ATP synthesis : The F1F0-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 “F0F1 ATPase” (ATP synthase) Removal of the headpieces gives vesicles that can’t make ATP Adding purified headpieces restores the ability to make ATP “Chemiosmotic hypothesis” (Peter Mitchell): H+ passage across membrane powers ATP synthesis How does it work? AO AO AO Clues: 1. Reversed coupling: ATP hydrolysis powers H+ flow. AO AO AO AO Add ATP M embrane vesicle containing ATP synthase + AOAO (outside) + + AO H+ + • Solution of acrydine orange (AO) AO (membrane-permeable fluorescent dye) AO+ • Add ATP, vesicle interior becomes acidic ATP ADP + Pi • AO AO+, trapped inside vesicle • High concentration of AO quenches fluorescence • 2. H+ flow in artificial vesicle powers ATP synthesis. Light H+ Bacteriorhodopsin H+ H+ H+ Add ADP H+ H+ H+ H+ ADP + Pi ATP 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) cytosol FCCP intermembrane space FCCP FCCP-H+ H+ H+ H+ H+ matrix H+ H+ (FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone) 4. Reversible oxygen exchange 18 O from H218O incorporated into HPO42- in the presence of the ATP synthase Without unidirectional H+ flow, ADP + Pi ATP + H2O occurs reversibly, implying that the application of energy is not at the formation of the ADP~P bond(!) 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 rotor rotation rotor rotation 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 Movie of the ATPase model -- Mechanical part of respiration. http://multimedia.mcb.harvard.edu/anim_mitochondria.html Model for H+-induced rotation from the textbook: What is the P:O ratio? ATP formed: 1/2 O2 (2 e-) taken up (NADH oxidized) For TCA cycle, early estimates: per NADH, 3; per succinate (FAD), 2 Best data: per NADH, 2.5; per succinate (FAD), 1.5 Book: 10 H+ per NADH, 4 H+ per ATP = 2.5 (Related to the Fo structure: 12 H+/Fo rotation, 3 ATP/Fo rotation = 4 H+/ATP; recall 10 H+/NADH; (10 H+/NADH)/(4 H+/ATP) = 2.5 ATP/NADH) Recent evidence: H+/ATP varies from 12/3 (4) in animals to 15/3 (5) in microbes, depending on the number of Fo subunits in the Fo ring in the membrane. [See Science 330:12 (1 Oct 10) or PNAS 107:16823] (How many ATP per FADH2?) 4 4 2 ATP from cytoplasmic NADH There is a problem with NADH from glycolysis: inner mitochondrial membrane is not permeable to NADH Glycerophosphate shuttle (animals): Electron input at Complex II FAD: expect 1.5 ATP/NADH Malate-aspartate shuttle (animals and plants) How much ATP/NADH? Summary Mitochondrial electron transport produces H+ gradient + ! H gradient rotates the ATP synthase ! ATP synthase rotation forces ATP synthesis (release) ! NADH reducing power from cytoplasm must be shuttled into the mitocondrion ! ATP/glucose ratio depends on shuttle and on Fo !