Biological oxidation
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Detail description of Biological oxidation, etc and ATP production process
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Biological oxidation
- 1. BIOLOGICAL OXIDATION Dr. Abhra Ghosh
- 2. • What is oxidation? - Addition of oxygen - Removal of hydrogen • What is biological oxidation? - Oxidation carried out by enzymes. Biological oxidations are restricted to three classes:
- 3. Fe+2 Fe+3 CH3CH2OH CH3CHO CH3CHO CH3COOH -e- -2H+, 2e- +O
- 4. Ared Box Cox Dred Energy Energy Biological oxidation is exergonic and endergonic reaction
- 5. Enzymes responsible for Biological oxidation: 1. Oxidation by direct action of oxygen a. Oxidases - Remove hydrogen from substrate - Oxygen acts as hydrogen acceptor H2O as final product Eg. – Cyto. oxi., Ascor. oxi H2O2 as final product Eg. Urate oxi., Xanthine oxi., Glucose oxi.
- 6. b. Oxygenases: - Incorporate O2 into their substrates. - Not associated with energy production. i) Dioxygenases – Incorporate both atoms of oxygen molecule into substrate Eg. Tryp. 2,3 dioxygenase L-tryptophan + O2 N-formyl-L-kynurenine
- 7. ii) Mono-oxygenase/Hydroxylase: - Incorporate one oxygen atom - Incorporated oxygen atom is present as hydroxyl group - Other oxygen atom gets converted into water Eg. Dopamine monooxygenase, Cyto. P450 mono-oxygenase
- 8. c) Hydroxyperoxidases: - Hydrogen peroxide acts as hydrogen acceptor - Formation of water Eg. Catalase, Peroxidase
- 9. Comparison between Catalase and Peroxidase Catalase Peroxidase Dissimilarities React directly with hyd. pero. Requires reduced glu. Km is higher Km is lower Present where L – amino acid oxidase acts Acts where small amount of hyd. pero. is formed – RB cells, lens Similarities Contains heam Contains heam Decompose hyd. pero. To water and oxygen Decompose hyd. pero. to water and oxygen
- 10. 2. Oxidation as a result of loss of hydrogen: - Remove hydrogen from the substrates - Classified into 4 type a) Aerobic dehydrogenases: - Flavoproteins acts a prosthetic group (FMN, FAD) - Donate the hydrogen to oxygen to form peroxide. Eg. Urate oxidase, Xanthine oxidase
- 11. b) Anaerobic dehydrogenase: - Indirect transfer of electrons to molecular oxygen - A carrier molecule is required i) Pyridine linked dehydrogenase: - Transfers a hydride ion to NAD+ or NADP - Second hydrogen is released as free H+
- 12. ii) Flavin – linked dehydrogenase: - Substrates get oxidized by removing 2H+ and 2e- - Form FMNH2 and FADH2 - They are reoxidised by giving the reducing equivalent to coenzyme Q
- 13. Difference between Aerobic and anaerobic dehydrogenases Aerobic dehydro. Anaerobic dehydro. Can react directly with oxygen Cannot react directly with molecular oxygen Transfers hydrogen/electrons to Fp which is autooxidisable Transfer hydrogen/electreon to NAD+ or Fp which is oxidized in ETC Hydrogen peroxide is produced Hydrogen peroxide is not produced ATP is never produced ATP is produced from NADH+H+ or FpH2 in ETC
- 14. 3. Iron – sulphur protein: - These re non-heam proteins having iron and Sulphur clusters. a) FeS: Single Fe, Co-ordinated with –SH of four cysteine residues b)Fe2S2: Two Fe, two inorganic S, linked with 2 –SH and two S groups. c) Fe4S4: Four Fe, four inorganic S, linked with 1 –SH and three S groups. d)Fe3S4: Three Fe, four inorganic S.
- 15. 4. Cytochromes: - Heam protein - Contains a single Fe atom – when in Fe+3 form – ferricytochrome and when in Fe+2 form – ferrocytochrome - They are identified by their absorption spectra – soret bands Eg. – Cyt c, Cyt c1, Cyt b, Cyt a-a3
- 16. Redox couple/Conjugate redox pair: The pair consisting of the oxidant and reductant forms of an oxidizing or reducing agent is known as a redox couple/conjugate redox pair. The standard redox potential E0 is the measure of tendency of a redox couple to donate or accept electron.
