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Oxidative phosphorylation and electron transport chain

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Oxidative phosphorylation and electron transport chain

  2. 2. Overview 2
  3. 3. introduction • Oxidative phosphorylation is the culmination of energy-yielding metabolism in aerobic organisms. • It is the final stage of cellular respiration in degradation of carbohydrates, fats and amino acids in which the energy of oxidation drives the synthesis of ATP. • Photophosphorylation is the means by which photosynthetic organisms capture the energy of sunlight and harness it to make ATP. • In eukaryotes, oxidative phosphorylation occurs in mitochondria, photophosphorylation in chloroplasts. 3
  4. 4. introduction • Oxidative phosphorylation involves the reduction of 02 to H2O with electrons donated by NADH and FADH2; it occurs equally well in Iight or darkness. • Photophosphorylation involves the oxidation of H2O to 02, with NADP+ as ultimate electron acceptor; it is absolutely dependent on the energy of light. • Despite their differences, these two highly efficient energy-converting processes have fundamentally similar mechanisms • Our current understanding of ATP synthesis in mitochondria and chloroplasts is based on the hypothesis, introduced by Peter Mitchell in 1961 4
  5. 5. • Each electron donor will pass electrons to a more electronegative acceptor, which in turn donates these electrons to another acceptor, a process that continues down the series until electrons are passed to oxygen, the most electronegative and terminal electron acceptor in the chain. • Passage of electrons between donor and acceptor releases energy, which is used to generate a proton gradient across the mitochondrial membrane by actively "pumping" protons into the intermembrane space, producing a thermodynamic state that has the potential to do work. • The entire process is called oxidative phosphorylation, since ADP is phosphorylated to ATP using the energy of hydrogen oxidation in many steps. • A small percentage of electrons do not complete the whole series and instead directly leak to oxygen, resulting in the formation of the free- radical superoxide, a highly reactive molecule that contributes to oxidative stress and has been implicated in a number of diseases and aging. 6
  6. 6. REDOX POTENTIAL • In reactions involving oxidation and reduction, the free energy change is proportionate to the tendency of reactants to donate or accept electrons. • Can be expressed as an oxidation-reduction or redox potential (E’0) • Usually compared with the potential of H electrode (0.0 V at pH 0.0) • However for biologic systems, is expressed at pH 7.0 at which hydrogen electrode is -0.42V. • Negative redox potential: lower affinity for electrons than hydrogen • Positive redox potential: higher affinity for electrons than hydrogen 7
  7. 7. Some Redox Potentials of Special Interest in Mammalian Oxidation Systems 8
  8. 8. Electron Transport Chain • An electron transport chain (ETC) is a series of complexes that transfer electrons from electron donors to electron acceptors via redox (both reduction and oxidation occurring simultaneously) reactions, and couples this electron transfer with the transfer of protons (H+ ions) across a membrane. • This creates an electrochemical proton gradient that drives the synthesis of adenosine triphosphate (ATP), a molecule that stores energy chemically in the form of highly strained bonds. • The molecules of the chain include peptides, enzymes and others. • The final acceptor of electrons in the electron transport chain during aerobic respiration is molecular oxygen although a variety of acceptors other than oxygen such as sulfate exist in anaerobic respiration. 9
  9. 9. Biochemical anatomy of a mitochondrion • Most eukaryotic cells have mitochondria, which produce ATP from products of the citric acid cycle, fatty acid oxidation, and amino acid oxidation. • At the mitochondrial inner membrane, electrons from NADH and FADH2 pass through the electron transport chain to oxygen, which is reduced to water. The electron transport chain comprises an enzymatic series of electron donors and acceptors 10
