2. Metabolic Biochemistry Lecture 8 Aug. 23, 2006 Oxidative Phosphorylation

3. Wild type SDHC

5. Outer membrane Inner membrane Intermembrane space Matrix

7. LNC Fig.19.7

9. the arrangement of the complexes in the inner membrane and the order of electron flow

10. In the presence of an inhibitor, all the complexes upstream of the block are reduced (blue), and all the complexes downstream of the block are oxidized: these determinations can be made by spectroscopic measurements with isolated mitochondria.

11. NADH MEASUREMENTS WITH AN OXYGEN ELECTRODE

12. LNC Fig.19.15 TMPD/ascorbate CN - rotenone malonate antimycin Glutamate/malate succinate Succinate dehydrogenase membrane bound enzyme of Krebs cycle Four integral membrane protein complexes Two mobile carriers: ubiquinone and cytochrome c

13. LNC Fig.19.8

14. oxidized Coenzyme Q or Q reduced Coenzyme Q or QH 2 LNC Fig.19.2 lipid-soluble polyisoprene chain

15. Heme + apoprotein cytochrome LNC Fig.19.3

16. Structure of cytochrome c

17. LNC Fig.19.4

18. NON-HEME IRON - SULFUR CENTERS [Fe 2 -S 2 ] [Fe 4 - S 4 ]

19. LNC Fig.19.5

20. [Fe 2 -S 2 ] LNC Fig.19.5

21. [Fe 4 -S 4 ] LNC Fig.19.5

22. A bacterial ferredoxin

23. NADH O2O2

24. LNC Fig.19.9 7 or 8 NADH + Q + H +  NAD + + QH 2

25. LNC Fig.19.10 Architecture of Succinate Dehydrogenase and Reactive Oxygen Species Generation Victoria Yankovskaya,1* Rob Horsefield,2* Susanna To¨rnroth,3* Ce´sar Luna-Chavez,1,4† Hideto Miyoshi,5 Christophe Le´ger,6‡ Bernadette Byrne,2 Gary Cecchini,1,4§ So Iwata2,3,7§ SCIENCE 31 JANUARY 2003 VOL 299, p.700 www.sciencemag.org [2Fe-2S] [3Fe-4S] [4Fe-4S] succinate Succinate + Q  fumarate + QH 2

26. Complex III 8-11 polypeptides two cytochromes, b and c 1 one iron-sulfur center LNC Fig.19-11 QH 2 + 2 cyt c (Fe +3 )  Q + 2 cyt c (Fe +2 ) (ignore the protons for the moment)

27. LNC Fig.19-11b Complex III 8-11 polypeptides two cytochromes, b and c 1 one iron-sulfur center

28. LNC Fig.19-12 The Q Cycle Net equation QH 2 + 2 cyt c ox + 2H + in  Q + 2 cyt c red + 4H + out 2 Fe +3 2 Fe +2

29. From AY Mulkidjanian BBA 1709: 5-34 (2005)

30. Cyt c 1 red + Cyt c ox  Cyt c 1 ox + Cyt c red Fe +3 Fe +2 

31. LNC Fig19-13a Complex IV - cytochrome oxidase 9 - 13 polypeptides cytochromes a and a 3 two copper centers 4 cyt c (Fe +2 ) + O 2 + 4H+  4 cyt c (Fe +3 ) + 2 H 2 O ( and protons pumped)

33. Complex IV (schematic) LNC Fig.19-14 4 cyt c red + O 2 + 4 H + 4 cyt c ox + 2 H 2 O Fe +2 Fe +3

35. LNC Fig.19.15 Succinate + FAD  fumarate + FADH 2 FAD FMN H+H+ H+H+ H+H+

36. Proton pumping and Storage of Free Energy

37. ++++++ ++++++ ------ ------ Matrix IMS inner membrane LNC 19-6

38.  G = 2.3 RT  pH + 1 x F x   pH = ~ 0.75  ~ 0.15 - 0.2 v = 200 mV  G = ~ +20 kJ/mol (H + ) The oxidation of NADH liberates ~ 220 kJ/mol (NADH) therefore, we can pump ~ 11 protons at 100% efficiency

39. tightly coupled vs uncoupled mitochondria

40. succinate

41. LNC 19-18a

44. Effect of antibiotics valinomycin and nigericin Valinomycin is a K + ionophore it breaks down the membrane potential Nigericin is a H + /K + antiporter it exchanges protons for potassium ions and thus converts a proton gradient into a K + gradient

45. ATP Synthase Complex V

46. F o F 1 ATP SYNTHASE F 1 :      F o :a b 2 c 9-12

59. The engine can turn in both directions: with ATP hydrolysis it turns in the opposite direction when compared with ATP synthesis driven by proton flux into the matrix

61. Interesting questions: How many protons enter per ATP produced (per 120 o turn)? How many c-subunits per  subunit?

63. A simple estimate 10 PROTONS pumped per NAD+ oxidized 10 PROTONS pumped per OXYGEN consumed 3 PROTONS pass through the ATP synthase per 120 o turn 1 ATP is made per 120 o turn Therefore: 3ATP per 360 o turn 3 ATP / 9 PROTONS 3 ATP per OXYGEN (or per NADH oxidized): P/O ratio = 3

64. Experimental measurements of P/O ratios: P/O = ~ 2.5 Is that a problem? NO! There are other ways for protons to go back to the matrix without passing through the ATPsynthase: COUPLING is not perfect

65. Thermoregulation Thermogenesis

66. (limited amount) In the presence of an uncoupler the P/O ratio is reduced to zero

68. LNC 19-17b Oligomycin is a highly specific inhibitor of the ATP synthase

71. Uncoupling protein, UCP

72. End of Lecture 8 August 23, 2006