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

Abstract

Cytochrome c oxidase (Cyt cO) 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 Zn 2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides Cyt cO with O 2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn 2+. The results show that addition of Zn 2+ at concentrations of ≤250 μM 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 Zn 2+ concentrations of ≤ 25 μM. Furthermore, proton pumping during the P 3 → F 3 transition was typically impaired with the “as isolated” Cyt cO, which was found to contain Zn 2+ ions at μM concentration. As has already been shown, Zn 2+ 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 ∼ 1 μM for this binding site. In conclusion, the results show that Zn 2+ ions bind on both sides of Cyt cO and that binding of Zn 2+ at the proton output side selectively impairs proton release during the P 3 → F 3 transition.