  10. 10. NADH GENERATION 11
  11. 11. Organization of the chain 12
  12. 12. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps • Complex I : NADH-Q oxidoreductase catalyzes transfer of e- s from NADH to CoQ. • Complex II : Succinate-Q reductase transfers e- s from succinate to CoQ. • Complex III : Q-cytochrome C oxidoreductase transfers e- s from ubiquinol ( reduced form of CoQ) to cytochrome C • Complex IV : cytochrome C oxidase transfers e- s from Cytochrome C to O2 13
  13. 13. 14
  14. 14. The 4 complexes consist of several different electron carriers. • Flavoproteins – Prosthetic group FMN or FAD • Heme containing proteins; Cytochromes subclassified – cytochrome b,c1,c,a & a3. a & a3 can directly react with molecular oxygen and are associated with copper in Complex IV. • Non-heme iron proteins; Iron-sulfur proteins contained bound inorganic Fe and S. • Copper 15
  15. 15. Two mobile e- carriers in ETC • Ubiquinone (Co Q) UQ is lipid-soluble can accept electrons from FMNH2 / FADH2 & transfer them to cytochrome. • Cytochrome C Cytochrome C is a water-soluble mobile electron carrier of the outer face of the inner membrane 16
  16. 16. PATHWAY OF ETC  Complex I :Electrons transferred from NADH to FMN, yielding FMNH2 .  Then from FMNH2 to iron sulfur centers.  Eventually 2 electrons are transferred to ubiquinone.  Electron movement accompanied by a net movement of protons from the matrix to the intermembrane space.  During oxidation of 1 NADH and transfer of 2 e- s to UQ by complex I , 4 protons are also pumped across the mitochondrial membrane.  UQ is lipid-soluble and shuttles electrons between ETC complexes along the inner mitochondrial membrane 17
  17. 17. 18
  18. 18. • Complex II ( succinate-UQ reductase/ succinate dehydrogenase) contains 4 pp chain; the 1st two constitute the SDH; a Krebs cycle enzyme that catalyzes: Succinate + FAD Fumarate + FADH2 SDH contains Fe-S centre and covalently bound FAD. eventually e- s are transferred to UQ 19
  19. 19. • Complex III (cytochrome b1 complex) transfers electrons from reduced UQ (UQH2) to cytochrome c. • Complex III contains b & c1 type of cytochromes and Fe-S protein called Rieske’s centre. UQH2  Cyt b Fe-S  Cyt c1  Cyt c • Cytochrome c is a water-soluble mobile electron carrier of the outer face of the inner membrane. • The transfer of e- s through Complex III is best explained by Q cycle mechanism in which 4 H+ are translocated across the IMM per 2 e- s transferred from UQH2 to Cyt c. 20
  20. 20. 21
  21. 21. • Complex IV catalyzes the transfer of electrons from Cyt c to O2 one at a time. • Complex IV contains Cyt a,a3 and 2 copper centres; CuA and CuB • Oxygen is tightly bound between heme a3 and copper during its reduction and released only after its complete reduction to water. Cyt c CuA  cyt a  a3-CuB. • Finally four electrons and four protons (from matrix) are passed to O2 to form H2O. • In addition to protons req. for water formation, cytochrome oxidase also pumps additional protons across the membrane; during the transfer of 2 electrons to O2 , 2 H+ s are released to the cytosol for every 4 H+ s taken up from the matrix. 22
  22. 22. 23
  23. 23. Flow of electrons through the respiratory chain complexes, showing the entry points for reducing equivalents from important substrates. 24
  24. 24. Release of free energy during electron transfer • The e- transfer from e- donor to e- acceptor is accompanied by the release of free energy. • e- s can be transferred in different forms, e.g. as hydride ions (:H- ) to NAD+ , as hydrogen atoms (.H) to FMN, CoQ & FAD or as electron (.e- ) to cytochromes. 25
  25. 25. Redox pairs/ Redox couple • The reduced and oxidised forms of the same carrier are together referred to as the redox pairs. E.g. NADH/ NAD+ , CoQH2/ CoQ etc. • Redox pairs differ in their tendency to lose e- s .This is a characteristic of a particular redox pair & quantitatively specified by a constant E0 , the standard reduction potential ( in Volts). • The e- s always flow from the –ve to +ve redox potential. 26
