ATP synthesis : The F 1 F 0 -ATPase

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

ATP synthesis depends on a peripheral membrane protein Inner mitochondrial (inside-out) vesicles capable of ATP synthesis--note the headpiece of the “F 0 F 1 ATPase” (ATP synthase) Removal of the headpieces gives vesicles that can’t make ATP Adding purified headpieces re- stores the ability to make ATP

AO AO + ATP ADP + Pi H+ AO AO + Add ATP

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

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

Note the rotor (base) and stator (head) and rotation Hypothesis: ADP + Pi ATP + H 2 O occurs on head; H + flow turns rotor; rotor/rotation stimulates ATP release F0 F1

rotor rotation rotor rotation

Figure 1 Observation system for the c subunit rotation in F0F1. Y Sambongi et al. Science 1999;286: Movie:

Movie of the ATPase model -- Mechanical part of respiration.

Model for H+-induced rotation from the textbook:

(How many ATP per FADH 2 ?) 442

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

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