ATP synthesis : The F 1 F 0 -ATPase

1. ATP synthesis : The F1F0-ATPase

2. Hypotheses on the mechanism of ATP synthesis in mitochondria: “Substrate level phosphorylation” -- coupling of ATP synthesis to an enzymatic reaction (as in glycolysis--requires a high-energy phosphate bond) Stress membrane conformation -- evidence: differences in membrane structure in mitochondria when provided substrate (pyruvate) or not “Chemiosmotic hypothesis” (Peter Mitchell): H+ passage across membrane powers ATP synthesis

3. ATP synthesis depends on a peripheral membrane protein Inner mitochondrial (inside-out) vesicles capable of ATP synthesis--note the headpiece of the “F0F1 ATPase” (ATP synthase) Removal of the headpieces gives vesicles that can’t make ATP Adding purified headpieces re- stores the ability to make ATP

4. AO AO AO AO AO AO AO Add ATP AO+ AO+ AO+ H+ AO+ AO+ ATP ADP + Pi

5. Light H+ Bacteriorhodopsin H+ H+ H+ Add ADP H+ H+ H+ H+ ADP + Pi ATP H+

6. Uncouplers — compounds that permeabilize the mitochondrial inner membrane to H+ inhibit ATP synthesis (and allow rapid oxidation of NADH and substrates like pyruvate) cytosol FCCP intermembrane space FCCP FCCP-H+ matrix H+ H+ H+ H+ H+ H+ (FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone)

8. Note the rotor (base) and stator (head) and rotation F1 F0 Hypothesis: ADP + Pi ATP + H2O occurs on head; H+ flow turns rotor; rotor/rotation stimulates ATP release

9. rotor rotation rotor rotation

10. Figure 1 Observation system for the c subunit rotation in F0F1. Y Sambongi et al. Science 1999;286:1722-1724 Movie: http://www.sciencemag.org/site/feature/data/1045705a.mov

11. Movie of the ATPase model -- Mechanical part of respiration. http://multimedia.mcb.harvard.edu/anim_mitochondria.html

12. Model for H+-induced rotation from the textbook:

13. 4 4 2 (How many ATP per FADH2?)

14. Electron input at Complex II FAD: expect 1.5 ATP/NADH

15. Malate-aspartate shuttle (animals and plants) How much ATP/NADH?