On the stoichiometry and thermodynamics of proton-pumping cytochrome c oxidase in mitochondria

Abstract

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 Ca 2+ 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.