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Chapter 21

Chapter 21. Electron Transport and Oxidative Phosphorylation. All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777. Outline.

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Chapter 21

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  1. Chapter 21 Electron Transport and Oxidative Phosphorylation All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 21.1 Electron Transport and Oxidative Phosphorylation at Mitochondrial Membrane • 21.2 Reduction Potentials • 21.3 - 21.7 Details of Electron Transport • 21.8, 21.9 Coupling and the ATP Synthase • 21.10 Inhibitors of Oxidative Phosphorylation • 21.11 Uncouplers Disrupt the Coupling • 21.12-21.14 Final Details

  3. An Overview • Electron Transport: Electrons carried by reduced coenzymes are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane • Oxidative Phosphorylation: The proton gradient runs downhill (favored direction) to drive the synthesis of ATP • It all happens in or at the inner mitochondrial membrane

  4. Reaction Summary Glucose + 2H2O + 10NAD+ + 2FAD + 4ADP + 4Pi 6CO2 + 10NADH + 10H+ + 2FADH2 + 4ATP NADH + H+ + 1/2O2 + 3ADP + 3Pi NAD+ + 3ATP + 4H2O Acetyl-CoA + 3NAD+ + FAD + ADP + Pi 2CO2 + 3NADH + 3H+ + FADH2 + ATP + CoA FADH2 + 1/2O2 + 2ADP + 2Pi FAD + 2ATP + 3H2O

  5. 21.2 Reduction Potentials High positive Eo' indicates a strong tendency to be reduced • Crucial equation: Go' = -nFEo' • Eo' = Eo'(acceptor) - Eo'(donor) • Electrons are donated by the half reaction with the more negative reduction potential and are accepted by the reaction with the more positive reduction potential: Eo’ positive, Go' negative • If a given reaction is written so the reverse is true, then the Eo' will be a negative number and Go' will be positive • Table 21.1 contains reduction potentials

  6. Example a-ketoglutarate + 2H+ + 2e- isocitrate Eo’ = -0.38V NAD+ + H+ + 2e- NADH Eo’ = - 0.32V Spontaneous reaction under standard conditions: NAD+ + isocitrate NADH + a-ketoglutarate DEo’ = Eo’ (e- acceptor) - Eo’ (e- donor) = -0.32V - (-0.38) = +0.06 DGo’ = -nF DEo’ = -11.58 kJ/mol

  7. 21.3 Electron Transport Figures 21.3 and 21.4 hold the secrets • Four protein complexes in the inner mitochondrial membrane • A lipid soluble coenzyme (UQ, CoQ) and a water soluble protein (cyt c) shuttle between protein complexes • Electrons generally fall in energy (negative to positive red. potential through the chain - from complexes I and II to complex IV

  8. 21.4 Complex I NADH-CoQ Reductase • Electron transfer from NADH to CoQ • More than 30 protein subunits - mass of 850 kD • Path: • NADH FMNFe-SUQFeSUQ • Four H+ transported out (i.e., from matrix to cytosol) per 2 e-

  9. 21.5 Complex II Succinate-CoQ Reductase • OR succinate dehydrogenase (from TCA cycle!) • also called flavoprotein 2 (FP2) - FAD covalently bound • four subunits, including 2 Fe-S proteins • Three types of Fe-S cluster: • 4Fe-4S, 3Fe-4S, 2Fe-2S • Path: succinateFADH22Fe2+UQH2 • Net reaction: • succinate + UQ fumarate + UQH2

  10. 21.6 Complex III CoQ-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 bL and bH • Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents • Study Figure 21.12 - the Q cycle • UQH2 is a lipid-soluble electron carrier • cyt c is a water-soluble electron carrier

  11. Cyt c - the mobile e- carrier

  12. 21.7 Complex IV Cytochrome c Oxidase • Electrons from cyt c are used in a four-electron reduction of O2 to produce 2H2O • Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - i.e., The End! • Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 copper sites • Structure is mostly known! • Complex IV also transports H+

  13. Coupling e- Transport and Oxidative Phosphorylation 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 (Equation 21.28)

  14. Free Energy Change for Chemiosmotic Coupling • DG = RT ln [H+out]/H+in] + FDY • DG = -2.303RT(pHout - pHin) + FDY

  15. 21.9 ATP Synthase Proton diffusion through the protein drives ATP synthesis! • Two parts: F1 and F0 (latter was originally "F-o" for its inhibition by oligomycin) • See Figure 21.25 and Table 21.3 for details • Racker & Stoeckenius confirmed Mitchell’s hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin • Paul Boyer’s binding change mechanism won a share of the 1997 Nobel in Chemistry

  16. F1-ATP Synthase

  17. 21.10 Inhibitors of Oxidative Phosphorylation • Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish! • Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe3+) of a3 • Oligomycin and DCCD are ATP synthase inhibitors

  18. 21.11 Uncouplers Uncoupling e- transport and oxidative phosphorylation • Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient • Uncouplers are hydrophobic molecules with a dissociable proton • They shuttle back and forth across the membrane, carrying protons to dissipate the gradient

  19. 21.12 ATP-ADP Translocase ATP must be transported out of the mitochondria • ATP out, ADP in - through a "translocase" • ATP movement out is favored because the cytosol is "+" relative to the "-" matrix • But ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in • So every ATP transported out costs one H+ • One ATP synthesis costs about 3 H+ • Thus, making and exporting 1 ATP = 4H+

  20. 21.13 What is the P/O Ratio?? i.e., How many ATP made per electron pair through the chain? • e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen • 4 H+ flow back into matrix per ATP to cytosol • 10/4 = 2.5 for electrons entering as NADH • For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen • 6/4 = 1.5 for electrons entering as succinate

  21. 21.14 Shuttle Systems for e - NADH from Glycolysis 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 • Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD • Malate-aspartate shuttle uses malate to carry electrons across the membrane

  22. Net Yield of ATP from Glucose It depends on which shuttle is used! • See Table 21.4! • 30 ATP per glucose if glycerol-3-P shuttle used • 32 ATP per glucose if malate-asp shuttle used • In bacteria - no mitochondria - no extra H+ used to export ATP to cytosol, so: • 10/3 = ~3ATP/NADH • 6/3 = ~ 2ATP/FADH2

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