1. Electron Transport Chain

2. Thermodynamics of Glucose Oxidation Glucose + 6 O 2 ——> 6 CO 2 + 6 H 2 O ∆G o’ = -2866 kJ/mol

3. Half-Reactions of Glucose Oxidation Glucose + 6 H 2 O ——> 6 CO 2 + 24 H + + 24 e – 6 O 2 + 24 H + + 24 e – ——> 12 H 2 O NADH and FADH 2

4. Sites of NADH and FADH 2 Formation

6. Mitochondrial Electron Transport Chain System of Linked Electron Carriers

7. Components of Electron Transport Process Reoxidation of NADH and FADH 2 Sequential oxidation-reduction of multiple redox centers (four enzyme complexes) Production of proton gradient across the mitochondrial membrane

8. Oxidative Phosphorylation Synthesis of ATP driven by free energy of electrochemical gradient

9. Coupling of Electron Transport and ATP Synthesis NOTE: ATP Synthesis in the Mitochondrion

10. The Mitochondrion Prokaryotic origin  Double membrane bound  Genome o Human: encodes 13 genes, all ETC subunits.

11. Mitochondrial Outer Membrane Permeable to molecules smaller than ~5 kD

12. Figure 9-23a X-Ray Structure of E. coli OmpF Porin

13. Figure 9-23b X-Ray Structure of E. coli OmpF Porin Trimer

14. Mitochondrial Intermembrane Space (IMS) [Metabolites] = Cytosolic Concentration Localized Compartmentation of H +

15. Mitochondrial Inner Membrane (Permeability Barrier) Transport

16. Types of Transport Nonmediated Transport (Diffusion) –H 2 O; O 2 ; CO 2 Mediated Transport –Passive-mediated Transport (facilitated diffusion) –Active Transport –Facilitated by Proteins: Carriers, Transporters, Translocases, or Permeases.

17. Kinetic Properties of Mediated Transport Saturation kinetics Speed and specificity Susceptibility to competitive inhibition Susceptibility to chemical inactivation

18. Stoichiometry of Mediated Transport

19. Entry of “NADH” into Mitochondria No NADH Transporter

20. Malate–Aspartate Shuttle

22. Glycerophosphate Shuttle

23. Transport of ADP, ATP, and Inorganic Phosphate (P i )

24. ADP-ATP Translocator ADP/ATP Exchanger Driven by electrochemical gradient

25. Phosphate Transport Driven by ∆pH

26. Phosphate Transporter H + (out) H 2 PO 4 – (out) H + (in) H 2 PO 4 – (in)

27. Electron Transport Electron Transport is an Exergonic Process

28. Standard Reduction Potentials

29. Standard Reduction Potential Difference ∆E o ’ = E o ’ (e – acceptor) – E o ’ (e – donor) ∆G o ’ = – nF∆E o ’ For negative  G need positive  E E (acceptor) > E (donor) Note: reduction potential is extremely pH sensitive E = E o’ + 0.06V*(7-pH)

30. What is the ∆E o ’ and ∆G o ’ for the Oxidation of NADH by O 2 ?

31. Electron Carriers Operate in Sequence

32. Electron Transport Complexes Complex I: NADH–Coenzyme Q Oxidoreductase Complex II: Succinate–Coenzyme Q Oxidoreductase Complex III: Coenzyme Q–Cytochrome c Oxidoreductase Complex IV: Cytochrome c Oxidase

33. Overview of Electron Transport in the Mitochondrion

34. Mobile Electron Carriers Coenzyme Q Cytochrome c

35. Coenzyme Q

36. Oxidation States of Coenzyme Q

37. Cytochromes Electron Transport Heme Proteins Fe 3+ + e – ——> Fe 2+

38. Hemes Note: isoprene side chain a b Iron-Protoporphyrin IX Like Mb and Hb c Note: Thioether Links

39. Cytochrome Spectra

40. Complex I (NADH–Coenzyme Q Oxidoreductase) Accepts Electrons from NADH NADH + CoQ(oxidized) ——> NAD + + CoQ(reduced) Protons translocated 4H+ (Matrix) ——> 4H+ (IMS)

41. Coenzymes of Complex I (Flavin Mononucleotide, FMN) Oxidation states like FAD

42. Coenzymes of Complex I (Iron-Sulfur Clusters) One-electron oxidation-reduction Conjugated System (Fe between +2 and +3)

