ATP synthesis ADP + Pi ATP + H2O Lei Li 01-20-2009

F1 complex of ATP synthase

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

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

Part I Chemiosmotic theory for ATP synthesis Peter D. Mitchell

Similarity between proton pump and water pump H2O

Proton pump provides energy for ATP synthesis

Succinate oxidation provides energy for ATP synthesis

Blocking electron transfer will shut down ATP synthesis X X

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

No electron transfer to O2, no ATP synthesis

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

Analogy between water pump and proton pump X X ΔE H2O

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

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

Maintain the electron transfer process after ATP synthesis is blocked X

Using water pump as an example H2O

Re-activate the electron transfer process by bringing back protons X

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

Chemical uncoupler resumes electron transfer process with no ATP synthesis conducted

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

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

Resuming the ATP synthesis without electron transfer to O2 X

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

Part II Structure of the ATP synthase Johns E. Walker

ATP synthase has two functional domains, Fo and F1

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

F1 has nine subunits with a α3β3γδε composition

F1 complex viewed from above

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

Overall structure of ATP synthase

Part III Reaction mechanism for ATP synthesis Paul D. Boyer

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

Experiment to test this rotational catalysis

Rotation of Fo has been experimentally observed

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

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

Without enzyme, ATP hydrolysis is heavily favored thermodynamically ATP + H2O ADP + Pi ΔGº = -30.5 kJ/mol ΔGº = – RT ln(Keq) Keq = ~ 105.3 Keq = vforward/vbackward vforward = 105.3 x vbackward The reactions is basically irreversible!

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

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

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

Bonus Material II the transportation of reactants

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

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

NADH transport by malate-aspartate shuttle

NADH transport by glycerol 3-phosphate shuttle

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