NADH 91 (13) [46,18] 281 (39) [20,8] 110 (22) [24,11] 537 (68) [23,14] succinate * 106 (15) [19,11] 156 (18) [17,7] 97 (7) [16,6] 144 (25) [10,6] glutamate 32 (4) [4,2] - - - glutamate + ATP 43 (8) [4,2] - - -
  • 242 Appendix 2B sp.011 AOX Respiratory Rate (nmol O2 min-1 mg-1 protein) substrate(s) state 2 state 3 state 4 uncoupled NADH 199 (27) [37,14] 328 (34) [8,3] 224 (13) [3,1] 531 (82) [14,13] NADH + OG 92 (10) [17,9] - - 396 (43) [19,9] NADH + AA 81 (17) [16,13] - - - succinate 142 (14) [17,4] 198 (13) [11,5] 136 (8) [11,4] 176 (15) [7,4] succinate + OG 83 (12) [3,3] 123 (21) [3,3] 72 (6) [2,2] - succinate + AA 84 (13) [10,4] - - - sp.011 AOX + T NADH 80 (13) [32,12] 169 (29) [7,4] 72 (9) [4,3] 398 (55) [23,11] NADH + OG 77 (12) [15,8] - - 350 (48) [14,7] succinate 83 (10) [16,5] 136 (16) [13,5] 87 (10) [11,5] 124 (20) [13,4] sp.011 pREP
  • 243 NADH 96 (13) [10,3] 245 (25) [8,3] - 543 (55) [10,3] succinate 100 (13) [3,3] 196 (24) [5,3] 107 (23) [2,2] - sp.011 wt NADH 91 (13) [46,18] 281 (39) [20,8] 110 (22) [24,11] 537 (68) [23,14] succinate 106 (15) [19,11] 156 (18) [17,7] 97 (7) [16,6] 144 (25) [10,6] Table 5.2 including data on number of mitochondrial isolations and amount of experimental traces. Respiratory rates in sp.011 AOX, AOX + T, wt and pREP mitochondria under various energetic conditions and various substrates. Format: substrate ( S.D.) [traces, preps]. Concentrations of chemicals added: NADH (1.8 mM), succinate (9 mM), octyl gallate (14 M), antimycin A (~40 nM). Uncoupled conditions are defined as respiratory activity in the presence of an uncoupler (2 µM CCCP in all cases), which dissipates the protonmotive force leading to maximal activation of the cytochrome pathway. The concentration of added ADP to induce a state 3 to state 4 transition was 0.2 mM in all cases. State 3 conditions were induced by pre-incubation of ADP (1 mM). All succinate rates are from mitochondria incubated in the presence of ATP (0.2 mM) and glutamate (9 mM). The energy status had no effect on AOX activity, therefore the sp.011 AOX rates when incubated in the presence of antimycin A are the same under all energetic conditions.
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