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 Zn(2+) 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 Zn(2+) concentration. Half-inhibition of proton pumping was observed at [Zn(2+)](i)=75 microM corresponding to about 5-6 Zn(2+) ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn(2+). Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn(2+) correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn(2+) ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase.