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Section 5: Bioenergetics

Section 5: Bioenergetics. 3. Oxidative phosphorylation: ATP synthesis. 10/14/05. The proton-motive force ( D p ). redox-driven pumping of H + results in D pH since H + is charged, effect includes net transfer of charge, i.e., this H + transport is electrogenic

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Section 5: Bioenergetics

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  1. Section 5: Bioenergetics 3. Oxidative phosphorylation: ATP synthesis 10/14/05

  2. The proton-motive force (Dp) • redox-driven pumping of H+ results in DpH • since H+ is charged, effect includes net transfer of charge, i.e., this H+ transport is electrogenic • combination of DpH & separation of charge (an electric mem-brane potential, Em) is known as the proton-motive force (Dp) • Em: ~ 140 millivolts; more positive outside • pH: ~ 1.4 pH units lower outside than matrix [H+]out ≈ 25 [H+]m • this Dp is a form of energy that is used to make ATP (slides 3-) H2O pHm - 1.4 +140 mV Em pHm 2e– +½O2 MATRIX 1 Alberts et al., Fig. 14-19

  3. Dp & ATP synthesis • this Dp equivalent to DG' of ~5.2 kcal/mol H+ • since 3 H+ used to synthesize 1 ATP, available free energy DG' » 3 × 5.2 » 15.6 kcal/mol ATP • sufficient to produce [ATP]/[ADP] >103 at equilibrium • actual ratio in resting muscle ~20 • device that couples Dpto ATP productioncalled ATP synthase(composed of F0 & F1 units) Alberts et al., Fig. 14-27 2

  4. ATP synthase TL O • F1: catalytic unit • subunits: 33 • each  subunit has 1 ATP synthesizing site •  subunits link  pairs to F0 • F0: transmembrane H+conducting channel • subunits: ab2c10 • H+ flow drives rotation of rotor: c10, , &  • rotation of g drives each b subunit in turn to release tightly bound ATP matrix Fig. 18.27 3

  5. subunit a c subunit H+ path through membrane cytosolichalf-channel asp Fig. 18.34 c ring & a subunit structure • each c subunit has 2 membrane-spanning a helices • midway along 1 helix: aspCOOH↔COO– • a subunit has 2 half-channels H+ path • H+ from cytosol diffuses via half-channel to asp on c ring subunit (c1) • this subunit can now move to interface membrane, allowingc ring to rotate • c9 now interfaces matrix half-channel, allowing H+ to diffuse into matrix matrixhalf-channel c ring subunit a c9 c1 4 Fig. 18.36

  6. H+ flow drives rotation of c ring matrix can rotate clockwise cannot rotate ineither direction 5 Fig. 18.35

  7. ATP synthase H+ TL O • F1: catalytic unit • subunits: 33 • each  subunit has 1 ATP synthesizing site •  subunits link  pairs to F0 • F0: transmembrane H+conducting channel • subunits: ab2c10 • H+ flow drives rotation of rotor: c10, , &  • rotation of g drives each b subunit in turn to release tightly bound ATP H H H H matrix H+ Fig. 18.27 3

  8. Binding-change mechanism of ATP synthesis ATPADP + Pi ATPADP + Pi • rotation of g drives release of tightly bound ATP • 3 active sites cycle through 3 structural states: O, open; L, loose-binding; T, tight-binding • at T site, ADP + Pi ATP, but ATP can’t dissociate • g rotation causes T  O, L  T, O L • as a result of the T  O structural change,ATP can now dissociate from what is now an O site structural changes of the 3 subunits cooperative & concerted Fig 21-28; cf. 18.32  L T O L b3 b3 ADP + Pi ADP + Pi drivesrotationof g ADP + Pi  b1 b1 120° rotation of g(counterclockwise) b2 b2  ATP O T Fig. 18.32 6 T O

  9. Transport across inner mitochondrial membrane • p also drives flow of substances across inner membrane • transported by specific carrier proteins • cotransport: coupled transport of 2 substances • symport: both move in same direction CH3CCOO– + H+O HPO4= + H+ • antiport:each movesin oppositedirection ADP-ATPexchange Alberts et al., Fig. 14-21 voltage gradient (Em)drives ADP-ATPexchange pH gradientdrives phosphateimport pH gradientdrives pyruvateimport 7

  10. Summary of ATP synthesis & translocation of ATP,ADP & Pi 8 Lehninger et al., Fig. 18-24

  11. ATP yield H+ pumped/e– pair H+ used NADH-Q reductase (I) 4 cytochrome reductase (III) 2 cytochrome oxidase (IV) 4 ATP-ADP-Pi transport 1 ATP synthesis 3 net ATP yield/e– pair: • via FADH2 (0 + 2 + 4)/(1 + 3) = 1.5 • via NADH (matrix) (4 + 2 + 4)/(1 + 3) = 2.5 energy efficiency (via NADH): (2.5 × 7.3)/53 = 34% NADH + ½O2 + 2.5ADP + 2.5Pi NAD+ + H2O + 2.5ATP 9

  12. Oxidative phosphorylation: control • Vmax of e– transport chain very high;coupling to H+ transport-ADP phosphorylation limits V • major control factor of Vox phos: [ADP] • Vox phos varies hyperbolically as the concentration of 1 of its substrates (ADP) varies • Vox phosµ [ADP] under resting conditions [ATP]/[ADP] ~20 • as [ADP] rises above its KM, slope diminishes, so that Vox phosapproaches a maximum(saturation) • ATP demand > supply Vox phos [ADP] rest sustainable exercise 10

  13. Inhibitors of oxidative phosphorylation • inhibition of e– transport chain • NADH-Q reductase • amytal blocks e– transfer • e– entry & transfer via FADH2 enzymes unaffected • cytochrome oxidase • CO: FeII form • CN–: FeIII form • in both cases, oxidase prevented from conversion to other form • inhibition of ATP synthesis • oligomycin inhibits synthase • because phosphorylation coupled to oxidation, e– transfer also inhibited 11

  14. Uncoupling • H+ gradient dissipated byleakage of H+ across inner membrane, bypassing synthase • result: Vphosphorylation¯, Voxidation­ • uncouplers • ionophores: small mobile car-riers of ions across membranese.g., dinitrophenol: H+ ionophore • thermogenin: transmembraneprotein in brown adipose tissue function: to generate heat • other mitochondrial proteins:variants may account for differences in weight gain/loss 12 Lehninger et al., Fig 19-28

  15. p, a widely used energy source Electric potential E Active Heat transport production PROTON GRADIENT p Flagellar NADPH rotation synthesis ATP ~P Fig. 21-37

  16. Next:Section 6:Carbohydrate metabolism

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