Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated to oxidoreductions of the oxygen-terminal respiratory carriers

Abstract

The kinetics and stoicheiometry of fast proton translocation associated to aerobic oxidation of the oxygen-terminal components of the mitochondrial respiratory chain have been analyzed by means of continuous- and stopped-flow techniques. 1. 1. In intact mitochondria the aerobic oxidation of the respiratory carriers situated on the oxygen side of the antimycin site was accompanied by synchronous release of protons. When the proton conductivity of the membrane was increased by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, oxidation of the terminal respiratory carriers was accompanied by stoicheiometric consumption of protons. This proton disappearance from the medium was, however, much slower than the oxidation of the respiratory carriers. 2. 2. In sonic sub-mitochondrial particles oxidation of the terminal respiratory carriers was accompanied by synchronous and stoicheiometric proton consumption. This proton uptake was practically unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone; its rate was markedly increased by valinomycin plus K +. 3. 3. The results presented provide functional evidence that cytochrome oxidase is a transmembranous molecule with haeme a 3 reacting with oxygen at the matrix side of the inner mitochondrial membrane and haeme a reacting with cytochrome c at the outer side. 4. 4. The fast proton release accompanying the oxidation of the terminal respiratory carriers in intact mitochondria appears to be associated to antimycin-insensitive oxidation of a hydrogen carrier.