- 17. In mitochondria, hydrogen/electron pass through carriers in seq of increasing order of red. potentials
- 18. When we integrate all the steps of biological oxidation, the free energy change between initial and final product is huge. G0’ = -nFE0’ = - 2X23.06X1.14 kcal/mol = - 52.6 kcal/mol Importance of step wise reaction of Electron transport chain:
- 19. ELECTRON TRANSPORT CHAIN This is the final common pathway in aerobic cells to transfer the electron generated from various substrate to oxygen Location: Inner membrane of mitochondria
- 22. Path of electrons from NADH, succinate, fatty acyl–CoA, and glycerol 3-phosphate to ubiquinone.
- 23. NADH + 5HN + + Q NAD+ + QH2 + 4HP + Exergonic reac. Endergonic reac.
- 26. Importance of Q-cycle: QH 2 passes two electrons to Q-cytochrome c oxidoreductase, but the acceptor of electrons in this complex, cytochrome c , can accept only one electron.
- 31. Synthesis of ATP: Reaction for flow of electron from NADH to O2 is – NADH+1/2 O2 + H+ ≒ H2O + NAD+ G0’ = - 52.6 kcal/mol Reaction for ATP synthesis is – ADP + Pi + H+ ≒ ATP + H2O G0’ = + 7.3 kcal/mol
- 32. Chemiosmotic hypothesis: - By Peter Mitchell - It states that electron transport and ATP synthesis are coupled by a PROTON GRADIENT ACROSS INNER MITOCHONDRIAL MEMB. - Transfer of e- causes transfer of H+ to the intermemb. space causing positive charge there ------------- (1) - This transfer also causes increase in H+ in the intermemb. space causing change in pH -------------(2)
- 33. So, (1) and (2) generates an electrical gradient and a chemical gradient respectively - This combining effect produces “PROTON MOTIVE FORCE” – ATP producing force
- 34. ATP Synthase or Complex V ] F0 F1
- 35. Rotational catalysis and binding change model of ATP synthesis L - form T - form O - form
- 36. How rotational catalysis occurs?
- 37. Inhibitors of ETC Site-I (Complex-I) • Rotenone: A fish poison and also insecticide. Inhibits transfer of electrons through complex-I-NADH-Q-reductase. • Amobarbital (Amytal) and Secobarbital: Inhibits electron transfer through NADH-Q reductase. • Piericidin A: An antibiotic. Blocks electron transfer by competing with CoQ. • Drugs: Chlorpromazine and hypotensive drug like guanethidine.
- 38. Site-II (Complex III) • Antimycin A • BAL (Dimer-Caprol) • Hypoglycaemic drugs Phenformin Site-III (Complex IV) • Cyanide • H2S • Azide • CO (Carbon monoxide): Inhibits Cyt. oxidase by combining with O2 binding site. ]Blocks electron transfer from cyt b to c1 ]Inhibits terminal transfer of electrons to molecular O2
- 39. Complex II: Succinate dehydrogenase FAD • Carboxin • TTFA • Malonate: A competitive inhibitor of succinate dehydrogenase Specifically inhibit transfer of reducing equivalent from succinate dehydrogenase]
- 40. Inhibitors of Complex V: • Oligomycin • Atractyloside
- 41. Uncouplers: • 2, 4 Dinitrophenol • Dicoumarol • Calcium • CCCP: Chlorocarbonyl cyanide phenylhydrazone • FCCP: Trifluorocarbonyl cyanide pheylhydrazone. • Valinomycin • Thyroxin • Thermogenin
- 43. ETC and oxidative phosphorylation is always coupled reaction
Editor's Notes
- Copper containing enzyme transfer the electron from the sustrate to cu+2 and gets reduced to Cu+. It later donates the electron to O2 and form Cu+2 again.
- This enzyme is also known as mixed function oxidase - because two atoms of an oxygen molecule is used for two different reaction.
- Soret bands can be showed by ferrocytochrome only – soret bands are lpha, beta, gamma Cyt a-a3 contains copper also in its active centre, Cyt a-a3 only binds with CO and CN-
- This huge amount of energy cannot be utilized by body if released in a single step. That’s why by step wise manner, the small change in energy can be entrapped in ATP
- Outer membrane is permeable to most of the molecules. Inner membrane is highly selectively permeable.
- Some of the transporter system in inner mitochondrial membrane and their inhibitors. Transporter systems in the inner mitochondrial membrane. (1) Phosphate transporter; (2) pyruvate symport; (3) dicarboxylate transporter; (4) tricarboxylate transporter; (5) α-ketoglutarate transporter; (6) adenine nucleotide transporter. N-Ethyl-maleimide, hydroxycinnamate, and atractyloside inhibit (–) the indicated systems
- Ubiquinone (Q) is the point of entry for electrons derived from reactions in the cytosol, from fatty acid oxidation, and from succinate oxidation (in the citric acid cycle). Electrons from NADH pass through a flavoprotein with the cofactor FMN to a series of Fe-S centers (in Complex I) and then to Q. Electrons from succinate pass through a flavoprotein with the cofactor FAD and several Fe-S centers (in Complex II) on the way to Q. Glycerol 3-phosphate donates electrons to a flavoprotein (glycerol 3-phosphate dehydrogenase) on the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase (the first enzyme of oxidation) transfers electrons to electron-transferring flavoprotein (ETF), from which they pass to Q via ETF : ubiquinone oxidoreductase.
- Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the Fe-S center N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons. The detailed mechanism that couples electron and proton transfer in Complex I is probably involves a Q cycle similar to that in Complex III in which QH2 participates twice per electron pair. Proton flux produces an electrochemical potential across the inner mitochondrial membrane (N side negative, P side positive).
- Electrons flow in Complex I from NADH through FMN and a series of iron–sulfur clusters to ubiquinone (Q), forming Q2−. The charges on Q2− are electrostatically transmitted to hydrophilic amino acid residues (shown as red (glutamate) and blue (lysine or histidine) balls) that power the movement of HL (a long horizontal helix) and bH (b -hairpin-helix connecting elements) components. This movement changes the conformation of the transmembrane helices and results in the transport of four protons out of the mitochondrial matrix.
- This complex (shown here is the porcine heart enzyme) has two transmembrane subunits, C and D; the cytoplasmic extensions contain subunits A and B. Just behind the FAD in subunit A is the binding site for succinate. Subunit B has three Fe-S centers, ubiquinone is bound to subunit B, and heme b is sandwiched between subunits C and D. Two phosphatidylethanolamine molecules are so tightly bound to subunit D that they show up in the crystal structure. Electrons move (blue arrows) from succinate to FAD, then through the three Fe-S centers to ubiquinone. The heme b is not on the main path of electron transfer but protects against the formation of reactive oxygen species (ROS) by electrons that go astray. Causes paraganglioma – a benign tumor of carotid body which senses the oxygen level in blood.
- The two cytochrome subunits of Q-cytochrome c oxidoreductase contain a total of three hemes: two hemes within cytochrome b , termed heme b L (L for low affinity) and heme b H (H for high affinity), and one heme within cytochrome c1 . These identical hemes have different electron affinities because they are in different polypeptide environments. For example, heme b L , which is located in a cluster of helices near the cytoplasmic face of the membrane, has lower affinity for an electron than does heme b H , which is near the matrix side. In addition to the hemes, the enzyme contains an iron–sulfur protein with a 2Fe-2S center. This center, termed the Rieske center, is unusual in that one of the iron ions is coordinated by two histidine residues rather than two cysteine residues. This coordination stabilizes the center in its reduced form, raising its reduction potential so that it can readily accept electrons from QH 2 . The path of electrons through Complex III is shown by blue arrows. The movement of various forms of ubiquinone is shown with black arrows. In the first stage (left), Q on the N side is reduced to the semiquinone radical, which moves back into position to accept another electron. In the second stage (right), the semiquinone radical is converted to QH2. Meanwhile, on the P side of the membrane, two molecules of QH2 are oxidized to Q, releasing two protons per Q molecule (four protons in all) into the intermembrane space. Each QH2 donates one electron (via the Rieske Fe-S center) to cytochrome c1, and one electron (via cytochrome b) to a molecule of Q near the N side, reducing it in two steps to QH2. This reduction also consumes two protons per Q, which are taken up from the matrix (N side). Reduced cyt c1 passes electrons one at a time to cyt c, which dissociates and carries electrons to Complex IV.
- Thus, each proton that is transported out of the matrix to the cytoplasmic side corresponds to 21.8 kJ/mol (5.2 kacal/mol) of free energy. Bovine cytochrome c oxidase is reasonably well understood at the structural level. Cytochrome c oxidase contains two heme A groups and three copper ions, arranged as two copper centers, designated A and B. One center, CuA , contains two copper ions linked by two bridging cysteine residues. This center initially accepts electrons from reduced cytochrome c. The remaining copper ion, Cu B , is bonded to three histidine residues, one of which is modified by covalent linkage to a tyrosine residue. The copper centers alternate between the reduced Cu 1 (cuprous) form and the oxidized Cu 2 1 (cupric) form as they accept and donate electrons. There are two heme A molecules, called heme a and heme a 3 , in cytochrome c oxidase. Heme A differs from the heme in cytochrome c and c1 in three ways. 1. Electrons from two molecules of reduced cytochrome c flow down an electron-transfer pathway within cytochrome c oxidase, one stopping at Cu B and the other at heme a 3 . With both centers in the reduced state, they together can now bind an oxygen molecule. 2. As molecular oxygen binds, it abstracts an electron from each of the nearby ions in the active center to form a peroxide bridge between them. 3. Two more molecules of cytochrome c bind and release electrons that travel to the active center. The addition of an electron as well as H to each oxygen atom reduces the two ion–oxygen groups to Cu B- OH and Fe - OH. 4. Reaction with two more H ions allows the release of two molecules of H 2 O and resets the enzyme to its initial, fully oxidized form.
- The four protons in this reaction come exclusively from the matrix. Thus, the consumption of these four protons contributes directly to the proton gradient. Recall that each proton contributes 5.2 kcal/mol to the free energy associated with the proton gradient; so these four protons contribute 20.8 kcal/mol, an amount substantially less than the free energy available from the reduction of oxygen to water. What is the fate of this missing energy? Remarkably, cytochrome c oxidase uses this energy to pump four additional protons from the matrix to the cytoplasmic side of the membrane in the course of each reaction cycle for a total of eight protons removed from the matrix.
- In oxidative phosphorylation ATP is produced by combining ADP and Pi with the energy generated by the flow of electrons from NADH to molecular oxygen in the electron transport chain. There are three sites in the respiratory chain where ATP is formed by oxidative phosphorylation. These sites have been proved by the free energy changes of the various redox couples. Since hydrolysis of ATP to ADP + Pi releases around 7.3 K. Cal/mole, the formation ATP from ADP + Pi requires a minimum of around 8 KCal/mole. The formation of ATP is therefore not possible at the sites where free energy released is less than 8 KCal/mole. Whenever two systems or redox couple of the respiratory chain differ from each other by 0.22 volts instandard redox potential (E’o), the free energy is sufficient to form ATP.
- Question is, how these two reactions are connected with each other?
- 0.14 volts higher in terms of electrical energy 1.4 units lower in terms of pH – chemical enerfy
- It is a large, complex enzyme resembling a ball on a stick. Much of the “stick” part, called the F 0 subunit, is embedded in the inner mitochondrial membrane. The 85-Å-diameter ball, called the F 1 subunit, protrudes into the mitochondrial matrix. The F 1 subunit contains the catalytic activity of the synthase. In fact, isolated F 1 subunits display ATPase activity. The F 1 subunit consists of five types of polypeptide chains (a 3 , b 3 , gamma, delta, and epsilon ) with the indicated stoichiometry. The a and b subunits, which make up the bulk of the F 1 , are arranged alternately in a hexameric ring; Both bind nucleotides but only the b subunits are catalytically active. Just below the a and b subunits is a central stalk consisting of the gamma and epsilon proteins. The subunit includes a long helical coiled coil that extends into the center of the a 3 b3 hexamer. The subunit breaks the symmetry of the 3 3 hexamer: each of the subunits is distinct by virtue of its interaction with a different face of gamma. Distinguishing the three b subunits is crucial for understanding the mechanism of ATP synthesis. The F 0 subunit is a hydrophobic segment that spans the inner mitochondrial membrane. F 0 contains the proton channel of the complex. This channel consists of a ring comprising from 8 to 14 c subunits that are embedded in the membrane. A single a subunit binds to the outside of the ring. The F 0 and F 1 subunits are connected in two ways: by the central gamma n epsilon stalk and by an exterior column. The exterior column consists of one a subunit, two b subunits, and the delta subunit.
- The F1 complex has three nonequivalent adenine nucleotide–binding sites, one for each pair of alpha and beta subunits. At any given moment, one of these sites is in the beta-ATP conformation (which binds ATP tightly), a second is in the beta-ADP (loose-binding) conformation, and a third is in the beta-empty (very-loose-binding) conformation. The proton-motive force causes rotationof the central shaft—the gamma subunit, shown as a green arrowhead—which comes into contact with each alpha-beta subunit pair in succession. This produces a cooperative conformational change in which the beta -ATP site is converted to the beta-empty conformation, and ATP dissociates; the beta-ADP site is converted to the beta-ATP conformation, which promotes condensation of bound ADP + Pi to form ATP; and the beta-empty site becomes a beta-ADP site, which loosely binds ADP + Pi entering from the solvent. This model, based on experimental findings, requires that at least two of the three catalytic sites alternate in activity; ATP cannot be released from one site unless and until ADP and Pi are bound at the other.
- It binds with the enzyme ATP synthase and blocks the proton channels. It thus prevents the translocation of H+ into the mitochondrial matrix, this leads to accumulation of H+ at higher concentration in intermembrane space. Since protons cannot be pumped out against steep proton gradients, electron transport stops (respiration stops). It is a glycoside, it blocks the translocase that is responsible for movement of ATP and ADP, across the inner mitochondrial membrane. Adequate supply to ADP is blocked thus preventing phosphoglation and ATP formation