  26. 26. Oxidative phosphorylation • The energy released during the electron transfer reactions is used to pump protons across the IMM. • This creates a proton & a charge gradient ( inside becomes more alkaline and more –vely charged)  electrochemical gradient. • The energy stored in electrochemical gradient is termed “ proton motive force” which is used to drive the ATP synthesis by movement of protons down the electrochemical gradient through the ATP synthase. • The process called chemiosmosis, was proposed originally by Prof. Peter Mitchel ( NP 1978) 27
  27. 27. • The chemiosmotic coupling theory explains how oxidative phosphorylation links the ETC and ATP synthesis.  The Chemiosmotic Theory 1. As electrons pass through the ETC, protons are pumped into the intermembrane space, generating proton motive force 2. Protons move back across the membrane through ATP synthase driving ATP formation 28
  28. 28. 29
  29. 29. Machinery for ATP synthesis; ATP synthase (Complex V)  ATP synthase is a multiprotein enzyme  Utilizes the electrochemical gradient across the IMM for ATP synthesis  Also c/a ATPase – isolated enzyme is capable of catalyzing hydrolysis of ATP ADP + Pi  Structure  Consists of 2 domains : F1 unit (ATP synthase) and F0 unit (transmembrane channel) 30
  30. 30. Structure of ATP synthase • F1 domain: - Peripheral enzyme complex bound to IMM. - Involved in catalysis of ATP synthesis - Consists of 5 non-identical subunits (α,β,ϒ,δ &ε) α,β binding site for ATP, ADP ϒ  forms central core δ  attach F1 to IMM • F0 domain - Embedded in the membrane - contains a transmembrane channel ( Proton pumped across the IMM during electron transfer flow back into the matrix through this. - 3 subunits : a,b,c 31
  31. 31. Synthesis of the ATP occurs on the surface of F1 domain • Rxn enz:ADP + Pi enz:ATP • The results of isotopic-exchange experiments unexpectedly revealed that enzyme-bound ATP forms readily on the surface of F1 domain even in the absence of a proton-motive force. • However, ATP does not leave the catalytic site unless protons flow through the enzyme. • Thus, the role of the proton gradient is not to form ATP but to release it from the synthase. • For the continuous synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP. 32
  32. 32. Binding change mechanism for ATP synthesis • Suggests that the 3 β subunits of the ATP synthase adopt different conformations that change during catalysis with only one β subunit acting as the catalytic site. • Conformations adopted by β subunit Open conformation (O) with a low affinity for ligands & is empty. Loose conformation (L) with a low affinity for ligands & is inactive. Tight conformation (T) with a high affinity for ligands & is active. ATP synthesis occurs on the surface of the β subunit in the T conformation. 33
  33. 33. Rotation catalysis is the key to binding change mechanism for ATP synthesis • Paul Boyer proposed the rotational catalysis mechanism in which the 3 active sites (β) of F1 take turns catalyzing ATP synthesis. • Passage of the protons through the F0 “pore” causes the cylinder of c subunits & the attached ϒ subunits to rotate about the long axis of ϒ which is perpendicular to the plane of the membrane. • With each 1200 rotation, β adopts an another conformation. (β- empty β-ADP β-ATP) OR ( OLT) In L conformation β binds to ADP & Pi In T conformation there is synthesis of ATP from ADP & Pi In O conformation there is release of ATP 34
  34. 34. 35 FIGURE 19–24 Binding-change model for ATP synthase. At any given moment, one of these sites is in the β–ATP conformation, a second is in the β-ADP conformation, and a third is in the β- empty conformation. The proton motive force causes rotation of the central shaft—the subunit, shown as a green arrowhead—which comes into contact with each subunit pair in succession. This produces a cooperative conformational change in which the β-ATP site is converted to the β-empty conformation, and ATP dissociates; the β-ADP site is converted to the β–ATP conformation, which promotes condensation of bound ADP Pi to form ATP; and the β-empty site becomes a β-ADP site, which loosely binds ADP Pi entering from the solvent.
