1. Lecture Connections 19 | Oxidative Phosphorylation and Photophosphorylation © 2009 Jim-Tong Horng

2. CHAPTER 19 Oxidative Phosphorylation and Photophosphorylation Electron transport chain in mitochondria Building up the the proton-motive force Synthesis of ATP in mitochondria Key topics:

3. Energy from Reduced Fuels is Used to Synthesize ATP in Animals Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell Electrons from reduced fuels used to reduce NAD + to NADH or FAD to FADH 2. In oxidative phosphorylation, energy from NADH and FADH 2 are used to make ATP

5. Oxidative Phosphorylation Electrons from the reduced cofactors NADH and FADH 2 are passed to proteins in the respiratory chain In eukaryotes, oxygen is the ultimate electron acceptor for these electrons Energy of oxidation is used to phosphorylate ADP

7. Chemiosmotic Theory How to make an unfavorable ADP + P i = ATP possible? Phosphorylation of ADP is not a result of a direct reaction between ADP and some high energy phosphate carrier Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient The electrochemical gradient is established by transporting protons against the electrochemical gradient during the electron transport

8. Chemiosmotic Energy Coupling Requires Membranes The proton gradient needed for ATP synthesis can be stably established across a topologically closed membrane –Plasma membrane in bacteria –Cristae membrane in mitochondria –Thylakoid membrane in chloroplasts Membrane must contain proteins that couple the “downhill” flow of electrons in the electron transfer chain with the “uphill” flow of protons across the membrane Membrane must contain a protein that couples the “downhill” flow of proton to the phosphorylation of ADP

9. Structure of a Mitochondrion The proton gradient is established across the inner membrane The cristae are convolutions of the inner membrane and serve to increase the surface area

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

11. Metabolic Sources of Reducing Power

12. 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 +

13. Electrons are funneled to universal electron acceptors

14. 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

15. Coenzyme Q or Ubiquinone Ubiquinone is a lipid-soluble conjugated dicarbonyl compound that readily accepts electrons Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side

17. Cytochrome c Cytochrome c is a soluble heme-containing protein in the intermembrane space Heme iron can be either ferrous(Fe 3+, oxidized) or ferric(Fe 2+, reduced) Cytochrome c carries a single electron from the cytochrome bc 1 complex to cytochrome oxidase

19. Iron-Sulfur Centers Found in several proteins of electron transport chain, including NADH:ubiquinone oxidoreductase Transfers one electron at a time

21. 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

22. NADH:Ubiquinone Oxidoreductase a.k.a. Complex I One of the largest macro-molecular assemblies in the mammalian cell Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes NADH binding site in the matrix side Non-covalently bound flavin mononucleotide (FMN) accepts two electrons from NADH Several iron-sulfur centers pass one electron at the time toward the ubiquinone binding site

24. The operation of respiratory complex I

25. NADH:Ubiquinone Oxidoreducase is a Proton Pump Transfer of two electrons from NADH to uniquinone is accompanied by a transfer of protons from the matrix (N) to the inter-membrane space (P) Experiments suggest that about four protons are transported per one NADH NADH + Q + 5H + N = NAD + + QH 2 + 4 H + P Reduced coenzyme Q picks up two protons

26. NADH:Ubiquinone Oxidoreducase is a Proton Pump Amytal (a barbiturate drug), rotenone (a plant product commonly used as an insecticide), and piericidin A (an antibiotic) inhibit electron flow from the Fe-S centers of Complex I to CoQ

28. Succinate Dehydrogenase a.k.a. Complex II FAD accepts two electrons from succinate Electrons are passed, one at a time, via iron- sulfur centers to ubiquinone that becomes reduced QH 2

29. 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

31. Cytochrome bc 1 Complex a.k.a. Complex III Uses two electrons from QH 2 to reduce two molecules of cytochrome c CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle

32. The Q Cycle Experimentally, four protons are transported across the membrane per two electrons that reach cyt. c Two of the four protons come from QH 2 The Q cycle provides a good model that explains how two additional protons are picked up from the matrix

34. Cytochrome Oxidase a.k.a. Complex IV Mammalian cytochrome oxidase is a membrane protein with 13 subunits Contains two heme groups Contains copper ions –Two ions (Cu A ) form a binuclear center –Another ion (Cu B ) bonded to heme forms Fe- Cu center

36. Cytochrome Oxidase Passes Electrons to O 2 Four electrons are used to reduce one oxygen molecule into two water molecules Four protons are picked up from the matrix in this process Four additional protons are passed from the matrix to the inter-membrane space by an unknown mechanism

37. Summary of the Electron Flow in the Respiratory Chain

38. Proton-motive Force The proteins in the electron transport chain created the electrochemical proton gradient by one of the three means: –actively transported protons across the membrane via poorly understood mechanisms –passed electrons to coenzyme Q that picked up protons from the matrix –took electrons from QH 2 and released the protons to the inter-membrane side

40. Chemiosmotic Model for ATP Synthesis Electron transport sets up a proton-motive force Energy of proton-motive force drives synthesis of ATP

42. Mitchell’s chemiosmotic hypothesis Proton gradient used to drive ATP synthesis Protons per electron pair –From succinate (FADH2) 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

43. 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 (FADH2): 1.5 ATP

44. 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. Proton diffusion through the protein drives ATP synthesis!

45. Mitochondrial ATP Synthase Complex The proton-motive force causes rotation of the central shaft  This causes a conformational change within all the three  pairs The conformational change in one of the three pairs promotes condensation of ADP and P i into ATP

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. There is no net flow of charge during symport of H 2 PO 4 and H +

49. ATP Yield From Glucose

50. 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

51. Malate aspartate shuttle Liver, kidney and heart

52. Glycerol 3-phosphate shuttle Skeletal muscle and brain

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

55. 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.

56. 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

57. Chapter 19: Summary The reduced cofactors pass electrons into the electron transport chain in mitochondria Stepwise electron transport is accompanied by the directional transport of protons across the membrane against their concentration gradient The energy in the electrochemical proton gradient drives synthesis of ATP by coupling the flow of protons via ATP synthase to conformational changes that favor formation of ATP in the active site In this chapter, we learned that:

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

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

60. 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