Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes.
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 pKa 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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011522:Protons
D008081:Liposomes; D011522:Protons; D003576:Electron Transport Complex IV; D008274:Magnesium; D002118:Calcium