Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase

Cytochrome e oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O-2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of <= 250 mu M to the outside of the vesicles did not alter the transition rates between intermediates P-R (P-3) -> F-3 -> O-4. However, proton pumping was impaired specifically during the P-3 -> F-3, but not during the F-3 -> O-4 transition at Zn2+ concentrations of <= 25 mu M. Furthermore, proton pumping during the P-3 -> F-3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at mu M concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P-3 -> F-3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a K-I of similar to 1 mu M for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P-3 -> F-3 transition. (c) 2006 Elsevier B.V. All rights reserved.