1. 1 Chapter 19 Oxidative Phosphorylation

2. 2 Chapter 16 Chapter 14 Chapter 19

3. 3 Electron transport and oxidative phosphorylation are membrane- associated processes Bacteria---- electron transport and oxidative phosphorylation are carried out at and across plasma membrane Eukaryotic cells---mitochondria (Plants---chloroplasts)

4. 4 1. TCA, fatty acid oxidation, electron transport and oxidative phosphorylation occur in mitochondria 2. Mammalian contains 800 to 2500 mitochondria, some types of cells have one or two, and some have as many as half million, human erythrocytes have none. 3. Outer membrane, intermembrane space, inner membrane, cristae, matrix 4. Genetic materials Mitochondria

5. 5 Permeable to Mr. 5,000 Porin 37 genes, 13 encoding ETC proteins (most mitochondrial proteins are encoded by nucleus and then transported into mitochondria)

6. 6 Electrons are funneled to universal electron acceptors Flavoproteins---FAD or FMN containing proteins (see below) NAD-linked dehydrogenase and NADP-linked dehydrogenase Reduced substrate+NAD + →Oxidized substrate+NADH + H + Reduced substrate+NADP + →Oxidized substrate+NADPH + H +

7. 7 Not metabolically interchangeable NADH : use the free energy of metabolite oxidation to synthesize ATP NADPH : use the free energy of metabolite oxidation for otherwise energy endergonic reductive biosynthesis NADH & NADPH

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9. 9 The Electron Transport Chain— An Overview The electron transport chain can be isolated in four complexes---- Complex I: NADH::Ubiquinone oxidoreductase, Complex II: Succinate dehydrogenase, Complex III: ubiquinone-cytochrome c oxidoreductase Complex IV: cytochrome c oxidase

10. 10 Flavoprotein--Tightly bound FMN or FAD, one or two e- carrier Coenzyme Q, also called ubiquinone (abbr. CoQ or Q)--one or two e- carrier, mobile within membrane cytochromes--- Fe 2+ /Fe 3+, one e- carrier –cytochrome a’s: Isoprenoid (15-C) on modified vinyl and formyl in place of methyl –cytochrome b’s: Iron-protoporphyrin IX –cytochrome c’s: Iron-protoporphyrin IX linked to cysteine iron-sulfur proteins--- one e- carrier, several types coppers--- one e- transfer, Cu+/Cu++ Electron Carriers

11. 11 Fig. 19-2

12. 12 Prosthetic groups of cytochromes Fig. 19-3

13. 13 Iron-sulfur centers Fe 2Fe-2S 4Fe-4S

14. 14 Determining the sequence of electron flow Standard reduction potential (table 19-2) Reducing the entire chain of carrier experimentally by providing an electron source but no electron acceptor (no O2). Inhibitors of electron transport chains. Page 1 of 3

15. 15 Reduction potentials Redox couples that have large positive reduction potential have a strong tendency to accept electrons, and the oxidized form of such a couple is a strong oxidizing agent. Redox couples with large negative reduction potentials have a strong tendency to undergo oxidation, and the reduced form of such a couple is a strong reducing agent.

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18. 18 Determining the sequence of electron flow Standard reduction potential (table 19-2) Reducing the entire chain of carrier experimentally by providing an electron source but no electron acceptor (no O2). Inhibitors of electron transport chains. Page 2 of 3

19. 19 1.Reducing the entire chain of carrier experimentally by providing an electron source but no electron acceptor (no O 2 ). When O 2 is suddenly introduced into the system, the rate at which each electron carriers oxidized (measured spectroscopically) shows the order in which the carriers function. 2. The carrier nearest O 2 (at the end of the chain) gives up its electrons first, the second carrier from the end is oxidized next, and so on.

