1. Oxidative Phosphorylation Endergonic Synthesis of ATP

2. Electron Transport and ATP Synthesis ATP Synthase (Complex V)

3. Coupling of Electron Transport and ATP Synthesis In intact mitochondria, electron transport requires simultaneous synthesis of ATP

4. Chemiosmotic Theory: The free energy from the ETC is coupled to ATP synthesis by the generation of a pH gradient across the mitochondrial inner membrane that is then used by ATP synthase Peter Mitchell Links Electron Transport to ATP Synthesis

5. Evidence Supporting The Chemiosmotic Theory Oxidative Phosphorylation requires an intact mitochondrial inner membrane Mitochondrial inner membrane is impermeable to ions — can maintain an electrochemical gradient Electron transport acidifies the cytosol Acidification outside mitochondrial inner membrane stimulates ATP synthesis Uncouplers — permeabilize the mitochondrial inner membrane –ETC continues, but ATP synthesis is inhibited UNCOUPLES ETC and oxidative phosphorylation

6. Electron Transport Generates a Proton Gradient (Proton Motive Force) ∆G = 2.3RT∆pH + ZF∆   = membrane potential ∆pH = 0.75 (inside higher) ∆G = ~21.5 kJ/mol

7. ATP Synthesis  

8. ATP Synthase Proton-pumping ATP Synthase F 1 F 0 –ATPase

9. Properties of ATP Synthase Multisubunit transmembrane protein Molecular mass = ~450 kD Functional units –F 0 : water-insoluble transmembrane protein (up to 8 different subunits) –F 1 : water-soluble peripheral membrane protein (5 subunits)

10. Structure of ATP Synthase (Cryoelectron Microscopy-Based Image) Matrix Inner Membrane IMS

11. F 1 Component of ATP Synthase Dissociated from F 0 by urea Catalyzes ATP hydrolysis (ATPase) but cannot synthesize ATP (F 1 -ATPase) Pseudo three-fold symmetry –Composition:  3  3  β- subunit catalyzes ATP synthesis

12. Ribbon Diagram of F 1 –ATP Synthase from Bovine Heart Mitochondria

13. F 0 Component of ATP Synthase Includes a transmembrane ring Composition (E. coli): a 1 b 2 c 9-12 Mitochondrial F 0 has additional subunits (function unclear)

14. Structure of E coli F 1 –c 10-15 Complex Composite Crystal StructureModel

15. F 1 –ATPase Three Interacting Catalytic Protomers (  )

16. Properties of F 1 Catalytic Protomers L state: binds substrates and products loosely T state: binds substrates and products tightly O state: open state does not bind substrate or product

17. ATP is Synthesized by the Binding Change Mechanism L = loose state T = tight state O = open state

18. Functions of Catalytic Protomers in ATP Synthesis L state: binds substrates (ADP and P i ) T state: formation of phosphoanhydride bond (ADP + P i —> ATP) O state: release of product (ATP) Proton translocation drives interconversion of states

19. Steps in ATP Synthesis ADP and P i bind to L site Energy-dependent conformational change –L —> T –O —> L –T —> O ATP synthesized at T site and ATP released from O site

20. Proton Translocation Drives Interconversion of States

21. F 1 F 0 –ATPase is a Rotary Engine: Movement of protons drives rotation Bind to c subunit  Exit through a subunit Stator (ab 2 –  3  3  ) Rotor (  –c 12 )

22. Rotation of F 1 F 0 –ATPase Protonation/ Deprotonation Rotation

23. Visualizing Rotation

25. P/O Ratio Relates the Amount of ATP Synthesized to the Amount of Oxygen Reduced

26. P/O Ratios Measured Using Isolated Mitochondria (only use of proton gradient) NADH: ~3 ATP/10 H + FADH 2 : ~2 ATP/6 H +

27. Mitochondrial Electron Transport Chain Complex II (FADH 2 )

28. Other Fates of Proton Gradient Dissipation (leakage) Consumption for other purposes (e.g. P i transport) 1H+/Pi  4H+/ATP

29. Actual ATP Yields Based on 4H+/ATP Demonstrated experimentally NADH: ~2.5 ATP FADH 2 : ~1.5 ATP

30. ATP from Glucose Glycolysis: 2 ATP + 2 NADH (= 5 ATP) = 7 ATP Pyruvate Dehydrogenase: 2 NADH (= 5 ATP) = 5 ATP Citric Acid Cycle: 6 NADH (= 15 ATP) + 2 FADH 2 (= 3 ATP) + 2 GTP (= 2 ATP) = 20 ATP TOTAL = ~32 ATP/Glucose

31. Thermodynamic Yield ATP/Glucose 32 ATP x ~45 kJ/mol = 1440 kJ Glucose —> CO 2 = 2866 kJ 1440/2866 = ~50%

32. Uncouplers Electron Transport and Oxidative Phosphorylation are Tightly Coupled

33. Tight Coupling Measuring O 2 consumption of isolated Mitochondria All ADP  ATP

34. Uncouplers Lipophilic Weak Acid Proton-transporting Ionophore 2,4-Dinitrophenol, FCCP, CCCP Valinomycin Protein Channels

35. Action of 2,4-Dintrophenol (Lipophilic weak acid)

36. Valinomycin Amphipathic Peptide Ring Hydrophillic Hydrophobic

37. Uncoupling in Brown Adipose Tissue Nonshivering Thermogenesis (regulated uncoupling of oxidative phosphorylation) Heat Generation

38. Uncoupling Protein (UCP1 or Thermogenin) Proton channel Inhibited by purine nucleotides –ADP and ATP –GDP and GTP Inhibition overcome by fatty acids

39. Mechanism of Hormonally-Induced Uncoupling of Oxidative Phosphorylation in Brown Adipose Tissue

41. Adult Humans: UCP2 and UCP3 May be related to “fast” or “slow” metabolism Possible targets for anti-obesity therapies Previous use of 2,4-dinitrophenol as dietary aid abandoned due to occasional lethality

42. Plants: Uncoupling Proteins Response to Cold Stress Increase Flower Temperature (vaporization of scent to attract pollinators)