1. Oxidative Phosphorylation

2. Definition It is the process whereby reducing equivalents produced during oxidative metabolism are used to reduce oxygen to water and produce ATP. It occurs in mitochondria, in eukaryotes.

3. Reducing equivalents: NADH and FADH 2 (nicotinamide adenine dinucleotide and flavin adenine dinucleotide). Oxidative processes: –glycolysis –oxidative decarboxylation of pyruvate to acetyl CoA –TCA cycle, and –fatty acid oxidation

4. The reducing equivalents transfer their electrons through electron transport chain in inner membrane of mitochondria to O 2, a terminal electron acceptor. Releases large amount of free energy, which pumps H + from the matrix across inner mitochondrial membrane, generating a transmembrane H + gradient.

5. Energy from the H + gradient drives the synthesis of ATP, catalyzed by the enzyme ATP synthase. Oxidation (electron transport chain) is coupled to phosphorylation (ATP synthesis) by this transmembrane H + fluxes. This combined process of electron transport and ATP synthesis is called oxidative phosphorylation.

6. Biochemical anatomy of a mitochondrion

7. Mitochondria Fig. 19-1 Lehninger Has two membranes Outer membrane - permeable to small molecules and ions Inner membrane - impermeable to most small molecules and ions Matrix - contains enzymes Cristae - infoldings of the inner membrane

8. Electron transport chain Electrons in NADH or FADH 2 are transferred to oxygen through a series of reactions called the electron transport chain. It is composed of electron transfer cofactors found in inner mitochondrial membrane.

9. Electron transfer cofactors Ubiquinone (coenzyme Q) Iron sulfur proteins Cytochromes

10. Ubiquinone UQ (coenzyme Q) Fig. 19-2 Lehninger UQ - a hydrophobic compound with benzene ring, can diffuse within inner mitochondrial membrane. UQ - can be reversibly reduced in two one- electron reductions forming the semiquinone radical and quinol.

11. Iron sulfur proteins Fig. 19-5 Lehninger Fe-S proteins : – contains iron bound to Cys in the protein. – iron undergoes reversible one-electron reduction from Fe 3+ to Fe 2+.

12. Cytochromes Fig. 19-3 Lehninger Complex organic molecule with Fe attached in the middle. Iron undergoes reversible one-electron reduction from Fe 3+ to Fe 2+.

13. Electron transport chain (Respiratory chain) The electron transport chain is organised into four multi-enzyme complexes: –complex I (NADH-Q reductase) –complex II (Succinate-Q reductase) –complex III (Cytochrome c reductase), and –complex IV (Cytochrome oxidase).

14. Oxidative Phosphorylation

15. Separation of electron carriers Fig. 19-7 Lehninger The electron carriers of the respiratory chain are separated by gentle treatment of the inner mitochondrial membrane with detergents. The resulting mixture is resolved by ion- exchange chromatography into different complexes.

16. Complex I - catalyzes electron transfer from NADH to ubiquinone Complex II - catalyzes electron transfer from succinate to ubiquinone Complex III - carries electrons from ubiquinone to cytochrome c Complex IV - transfers electrons from cytochrome c to O 2

17. Complex I(NADH-Q reductase) –Fig. 19-9 Lehninger –Mass: 850 kDa –no. of sub units: 42 –prosthetic groups: FMN, Fe-S –Overall reaction: –NADH + H + + UQ -----> NAD + + UQH 2 –Oxidised UQ accepts hydride ion (H - ) (two e - and one H + ) from NADH and a H + from the solvent water in the matrix.

18. Flow of electrons NADH FMN Reduced Fe-S UQ NAD + FMNH 2 Oxidised Fe-S UQH 2 NADH ---> FMN ---> Fe-S ---> UQ Flow of 2e - from NADH to UQH 2 leads to the pumping of 4H + from matrix to cytosolic side of inner membrane.

19. Ubiquinol, UQH 2 (reduced form) diffuses in the membrane from complex I to complex III where it is oxidised to ubiquinone, UQ. Flow of e - from complex I ---> ubiquinone - --> complex III is accompanied by movement of protons from mitochondrial matrix to the outer side (cytosolic) of the inner membrane (the intermembrane space).

20. Complex II (succinate dehydrogenase) Fig. 19-8 Lehninger Mass : 140 kDa No. of sub units : 5 Prosthetic groups : FAD, Fe-S Flow of electrons : succinate ---> FADH 2 ---> Fe-S ---> UQ

21. Succinate -----> Fumarate FADH 2 FADH 2 carries electrons from reactions by (i) glycerol phosphate dehydrogenase and (ii) fatty acyl CoA dehydrogenase and transfer it to UQ.  G 0’ standard free energy change is too small, so no H + is pumped out.

22. Complex III cytochrome c reductase Fig. 19-10, 19-11 Lehninger Mass : 250 kDa No. of sub units : 11 Prosthetic groups : heme, Fe-S Flow of electrons : UQ ---> cyt b ---> Fe-S ---> cyt c 1 ---> cyt c catalyses the transfer of electrons from UQ to cytochrome c.

