Zinc ions inhibit oxidation of cytochrome c oxidase by oxygen

Abstract Cytochrome c oxidase is a membrane-bound enzyme that catalyses the reduction of O 2 to H 2 O and uses part of the energy released in this reaction to pump protons across the membrane. We have investigated the effect of addition of Zn 2+ on the kinetics of two reaction steps in cytochrome c oxidase that are associated with proton pumping; the peroxy to oxo-ferryl (P r →F) and the oxo-ferryl to oxidised (F→O) transitions. The Zn 2+ binding resulted in a decrease of the F→O rate from 820 s −1 (no Zn 2+ ) to a saturating value of ∼360 s −1 with an apparent K D of ∼2.6 μM. The P r →F rate (∼10 4 s −1 before addition of Zn 2+ ) decreased more slowly with increasing Zn 2+ concentration and a K D of ∼120 μM was observed. The effects on both kinetic phases were fully reversible upon addition of EDTA. Since both the P r →F and F→O transitions are associated with proton uptake through the D-pathway, a Zn 2+ -binding site is likely to be located at the entry point of this pathway, where several carboxylates and histidine residues are found that may co-ordinate Zn 2+ .