20. 20 Determining the sequence of electron flow Standard reduction potential (table 19-2) Reducing the entire chain of carrier experimentally by providing an electron source but no electron acceptor (no O2). Inhibitors of electron transport chains. Page 3 of 3

21. 21 FIGURE 19–6 Method for determining the sequence of electron carriers. This method measures the effects of inhibitors of electron transfer on the oxidation state of each carrier. In the presence of an electron donor and O 2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), and those after the block become oxidized (pink).

22. 22 Electron carriers function in multienzyme complexes The electron transport chain can be isolated in four complexes---- Complex I: NADH::Ubiquinone oxidoreductase, Complex II: Succinate dehydrogenase, Complex III: ubiquinone-cytochrome c oxidoreductase Complex IV: cytochrome c oxidase

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24. 24 Complex I: NADH::Ubiquinone oxidoreductase, (NADH dehydrogenase) Transfer one pair of e- from NADH to coenzyme Q Reaction steps: (1) transfer 2e- from NADH to [FMN] (2) transfer e- from [FMNH2] to Fe-S cluster, (3) transfer e- from Fe-S cluster to coenzyme Q. Four H+ transported per 2 e- passes from NADH to UQ. Coenzyme Q is a mobile electron carrier which shuttles e- from Complexes I and II to III. P (for positive, intermembrane space) face and N (for negative, matrix) face Protons pumped from matrix to cytosol

25. 25 Inhibitors: Amytal, rotenone and pieridin A Fig. 19-9

26. 26 The operation of respiratory complex I

27. 27 Complex II: succinate-coenzyme Q reductase (succinate dehydrogenase) The only TCA cycle enzyme that is an integral membrane protein, also called flavoprotein (FP2). Succinate →fumarate + 2H + + 2e- UQ + 2H + + 2e- → UQH2 Components: FAD and Fe-S centers No proton pumped Similar complexes –Glycerolphosphate dehydrogenase: Reduces CoQ: No protons pumped –Fatty acyl-CoA dehydrogenase:Reduces CoQ: No protons pumped

28. 28 Fig. 19-8

29. 29 The operation of respiratory complex II

30. 30 Complex III: Coenzyme Q-cytochrome c reductase CoQ passes electrons to cyt c (and pumps H + ) in a unique redox cycle known as the Q cycle The principal transmembrane protein in complex III is the b cytochrome - with hemes b L and b H Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents Study Figure 19.11 - the Q cycle UQH 2 is a lipid-soluble electron carrier cyt c is a water-soluble electron carrier

31. 31 Q cycle First half reaction: A molecule UQH2 diffuses to Qp site on Complex III, an e- from UQH2 is transferred to the Rieske protein and then to cytochrome c1. This releases two H+ to the cytosol and leaves UQ. -. The second e- is then transferred to the b L heme, converting UQ. - to UQ: Second half reaction: oxidation of UQH2 at Q N site, one e- being passed to cytochrome c1 and the other transferred to heme b L and then to b H.

32. 32 Fig. 19-12

33. 33 The operation of respiratory complex III

34. 34 Complex IV: cytochrome c oxidase Electrons from cyt c are used in a four- electron reduction of O 2 to produce 2H 2 O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! Cytochrome c oxidase utilizes 2 hemes (a and a 3 ) and 2 copper sites Structure is now known - mostly! Complex IV also transports H +

35. 35 Path of e- through complex IV Fig. 19-14

36. 36 The operation of respiratory complex IV

37. 37 Summary of the flow of electrons and protons through the four complexes of the respiratory chain Fig. 19-15

38. 38 Coupling e - Transport and Oxidative Phosphorylation (ATP synthesis) This coupling was a mystery for many years Many biochemists squandered careers searching for the elusive "high energy intermediate" Peter Mitchell proposed a novel idea - a proton gradient across the inner membrane could be used to drive ATP synthesis Mitchell was ridiculed, but the chemiosmotic hypothesis eventually won him a Nobel prize Be able to calculate the  G for a proton gradient (Figure 19-15)

