2. Pyruvate Carboxylase

5. Reversing the final steps

6. Inverse regulation of glycolysis and gluconeogenesis

7. Hexokinase IV in liver Hexokinase I in muscle HxkIV Km = 10 mM

8. When blood glucose drops below 5 mM, F6P inhibits it. This way liver does not compete with muscle for glucose

9. Regulation of phosphofructokinase

17. Electron transport and oxidative phosphorylation Glucose is completely oxidized to CO 2 through the enzymatic reactions of glycolysis and the citric acid cycle. The redox equation for this process is: C 6 H 12 O 6 + 6O 2 ---> 6CO 2 + 6H 2 O ΔG°’ = -2823 kJ.mol -1 Which can be represented by two half reactions: C 6 H 12 O 6 + 6H 2 O ---> 6CO 2 + 24H + + 24e - glucose is oxidized 6O 2 + 24H + + 24e - ---> 12H 2 O molecular oxygen is reduced In living systems the electron transfer process connecting these two half reactions occurs through a multistep pathway that harnesses the liberated free energy to form ATP.

18. The sites of electron transfer that form NADH and FADH 2 in glycolysis and the citric acid cycle are represented in the figure. The 12 electron pairs involved in glucose oxidation are not transferred directly to O 2. Rather they are transferred to coenzymes NAD + and FAD to form NADH and FADH 2 10 NADH : 20 e - 2 FADH 2 : 4 e -

19. The electrons are extracted from the cofactors by reoxidation and then join the electron-transport chain, in this process, protons are expelled from the mitochondrion. The free energy stored in the resulting pH gradient drives the synthesis of ATP from ADP and P i (inorganic phosphate) through oxidative phosphorylation. Reoxidation of NADH ~ 3 ATP Reoxidation of FADH 2 ~ 2 ATP A total of 38 ATP are produced per each molecule of glucose completely oxidized to CO 2 and H 2 O (including the 2 ATP made in glycolysis and the 2 ATP made in the citric acid cycle)

20. NAD + and FAD coenzymes are reduced during glucose oxidation

21. Mitochondria is the site of eukaryotic oxidative metabolism 0.5  m in diameter and 1  m long (about the size of a bacterium) The outer membrane contains porin, a protein that forms pores and allows free difussion of up to 10 kD molecules The inner membrane is a lot more dense and is permeable only to O 2, CO 2 and H 2 O. Contains numerous transport proteins that control metabolite passage.

23. Mitochondrion is not a regular shaped organelle it is a dynamic organelle that is reticulated throughout the cell

24. Electrons enter the electron transport chain onto Q Fatty acid metabolism Glycerol phosphate shuttle Succinate dehydrogenase NADH:Q Oxidoreductase

25. Reduced state has more protons than the oxidized state!

26. Redox loops pumps out four protons!

27. The glycerophosphate shuttle mainly occurs in rapidly metabolizing tissues.

29. NADH from TCA cycle are generated in mitochondrial matrix

30. NADH from glycolysis are generated in cytoplasm Problem: No way to transport NADH into the mitochondrion to be reoxidized! Solution: Use the malate-aspartate shuttle

32. Complex I: NADH:CoQ oxidoreductase ∆E = 0.360 V ∆G = -69.5 kJ/mol NADH + H + + CoQ (ox) + 4H + (in) ---> NAD + + CoQH 2(red) + 4H + (out) -0.32 -0.30 -0.030 +0.045 -0.25

33. Ubiquinone+0.045 -0.30 -0.32

34. What is FMN? Flavin mononucleotide

35. What are FeS clusters?

36. Complex II: succinate dehydrogenase Succinnate:CoQ oxidoreductase FADH 2 + CoQ (ox) ---> FAD + CoQH 2(red) ∆E = 0.085 V ∆G = -16.4 kJ/mol

37. -0.031 -0.040 -0.030 -0.245 -0.060 -0.080 +0.045

38. Succinate -0.031

39. Heme b

41. Complex III CoQH 2(red) + 2cyt c (ox) + 2H + (in) ---> CoQ (ox) + 2cyt c (red) + 4H + (out) ∆E = 0.190 V ∆G = -36.7 kJ/mol