  35. 35. Transporters on IMM • Transport cytosolic ADP and Pi across the IMM to mitochondrial matix. & newly synthesized ATP to the cytosol. Adenine nucleotide translocase : Catalyses 1:1 exchange of ATP for ADP Phosphate translocase: transports cytosolic phosphate into the matrix along with a proton in 1:1 ratio Both require the proton gradient to transport these molecules. 36
  36. 36. ATP production and P:O ratio P- referring to a high energy phosphate bond being synthesized. O- referring to an atom of O being reduced. P:O ratio is defined as the number of ATPs generated during the transfer of 2 electrons through a segment of ETC. 3 energy conserving segments on ETC ( Phosphorylation sites) This results in the release of sufficient energy for synthesis of ATP Site I : between NADH & UQ Site II: between UQH2 & Cytc Site III: between Cyt c & O2 37
  37. 37. 3 sites correspond to Complex I, III & IV of the ETC. Since complex I is bypassed when substrate is succinate ( with FADH2 ) only 2 ATPs are generated ( P:O ratio=2) for NADH P:O ratio =3 However, since 10 protons are pumped across IMM during transfer of 2 electrons from NADH to O2 & 4 protons are req. for synthesis of ATP. So, Actual P:O ratio for NADH = 2.5 for FADH2 = 1.5 38
  38. 38. Regulation of the oxidative phosphorylation • Oxidative phosphorylation is regulated by cellular energy demands. • The intracellular [ADP] and the mass-action ratio [ATP]/([ADP][Pi]) are measures of a cell’s energy status. Normally this ratio is very high, so the ATP-ADP system is almost fully phosphorylated. • When the rate of some energy-requiring process (protein synthesis, for example) increases, the rate of breakdown of ATP to ADP and Pi increases, lowering the mass-action ratio. • With more ADP available for oxidative phosphorylation, the rate of respiration increases, causing regeneration of ATP. This continues until the mass-action ratio returns to its normal high level, at which point respiration slows again. 39
  39. 39. Many poisons inhibit the respiratory chain • Barbiturates such as amobarbital inhibit electron transport via Complex I by blocking the transfer from Fe-S to Q. At sufficient dosage, they are fatal in vivo. • Antimycin A and dimercaprol inhibit the respiratory chain at Complex III • The classic poisons H2S, carbon monoxide, and cyanide inhibit Complex IV and can therefore totally arrest respiration • Malonate is a competitive inhibitor of Complex II. • Atractyloside inhibits oxidative phosphorylation by inhibiting the transporter of ADP into and ATP out of the mitochondrion • The antibiotic oligomycin completely blocks oxidation and phosphorylation by blocking the flow of protons through ATP synthase 40
  40. 40. Electron Transport Inhibitors  Several molecules specifically inhibit the electron transport process  When electron transport is inhibited, O2 consumption is reduced or eliminated  Important for understanding the correct order of ETC components • CN– = cyanide ; CO = carbon monoxide; H2S = hydrogen sulfide ; NaN3 = sodium azide ; FMN = flavin mononucleotide ;FAD = flavin adenine dinucleotide ; CoQ = coenzyme Q; Cyto = cytochrome 41
  41. 41. 42
  42. 42. 43
  43. 43. Uncouplers • Oxidation & phosphorylation are coupled processes. They can be uncoupled from each other by certain compunds called uncouplers. • Primary action of the uncouplers - increase permeability of the IMM to protons. • As a result, relatively free movement of the protons across the IMM occurs, which prevent building of the electrochemical gradient stops ATP generation. • E.g. Of uncouplers DNP : lipophilic ( freely move across IMM) can carry a proton along with, so prevent builing up of proton gradient. Other e.g. Pentachorophenol, dinitrocresol, trifluorocarbonylcyanide 44
  44. 44. Significance of uncoupling • Uncoupling proteins (UCP) occur in the inner mitochondrial membrane of mammals, including humans. • Brown adipose tissue which are specialised to carry out oxidation uncoupled from phosphorylation. • When fats are oxidised in the brown adipose tissue, the energy liberated is not trapped as ATP instead released as heat. • UCP1, also called thermogenin is responsible for the activation of fatty acid oxidation and heat production in the brown adipocytes of mammals. • Brown fat, unlike the more abundant white fat, wastes almost ninety percent of its respiratory energy for thermogenesis in response to cold, at birth, and in hibernating animals. 45