43. Thermodynamics of Complex I

44. Hydrophilic Domain of Complex I from Thermus thermophilis Electrons follow a multistep path ~ matrix ~ cytoplasm

45. Figure 9-22 Structure of Bacteriorhodopsin

46. Proton Wire 1)Deprotonation of Schiff base and protonation of Asp 85 2)Proton release to the extracellular surface 3)Reprotonation of the Schiff base and deprotonation of Asp 96 4)Reprotonation of Asp 96 from the cytoplasmic surface 5)Deprotonation of Asp 85 and reprotonation of the proton release site

47. Complex II (Succinate–Coenzyme Q Oxidoreductase) Contributes Electrons to Coenzyme Q Succinate + CoQ(oxidized) ——> Fumarate + CoQ(reduced) Does not pump protons

48. Composition of Complex II Succinate Dehydrogenase –FAD [4Fe-4S] cluster [3Fe-4S] cluster [2Fe-2S] cluster Cytochrome b 560

49. Thermodynamics of Complex II

50. E. coli Complex II Cytoplasm ~matrix Plasma Membrane ~IM Periplasm ~cytoplasm

51. Complex II (Linear Chain of Redox Cofactors) Cytochrome b 560 scavenges electrons to prevent formation of reactive oxygen species

52. Complex III (Coenzyme Q–Cytochrome c Oxidoreductase) Translocates Protons via the Q Cycle CoQ(reduced) + 2 Cytochrome c (oxidized) ——> CoQ(oxidized) + 2 Cytochrome c (reduced)

53. Oxidation States of Coenzyme Q

54. Composition of Complex III Cytochrome b 562 (b H – high potential) Cytochrome b 566 (b L – low potential) Cytochrome c 1 [2Fe–2S] cluster (ISP)

55. Thermodynamics of Complex III

56. Yeast Complex III

57. The Q Cycle (Electrons from CoQH 2 follow two paths)

58. Cycle 1 IMS Matrix

59. Steps in Cycle 1 CoQH 2 supplied by Complex I from matrix side CoQH 2 diffuses to IMS side and binds to Q o site CoQH 2 transfers one electron to ISP and releases 2 H + into IMS yielding CoQ – ; ISP reduces cytochrome c 1 CoQ – transfers electron to cytochrome b L yielding CoQ CoQ diffuses to the matrix side and binds to Q i site Cytochrome b L transfers electron to cytochrome b H CoQ in Q i site reduced to CoQ – by cytochrome b H

60. Summary of Cycle 1 CoQH 2 + Cytochrome c 1 (Fe 3+ ) ——> CoQ – + Cytochrome c 1 (Fe 2+ ) + 2 H + (IMS)

61. Cycle 2

62. Steps in Cycle 2 CoQH 2 supplied by Complex I from matrix side CoQH 2 diffuses to IMS side and binds to Q o site CoQH 2 transfers one electron to ISP and releases 2 H + into IMS yielding CoQ – ; ISP reduces cytochrome c 1 CoQ – transfers electron to cytochrome b L yielding CoQ CoQ diffuses to the matrix side (to Complex I) Cytochrome b L transfers electron to cytochrome b H CoQ – in Q i site reduced to CoQH 2 by cytochrome b H (2 H + from Matrix side)

63. Summary of Cycle 2 CoQH 2 + CoQ – + Cytochrome c 1 (Fe 3+ ) + 2 H + (matrix) ——> CoQ + CoQH 2 + Cytochrome c 1 (Fe 2+ ) + 2 H + (IMS)

64. Overall Summary of Q Cycles CoQH 2 + 2 Cytochrome c 1 (Fe 3+ ) + 2 H + (matrix) ——> CoQ + 2 Cytochrome c 1 (Fe 2+ ) + 4 H + (IMS)

65. Complex IV (Cytochrome c Oxidase) Reduces Oxygen to Water 4 Cytochrome c (reduced) + 4 H + + O 2 —— > 4 Cytochrome c (oxidized) + 2 H 2 O

66. Composition of Complex IV Homodimer (2x 13 subunits) Subunits I, II, and III: encoded by mitochondrial DNA Subunits IV–XIII: encoded by nuclear DNA

