Understanding the Chemiosmotic Theory: ATP Synthesis

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate pyruvate dehydrogenase (ox-decarbox; S-ester) Krebs’ Cycle Exam-1 material Exam-2 material Chemiosmotic theory: Phosphorylation Ox/Phos is Coupled ATPase Mitchell Hypothesis Binding-Change Model Connection to the protonmotive force How did he figure it out? Overview 8 Steps Citrate Synthase (C–C) Aconitase (=, -OH) Isocitrate dehydrogenase (ox-decarbox; =O) Ketoglutarate dehydrogenase (ox-decarbox; S-ester) Succinyl-CoA synthetase (sub-level phos) Succinate dehydrogenase (=) Fumarase ( -OH) Malate dehydrogenase (=O) Energetics Regulation Summary Oxidative Phosphorylation Energetics (–0.16 V needed for making ATP) Mitochondria Transport (2.4 kcal/mol needed to transport H+ out) Electron transport: Oxidative Discovery Four Complexes Complex Complex Complex Complex I: NADH à CoQH2 II: Succinate à CoQH2 III: CoQH2 à Cytochrome C (Fe2+) IV: Cytochrome C (Fe2+) à H2O Oxidative Phosphorylation Glycogenolysis Glycolysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative These are tightly COUPLED 1 Electron Transport Summary of the Electron Flow in the Respiratory Chain DG’ of H+ transport = 2.4 kcal/mol As a consequence, it will take ~3 protons per ATP. This scheme is for 2e– coming down from 1 NADH or 1 FADH2 Difference in number of protons transported is reflected in differences in ATP synthesized=P/O ratios: Complex I à Complex IV 1NADH + 11H+(N) + ½O2 à NAD+ + 10H+(P) + H2O Substrate Pyruvate 3 Complex II à Complex IV FADH2 + 6H+(N) + ½O2 à FAD + 6H+(P) + H2O Acetyl CoA 3 NADH 3 Succinate 2 P/O ratio Phosphorylation (The Chemiosmotic Theory) 2 The Chemiosmotic Theory Oxidative Phosphorylation Relationship of ETC and ATP Synthesis The Chance/Williams experiment showed that fuel oxidation is COUPLED to ATP synthesis Here, same experiment done the other way: • As described, ATP synthesis requires electron transport. • But electron transport does not require ATP synthesis. 3 Oxidative Phosphorylation Relationship of ETC and ATP Synthesis The Chance/Williams experiment showed that fuel oxidation is COUPLED to ATP synthesis Here, same experiment done the other way: • As described, ATP synthesis requires electron transport. • But electron transport does not require ATP synthesis. Phosphorylation How Does Oxidative Phosphorylation Form ATP? • Before the 1970’s, it was thought that the ET chain used the energy of redox to make a ”high-energy” intermediate for “substrate-level phosphorylation, as occurs in glycolysis and TCA cycle. • It was clear that one of the most abundant proteins on the inner-mitochondrial membrane had a curious mushroom-shaped structure. ATPase V • It was purified, along with the other Complexes I-IV, initially called Complex V. 4 Phosphorylation How Does Oxidative Phosphorylation Form ATP? • Complex V, when purified separately from the membrane is an effective ATPase enzyme, hence it was initially called the F1ATPase. F1-ATPase • The F1 part had 9 subunits (a3b3gde) F0 • When more careful purifications were performed, it was clear that its activity was closely coupled to an intact inner membrane with little activity. F1 • When membrane proteins were isolated, the F0 part • Inhibited by had 15 subunits of 3 venturicidin or different proteins (a, b2, c10). oligomycin Phosphorylation How Does Oxidative Phosphorylation Form ATP? • Complex V, when purified separately from the membrane is an effective ATPase enzyme, hence it was initially called the F1ATPase. • The F1 part had 9 subunits (a3b3gde) • When more careful purifications were performed, it was clear that its activity was closely coupled to an intact inner membrane with little activity. • When membrane proteins were isolated, the F0 part • Inhibited by had 15 subunits of 3 venturicidin or different proteins (a, b2, c10). oligomycin 5 Phosphorylation Contains two functional units: • F1 • • soluble complex in the matrix individually catalyzes the hydrolysis of ATP • F0 • • • • integral membrane complex transports protons from IMS to matrix, dissipating the proton gradient energy transferred to F1 to catalyze phosphorylation of ADP. This mechanism was only fully accepted in late 1970s. Phosphorylation Hypothesis: The Respiratory Chain and Hypothesis: The Respiratory Chain Produce ATP by a substrate-levelATP Synthase Produce ATP by a VS. phosphorylation mechanism Chemiosmotic Mechanism Peter Mitchell 1920-1992 The search was for a compound, like 1,3 bisphosphoglycerate, that would be used by the ATP Synthase to make ATP. Peter Mitchell, 1920-1992 1978 Nobel Prize for Chemistry 6 Phosphorylation Chemiosmotic Theory Ø ADP + Pi à ATP is highly thermodynamically unfavorable. • How do we make it possible? • Phosphorylation of ADP is not a result of a direct reaction between ADP and some high-energy phosphate carrier. • The energy released by the exergonic flow of electrons to oxygen in electron transport is used to transport protons against the electrochemical gradient. Secondary active transport principles are at work. • Energy needed to phosphorylate ADP is provided by the flow of protons down this electrochemical gradient. This can be calculated. • If all that was needed was a proton gradient, could one be established without the ET chain and still drive ATP biosynthesis? Phosphorylation Chemiosmotic Theory • The proteins in the electron-transport chain created the electrochemical proton gradient (proton-motive force (PMF); a chemical AND electrical gradient) by one of three means: – actively transporting (pumping) protons across the membrane • Complex I and Complex IV – releasing protons into the intermembrane space • oxidation of QH2 at Complex III – chemically removing protons from the matrix • reduction of CoQ in Q-cycle (Complex I, II, & III) • reduction of oxygen (Complex IV) DG’ = 1.0 + 1.4 = 2.4 kcal/mol As a consequence, it will take ~3 protons per ATP. But, how many precisely? 7 Phosphorylation Chemiosmotic Theory Mitchell’s experiment: chemical AND electrical Nobel Prize in Chemistry 1978 For 3 protons: DG’ = 3.0 + 4.2 = 7.2 kcal/mol (if there is also a sufficient membrane potential) Phosphorylation Chemiosmotic Theory for ATP Synthesis: • Electron transport chain sets up a proton-motive force. • Energy of proton-motive force drives synthesis of ATP. 8 Phosphorylation Chemiosmotic Theory Chemiosmotic Energy Coupling Requires Membranes • The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions. – plasma membrane in bacteria – inner membrane in mitochondria – thylakoid membrane in chloroplasts • The membrane must contain proteins that couple the “downhill” flow of electrons in the electron-transfer chain with the “uphill” pumping of protons across the membrane. • The membrane must contain a protein that couples the “downhill” flow of protons to the phosphorylation of ADP. k–1 k–1 Phosphorylation Mechanism: BINDING –Isotope studies The F1 catalyzes ADP + Pi – 18O from water is quickly incorporated into Pi ATP + H2O –Kinetic studies Both ATP hydrolysis and ATP synthesis have occurred several times during the incubation (DG’°= –0.5 kcal/mol) –Structural studies – Compare to >105 for in solution (from DG’°= +7.3 kcal/mol) 9 Phosphorylation –MORE Structural studies Mechanism: RELEASE • Hexamer arranged in three αβ dimers • Dimers can exist in three different conformations: – open: empty – loose: binding ADP and Pi – tight: catalyzes ATP formation and binds product T=tight O=open L L=loose T WHERE does the energy come from for the release? Called the BindingChange Model O – This is where ALL the energy (in Dconf. from tight to of PMF is used open; thus, releasing ATP) to release ATP! Phosphorylation Mechanism Binding-Change Model WHAT drives the g-subunit’s motion? John E. W alker Nobel Prize in Chemistry 1997 ATP cannot be released from one site unless and until ADP and Pi are bound at the other…………the g-subunit cannot move if the a/b-dimer is in the open conformation: without substrates IT ALL GRINDS TO A HALT! 10