Zn2+ binding to the cytoplasmic side of Paracoccus denitrificans cytochrome c oxidase selectively uncouples electron transfer and proton translocation

Using a combination of stopped-flow spectrophotometric proton pumping measu rements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn2+ ions on the proton transfer propert ies of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclose d in the interior of cytochrome c oxidase containing liposomes, the H/e sto ichiometry was found to gradually decrease with increasing Zn2+ concentrati on. Half-inhibition of proton pumping was observed at [Zn2+](i) = 75 muM co rresponding to about 5-6 Zn2+ ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteol iposomes upon incorporation of Zn2+. Time-resolved potential measurements o n a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn2+ correspond to phases that have been attributed to pro ton uptake from the cytoplasmic side and to proton pumping. We conclude tha t Zn2+ ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase. (C) 2001 Federation of European Biochemica l Societies. Published by Elsevier Science B.V. All rights reserved.