42. +0.045 +0.030 -0.030 +0.280 +0.215 +0.235

43. CoQH 2 + cyt c 1(ox) ---> CoQ - + cyt c 1(red) + 2H + (out) CoQH 2 + CoQ - + cyt c 1(ox) + 2H + (in) ---> CoQ + CoQH 2 + cyt c 1(red) + 2H + (out) Cycle I Cycle II CoQH 2 + 2cyt c 1(ox) + 2H + (in) ---> CoQ + 2cyt c 1(red) + 4H + (out) Net Reaction 4 protons pumped instead of 2

44. Ubiquinone = +0.045

45. Cytochrome c: an electron bottle neck

46. Complex IV ∆E = 0.580 V ∆G = -112 kJ/mol 4 cytochrome c 2+ + O 2 + 8H + (in) => 4 cytochrome c 3+ + 2H 2 O + 4H + (out)

47. +0.235 +0.245 +0.385 +0.340 +0.815

49. 2H +

50. If 2 electrons enter at complex I 4 + 4 + 2 = 10 protons pumped out If 2 electrons enter at complex II or Glycerol dehydrogenase or fatty acid metabolism 4 + 2 = 6 protons pumped out

52. Proton concentration gradient pH is lower in intermembrane space than in the mitochondrial matrix G A - G A 0 ’ = RT ln [A] A (out) A (in) ∆G A = G A(in) - G A(out) = RT ln [A] in [A] out ()

53. ∆G A = RT ln [A] in [A] out ( ) + Z A F ∆  If the solute is charged there is another aspect of the equation: electrical potential Membrane potential = ∆  =  (in) -  (out) Free energy is a combination of chemical and electrical potential

54. ∆G A = RT ln [A] in [A] out ( ) + Z A F ∆  ∆G = 2.3RT [pH (in) - pH (out) ] + Z F ∆  ∆  = 0.168V = 0.168 JC -1 ∆pH = 0.75 ∆G = 21.5 kJmol -1 F = 96,485 Cmol -1 Z = +1 ∆G of ATP synthesis = 40 to 50 kJmol -1 [≈ 210,000Vcm -1 !!!!]

55. ATP synthase allows protons to flow back in Harnesses the free energy in the process

58. The gamma subunit: the rotor

66. Per glucose 10 NADH : 20 e - 2 FADH 2 : 4 e - If 2 electrons enter at complex I 4 + 4 + 2 = 10 protons pumped out If 2 electrons enter at complex II or Glycerol dehydrogenase 4 + 2 = 6 protons pumped out Per glucose 120 protons 120/3 = 40 + 4ATP from glycolysis and TCA 44 ATP! Why only 38?

67. H + transported -0.3ATP 2.7 ATP/ NADH ATP synthase is nearly 100% efficient: so why do you not get 1ATP per 3 H + ?

68. H + transported -0.3ATP 2.4 ATP/ NADH

69. What about fatty acid biosynthesis, succinate dehydrogenase and glycerol phosphate shuttle? 1.5 ATP per pair of electrons...

70. How is electron transport regulated? What is uncoupling?

74. The c-subunits of F 0 ATPase pH 8 pH 5

76. Electrons sometimes leak out of the chain onto molecular oxygen. As much 5% leak out onto oxygen Incompletely reduced oxygen is toxic Reactive oxygen species Superoxide O 2 - Peroxide O 2 2- Hydroxyl radical OH Where does this occur?

77. Superoxide comes from Q not Complex IV

79. Why does this happen? The ox-tox hypothesis Oxygen is soluble in membranes and it can oxidize lipids, albeit slowly. Too much oxygen is toxic. Mitochondria detoxify oxygen by reducing it to water. This is how they were beneficial

80. What about superoxide and peroxide? Maybe these are allowed to leak out as signal molecules that apprise the cell of the energetic state…. Maybe the generation of superoxide, which is not soluble in membrane is a way to detoxifying oxygen……

81. Mitochondria and apoptosis Cytochrome c is released from intermembrane space