1. ATP synthesis
ADP + Pi
ATP + H2O
Lei Li

2. F1 complex of ATP synthase

3. ATP: the perfect energy currency for the Cell
The Nobel Prize in Chemistry 1978
The Nobel Prize in Chemistry 1997
"for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory"
"for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)"
Peter D. Mitchell
Paul D. Boyer
John E. Walker

4. Outline of today’s lecture
Peter D. Mitchell
(1) Chemiosmotic theory
(2) Structure of ATP synthase
Johns E. Walker
(3) Reaction mechanism
— rotational catalysis
Paul D. Boyer

5. Part I Chemiosmotic theory for ATP synthesis
Peter D. Mitchell

6. Similarity between proton pump and water pump
H2O

7. Proton pump provides energy for ATP synthesis

8. Succinate oxidation provides energy for ATP synthesis

9. Blocking electron transfer will shut down ATP synthesisX X

10. Cyanide extracts iron away from cytochromes, thus shut down the electron transfer to O2

11. No electron transfer to O2, no ATP synthesis

12. Blocking H+ transfer channel, electron transfer to O2 will be also quenched? X

13. Analogy between water pump and proton pumpX X ΔE
H2O

14. Blocking H+ flow by venturicidin or oligomycin disrupts electron transfer to O2

15. Oligomycin and venturicidin bind Fo of ATP synthase and stop the proton flow

16. Maintain the electron transfer process after ATP synthesis is blockedX

17. Using water pump as an example
H2O

18. Re-activate the electron transfer process by bringing back protonsX

19. Chemical uncouplers are weak acids with hydrophobic properties
pKa = 4.08
pKa = 6.2

20. Chemical uncoupler resumes electron transfer process with no ATP synthesis conducted

21. Electron transfer process can also be rescued by addition of ionophores
Na+,K+
or Ca2+ X
ΔG = (5.70 kJ/mol)ΔpH + (96.5 kJ/V●mol)ΔΨ
ΔΨ ~ 0.15 – 0.20V, ΔpH ~ 0.75

22. Using ionophore (valinomycin, for instance) to transfer inorganic ions into N side to decrease ΔΨ
Na+, K+, Ca2+ …
valinomycin

23. Resuming the ATP synthesis without electron transfer to O2X

24. ATP synthesis without an oxidizable substrate
pH 9
pH 9
pH 7

25. Part II Structure of the ATP synthase
Johns E. Walker

26. ATP synthase has two functional domains, Fo and F1

27. F1 was first identified and purified by Efraim Racker in 1960s

28. F1 has nine subunits with a α3β3γδε composition

29. F1 complex viewed from above

30. Fo has three types of subunits a, b and c in the proportion of ab2c10-12

31. Overall structure of ATP synthase

32. Part III Reaction mechanism for ATP synthesis
Paul D. Boyer

33. Binding model change for ATP synthesis
ADP + Pi
ATP + H2O

34. Experiment to test this rotational catalysis

35. Rotation of Fo has been experimentally observed

36. * Summary Theory for ATP synthesis Crystal structure of ATP synthase
Chemiosmotic model
Crystal structure of ATP synthase
Reaction mechanism for ATP synthesis
Rotational catalysis *

37. The reversibility of ATP synthesis/hydrolysis reaction
Bonus material I
The reversibility of ATP synthesis/hydrolysis reaction
(The magic of enzyme!!)

38. Without enzyme, ATP hydrolysis is heavily favored thermodynamically
ATP + H2O
ADP + Pi
ΔGº = kJ/mol
ΔGº = – RT ln(Keq)
Keq = ~ 105.3
Keq = vforward/vbackward
vforward =
105.3 x
vbackward
The reactions is basically irreversible!

39. On ATP synthase surface, the ATP hydrolysis reaction is readily reversible
k 1
Enz – (ATP + H2O)
Enz – (ADP + Pi)
k -1
k 1
10 s-1
Keq’ = = =
0.42
k -1
24 s-1

40. Mechanistic details for the observed O18 isotope exchange reactions
ATP
ADP Pi
ATP
ADP Pi
ADP Pi

41. ATP synthase binds ATP much more tightly than ADP
Kd ≤ 10-12M
Kd ≈ 10-5M

42. Bonus Material II the transportation of reactants

43. NADH, ATP, ADP, Pi need to be transported across the membrane

44. ATP, ADP and Pi transports through adenine nucleotide and phosphate translocases

45. NADH transport by malate-aspartate shuttle

46. NADH transport by glycerol 3-phosphate shuttle

47. Stoichiometries of substrate oxidation and ATP synthesis
4 protons must flow into the matrix to generate 1 ATP