2. Metabolic Biochemistry Lecture 8 Aug. 17, 2005 Oxidative Phosphorylation Other Pathways of Carbohydrate Metabolism

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

5. Proton pumping and Storage of Free Energy

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

7.  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

8. tightly coupled vs uncoupled mitochondria

9. succinate

10. LNC 19-18a

13. 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

14. ATP Synthase Complex V

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

29. 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

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

33. 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 360o turn 3 ATP / 9 PROTONS 3 ATP per OXYGEN (or per NADH oxidized): P/O ratio = 3

34. 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

35. Thermoregulation Thermogenesis

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

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

41. Uncoupling protein, UCP

42. LNC 19-28 We have > 6 genes for different isozymes of uncoupling proteins”: UCP-1, UCP-2, UCP-3, ….. they are differentially expressed In different tissues and cells

43. Mills, et al., NATURE|VOL 426 | 27 NOVEMBER 2003| The recreational use of amphetamine type stimulants can produce a marked and sometimes lethal increase in body temperature. Here we show that mice deficient in a mitochondrial protein known as UCP-3 (for ‘uncoupling protein-3’) have a diminished thermogenic response to the drug MDMA (3,4 methylenedioxymethamphetamine, nicknamed ‘ecstasy’) and so are protected against this dangerously toxic effect. Our findings indicate that UCP-3 is important in MDMA-induced hyperthermia and point to a new therapeutic direction for solving an increasing public health problem.

44. QH 2 can also be oxidized directly through a cyanide-insensitive oxidase LNC box 19-1 Alternative NADH oxidases exist in plant mitochondria No proton pumping and hence no ATP synthesis The free energy from the oxidation of NADH appears as HEAT

45. Eastern skunk cabbage

46. Amorphophallus konjac Voodoo Lilly

47. Seymour et al. NATURE 426, 243-244 (2003) …..heat produced by the flower constitutes an important energy reward to pollinators, allowing them to feed and mate at a fraction of the energy cost that would be required outside the flower.

48. End of lecture 8 August 17, 2005