Characteristics of the redox‐linked proton ejection in beef‐heart cytochrome c oxidase reconstituted in liposomes

Abstract

In this paper a study is presented of the characteristics of redox‐linked proton ejection exhibited by isolated beef‐heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented ‘right‐side out’ as in the mitochondrial membrane. The effects on the H⁺/e⁻ stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K⁺ to ensure charge compensation produced H⁺/e⁻ ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H⁺/e⁻ ratios around 0.3. The H⁺/e⁻ ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0–7.7, which decreased to negligible values at acidic pH with apparent pK_a of 6.7–6.3. Mg²⁺ and Ca²⁺ caused a marked depression of the H⁺/e⁻ ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox‐linked proton ejection in reconstituted cytochrome c oxidase.