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Cytochrome c oxidase; Proton pumping; Zn2+ binding; Black lipid membrane; Proton slip
Abstract:
Using a combination of stopped-flow spectrophotometric proton pumping measurements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn2+ ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/e stoichiometry was found to gradually decrease with increasing Zn2+ concentration. Half-inhibition of proton pumping was observed at [Zn2+]i=75 μM corresponding to about 5–6 Zn2+ ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn2+. Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn2+ correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn2+ ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase.