39. 39 Fig. 19-17

40. 40 Mitchell’s chemiosmotic hypothesis Proton gradient used to drive ATP synthesis Protons per electron pair –From succinate 6 –From NADH 10 Four protons per ATP –ADP uptake: 1 proton –ATP synthesis: 3 protons One oxygen atom consumed per electron pair P/O: the number of ATP produced per atom of oxygen consumed. Used as an index of oxidative phosphorylation. –From NADH: 2.5 –From succinate: 1.5

41. 41 P/O ratio ADP + P i  ATP P/O: the number of ATP produced per atom of oxygen consumed. Used as an index of oxidative phosphorylation. –From NADH: 2.5 ATP –From succinate: 1.5 ATP

42. 42 ATP synthase Two parts: F 1 and F  (latter was originally "F-o" for its inhibition by oligomycin) ---F 1 : ATP synthesis,  ---F o : Proton channel in inner membrane, ab 2 c 10-12 Binding change mechanism: the three catalytic b subunits are intrinsically identical but are not functionally equivalently at any particular moment where they cycle through three conformational states. 18 O exchange experiment: the role of proton gradient is not to form ATP but to release it from the enzyme Proton diffusion through the protein drives ATP synthesis!

43. 43 Mitochondrial ATP synthase complex Fig. 19-23

44. 44 Mitochondrial ATP synthase complex Fig. 19-23

45. 45 Catalytic mechanism of F 1 : 18 O exchange experiment Figure 19-21

46. 46 Binding change model for ATP synthesis Fig. 19-24

47. 47 Adenine nucleotide and phosphate translocase ATP must be transported out of the mitochondria Adenine nucleotide translocase ATP-ADP antiporter located in inner mitochondrial membrane But ATP 4- out and ADP 3- in is net movement of a negative charge out - equivalent to a H + going in The proton-motive force drives ATP-ADP exchange Phosphate translocase Symport of H 2 PO 4 - and H + located in inner mitochondrial membrane Consume some of the energy of e- transport (H + moved from P to the N side of the inner membrane)

48. 48 Fig. 19-26 There is no net flow of charge during symport of H 2 PO 4 and H +

49. 49 Shuttle Systems for e - Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane What to do?? "Shuttle systems" effect electron movement without actually carrying NADH Glycerol 3-phosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD Malate-aspartate shuttle uses malate to carry electrons across the membrane

50. 50 Malate aspartate shuttle Liver, kidney and heart Fig. 19-27

51. 51 Glycerol 3-phosphate shuttle Skeletal muscle and brain Fig. 19-28

52. 52 Net Yield of ATP from Glucose It depends on which shuttle is used!

53. 53 Mitochondria are from mother side Fig. 19-32

54. 54 Mutations in mitochondria genes cause human disease Leber’s hereditary optic neuropathy(LHON) –Central nerve system defect, including optic nerves, causing bilateral loss of vision in early childhood –A single base change in the mitochondrial gene in complex I –A single base change in the mitochondrial gene in cyto. b in complex III Myoclonic epilepsy and ragged-red fiber disease (MERRF) –Leucyl-tRNA defect –Characterized by uncontroled muscular jerking –Paracrystalline structure –Also one of the causes of adult-onset diabetes mellitus Aging –DNA damage

55. 55 Inhibitors of electron transport and oxidative phosphorylation Complex IV Complex III Complex I or III

56. 56 Uncouplers: Stimulate e- transport: short circuit proton gradient: Block ATP production Carry protons from cytosolic surface of the membrane and then carry them back to matrix. Proton ionophores: Lipid soluble substance with dissociable proton, eg., 2,4 dinitrophenol (DNP) and FCCP. Thermogenin: uncoupler protein: Generates heat using proton gradient.

57. 57 Fig. 19-20

58. 58 Heat generation by uncoupled mitochondria Brown fat thermogenesis Brown fat, rich in mitochondria and cytochromes Thorax near shoulders Hibernating animals · Produces heat instead of ATP Protein thermogenin is an “uncoupler” (uncoupling protein) Creates a H+ pore in inner mitochondrial membrane