  45. 45. Inherited defects in oxidative phosphorylation • 13 of the approximately 100 polypeptides required for oxidative phosphorylation are coded for by mitochondrial DNA • whereas the remaining mitochondrial proteins are synthesized in the cytosol and transported into mitochondria. • mtDNA has a mutation rate about ten times greater than that of nuclear DNA. • mtDNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg. • Tissues with the greatest ATP requirement (for example, CNS, skeletal and heart muscle, kidney, and liver) are most affected by defects in oxidative phosphorylation. • Mutations in mtDNA are responsible for several diseases, including some cases of mitochondrial myopathies - Leber's hereditary optic neuropathy, a disease in which bilateral loss of central vision occurs as a result of neuroretinal degeneration, including damage to the optic nerve. 46
  46. 46. Thank-you 47

Editor's Notes

  • This proton gradient is largely but not exclusively responsible for the mitochondrial membrane potential (ΔΨM). It allows ATP synthase to use the flow of H+ through the enzyme back into the matrix to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. Complex I (NADH coenzyme Q reductase; labeled I) accepts electrons from the Krebs cycle electron carrier nicotinamide adenine dinucleotide (NADH), and passes them to coenzyme Q (ubiquinone; labeled Q), which also receives electrons from complex II (succinate dehydrogenase; labeled II). Q passes electrons to complex III (cytochrome bc1 complex; labeled III), which passes them to cytochrome c (cyt c). Cyt c passes electrons to Complex IV (cytochrome c oxidase; labeled IV), which uses the electrons and hydrogen ions to reduce molecular oxygen to water.
  • Together, oxidative phosphorylation and photophosphorylation account for most of the ATP synthesized by most organisms most of the time
  • The electrons flow from electronegative potential (-0. 32) to electropositive potential (+0.82)
  • The mitochondrial matrix, enclosed by the inner membrane, contains the pyruvate dehydrogenase complex and the enzymes of the citric acid cycle, the fatty acid B-oxidation pathway, and the pathways of amino acid oxidation-all the pathways of fuel oxidation except glycolysis, which takes place in the cytosol.
  • Malate aspartate shuttle: occurs in liver, kidney and heart
    Glycerol 3 phosphate shuttle: occurs in skeletal and heart muscle
  • The citric acid cycle enzyme succinate dehydrogenase, which generates FADH2 with the oxidation of succinate to fumarate is part of the succinate-Q reductase complex (Complex II), an integral membrane protein of the inner mitochondrial membrane. FADH2 does not leave the complex. Rather, its electrons are transferred to Fe-S centers and then to Q for entry into the electron-transport chain.
  • Complex I is a large L-shaped multisubunit protein that catalyzes electron transfer from NADH to Q, coupled with the transfer of four H+ across the membrane:
  • Reduced QH2 (lipid soluble) freely diffuses within the membrane
    NADH oxidized to NAD+ by reducing FMN to FMNH2
  • 3 major enzyme systems that transfer their electrons to complex II are succinate dehydrogenase, fatty acyl CoA dehydrogenase and mitochondrial glycerol phosphate dehydrogenase. no protons are transported to the intermembrane space in this pathway. Therefore, the pathway through complex 2 contributes less energy to the overall electron transport chain process.
  • Q and cyt c are mobile components of the system as indicated by the dotted arrows. The flow through Complex III (the
    Q cycle) is shown in more detail in Figure 13–6. (cyt, cytochrome; ET F, electron transferring flavoprotein; Fe-S, iron-sulfur protein; Q, coenzyme Q
    or ubiquinone.)
  • Much information about the respiratory chain has been obtained by the use of inhibitors, and, conversely, this has provided knowledge about the mechanism of action of several poisons (Figure 13–9). They may be classified as inhibitors of the respiratory chain, inhibitors of oxidative phosphorylation, or uncouplers of oxidative phosphorylation
  • Uncouplers impede ATP generation but have no effect on the electron transfer. Some endogenous compounds (free fatty acids, bilirubin, thyroxine) can act as uncouplers at concentration well above the physiological range.

  • Thermogenin (or the uncoupling protein) is a physiological uncoupler found in brown adipose tissue that functions to generate body heat, particularly for the newborn and during hibernation in animals.