On the stoichiometry and thermodynamics of proton-pumping cytochrome c oxidase in mitochondria.
Different approaches have been used to evaluate the stoichiometry of proton translocation linked to cytochrome c oxidase in rat liver mitochondria. A mathematical model was designed that successfully describes the kinetics of redox-linked proton translocation provided that the rate of electron transfer is not too high. With ascorbate as reductant, an essentially pH-independent (in the pH range 6--8.5) proton ejection stoichiometry (H+/e-) is obtained from either initial rates of H+ ejection (0.86 +/- 0.12), or the model (0.87 +/- 0.14). Similar results are obtained with either ferrocyanide, N.N.N',N'-tetramethyl-p-phenylenediamine or externally added cytochrome c mediating between ascorbate and cytochrome c in rotenone- and antimycin-inhibited mitochondria. Oxygen pulse experiments with ferrocytochrome c as substrate show fully uncoupler-sensitive redox-linked proton ejection with a stoichiometry of 0.78 +/- 0.14. With murexide to measure Ca2+ uptake during oxidation of ferrocyanide, we found a stoichiometry of two positive charges taken up/electron transferred, confirming earlier findings. These results provide strong evidence that cytochrome c oxidase functions as a redox-linked proton pump with a stoichiometry of one H+ ejected and two charges translocated/electron transferred. The thermodynamic consequences of the proton pump are discussed and a maximal P/O ratio of 1 1/3 for 'site 3' is predicted in agreement with state 4 redox potentials and phosphate potential.
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D010084:Oxidation-Reduction; D051381:Rats; D012402:Rotenone; D013816:Thermodynamics
D012402:Rotenone; D000968:Antimycin A; D003576:Electron Transport Complex IV