67. Bovine Heart Cytochrome c Oxidase

68. Redox Centers in Cytochrome c Oxidase Cytochrome a Cytochrome a 3 Cu B Cu A center (two Cu-atoms)

69. Organization of Redox Centers in Cytochrome c Oxidase Above Membrane Surface Membrane

70. Electron Transfer in Cytochrome c Oxidase Cytochrome c —> Cu A Center —> Cytochrome a —> Cytochrome a 3 –Cu B Binuclear Complex —> O 2

71. Cytochrome c Oxidase Catalyzes a Four-Electron Redox Reaction 4 Cytochrome c (reduced) + 4 H + + O 2 —— > 4 Cytochrome c (oxidized) + 2 H 2 O

72. Source of Four Electrons Heme a 3 (Fe 2+ —> Fe 4+ ): 2 electrons Cu B (Cu 1+ —> Cu 2+ ): 1 electron Tyrosine 244: 1 electron –Covalent link to His 240 –Tyr–OH —> Tyr–O

73. Heme a 3 –Cu B Binuclear Complex in Cytochrome c Oxidase

74. Proposed Reaction Sequence for Cytochrome c Oxidase

75. Protons in Cytochrome c Oxidase Chemical or Scalar Protons (4) –From matrix –Used in reduction of O 2 —> 2 H 2 O Pumped or Vectorial Protons (4) –Matrix —> IMS

76. Summary of Proton Utilization in Cytochrome c Oxidase 8 H + (matrix) + O 2 + 4 Cytochrome c (Fe 2+ ) ——> 4 Cytochrome c (Fe 3+ ) + 2 H 2 O + 4 H + (IMS)

77. Complex Proton Channels in Cytochrome c Oxidase K-channel (lysine) H + (matrix) —> Tyr 244 —> H 2 O D-channel (aspartate) H + (matrix) —> Heme a 3 –Cu B —> H + (IMS) [pumped protons]

78. Summary of Electron Transport Complex I  Complex IV 1NADH + 11H + (matrix) + ½O 2 ——> NAD + + 10H + (IMS) + H 2 O Complex II  Complex IV FADH 2 + 6H + (matrix) + ½O 2 ——> FAD + 6H + (IMS) + H 2 O ~3H+/ATP

79. Thermodynamics of Electron Transport Complexes

80. Standard Reduction Potentials of Electron Transport Chain Components

81. Mitochondrial Electron Transport Chain

82. Complex I (NADH–Coenzyme Q Oxidoreductase) NADH + CoQ (oxidized) —— > NAD + + CoQ (reduced) ∆E o ’ = + 0.360 V ∆G o ’ = – 69.5 kJ/mol

83. Complex II (Succinate–Coenzyme Q Oxidoreductase) Succinate + E – FAD —— > Fumarate + E – FADH 2 E – FADH 2 + CoQ (oxidized) —— > E – FAD + CoQ (reduced) ∆E o ’ = + 0.085 V ∆G o ’ = – 16.4 kJ/mol

84. Complex III (Coenzyme Q–Cytochrome c Oxidoreductase) CoQ (reduced) + 2 Cytochrome c (oxidized) —— > CoQ (oxidized) + 2 Cytochrome c (reduced) ∆E o ’ = + 0.190 V ∆G o ’ = – 36.7 kJ/mol

85. Complex IV (Cytochrome c Oxidase) 4 Cytochrome c (reduced) + 4 H + + O 2 —— > 4 Cytochrome c (oxidized) + 2 H 2 O ∆E o ’ = + 0.580 V ∆G o ’ = – 112 kJ/mol

86. Electron transport chain Complex I  Complex IV 2NADH + 2 H + + O 2 —— > 2NAD+ + 2 H 2 O ∆E o ’ = + 1.130 V ∆G o ’ = – 218 kJ/mol Complex II  Complex IV 2FADH 2 + O 2 —— > 2FAD + 2 H 2 O ∆E o ’ = + 0.855 V ∆G o ’ = – 165 kJ/mol

87. ATP Synthesis from NADH ATP synthesis: ∆G o ’ = 30.5 kJ/mol Standard Biochemical Conditions: Efficiency = = ~35% (FADH 2 is ~30%) Physiological conditions ~70% efficiency