23. Summary of flow of electrons UQ cyt b UQH cyt c 1 Fe-S cyt c (+2) (+3) UQH cyt b UQH 2 cyt c 1 Fe-S cyt c (+3) (+2) UQH 2 + 2cyt c (Fe 3+ ) + 2H + ---> UQ + 2cyt c (Fe 2+ ) + 4H +

24. Net effect: UQH 2 is oxidised to UQ (H + released), cyt c is reduced, and movement of protons from matrix to intermembrane space (4H + ).

25. Complex IV, cytochrome oxidase Fig. 19-13 Lehninger Mass : 160 kDa No. of sub units : 13 Prosthetic groups : heme, Cu A, Cu B. Flow of electrons: cyt a ---> cyt a 3 ---> O 2 transfers electrons from cyt c to O 2 to produce H 2 O.

26. Overall reaction: 4cyt c(Fe 2+ ) + 8H + + O 2 ---> 4cyt c(Fe 3+ ) + 4H + + 2H 2 O

27. Summary of flow of electrons and protons through 4 complexes Fig. 19-14 Lehninger Complexes I & II : transfers e - to UQ UQH 2 passes e - to complex III Complex III passes e - to cytochrome c Complex IV transfers e - from reduced cytochrome c to O 2 e - flow through the complexes is accompanied by H + flow from the matrix to intermembrane space.

28. Inhibitors of ox. Phos.

29. Coupling of electron transport with ATP synthesis Oxidative phosphorylation is a combination of two distinct activities: –(i) The flow of electrons from NADH (or FADH 2 ) to oxygen via electron transport chain. NADH + H + + 1/2O 2 ---> NAD + + H 2 O  G ’o = -nF  E ’o = -220kJ/mol –(ii) The phosphorylation of ADP to ATP. ADP + P i ---> ATP + H 2 O  G ’o = +31kJ/mol

30. Flow of e - from NADH to O 2 Half-reaction (redox reaction) NAD + + H + + 2e - ---> NADH, E ’o = -0.32V 1/2O 2 + 2H + + 2e - ---> H 2 O, E ’o = +0.816V  E ’o = 1.14V  G ’o =-2(96.5kJ/V.mol)(1.14V)=-220kJ/mol Net reaction: exergonic This energy is used to pump H + out of the matrix

31. Phosphorylation of ADP to ATP This reaction is catalyzed by an enzyme in the inner mitochondrial membrane, ATP synthase. The large energy from reaction (i) is collected in the form of proton gradient.

32. Evidence for the coupling of e - transport and ATP synthesis Fig. 19-17 Lehninger Isolated mitochondria are suspended into buffered medium with an O 2 electrode to monitor O 2 consumption. At intervals, samples are assayed for the presence of ATP.

33. Addition of... ADP + P i : no O 2 consumption (no electron flow) and no ATP synthesis. Succinate : e - transported to O 2, H + gradient is generated and ATP is synthesized. CN - : no electron flow, no O 2 consumption and no ATP synthesis. Electron transfer and ATP synthesis are obligatorily coupled, i.e. neither reaction occurs without the other.

34. Addition of... Succinate : no electrons transported (obligatorily coupled), no ATP synthesis. ADP + P i : electrons transported to O 2, and ATP synthesized. Oligomycin : inhibitor of ATP synthase, no ATP synthesis and no e - transported to O 2. Dinitrophenol (DNP) : uncoupler, electrons transported to O 2, no ATP synthesis.

35. Oligomycin : –inhibitor of ATP synthase, flow of protons into matrix is blocked –PMF builds up –when  G for pumping H + out of the matrix is >  G relesed by the transfer of electrons from NADH to O 2. –Electron flow stops and equilibrium is attained.

36. Fig. 19-18 Lehninger DNP, dinitrophenol : –uncoupler, has a dissociable proton and is hydrophobic. –carries proton across inner mitochondrial membrane, dissipates the proton gradient

37. ATP synthesis ATP synthesis is coupled to the dissipation of the proton gradient created during electron transport. The proton flow back into the matrix through the proton pore in ATP synthase. ADP + P i + nH + ---> ATP + H 2 O + nH + intermembrane matrix

38. ATP synthase, complex V ATP synthase is an F-type ATPase. ATP synthase catalyzes the formation of ATP from ADP and P i accompanied by the flow of protons from the intermembrane space to the matrix. ATP synthase has two functional domains: F 0 and F 1.

39. F o - integral membrane complex –sensitive to oligomycin –has a proton pore F 1 - peripheral membrane complex –essential for oxidative phosphorylation –binding site for ATP and ADP

40. Chemiosmotic model Fig. 19-16 Lehninger Proposed by Peter Mitchell (1960) The chemiosmotic theory proposed that ATP synthesis is coupled to the dissipation of the proton gradient created during electron transport.

41. Synthesis of ATP depends on: –(i) Generation of proton gradient as the result of electron transfer through complexes I,III,& IV. –(ii) The inner membrane is impermeable to protons. –(iii) Protons move from the intermembrane space to the matrix space through a membrane pore in ATP synthase and this movement provides the driving force for ATP synthesis

42. (i) produces both chemical gradient,  pH and electrical gradient,  (ii) protons reenter the matrix only through F o. PMF drives protons back into the matrix and provides the energy for ATP synthesis. ATP synthesis is catalyzed by the F 1 complex associated with F o.