Inhibition of proton transfer in cytochrome c oxidase by zinc ions: delayed proton uptake during oxygen reduction

AbstractWe have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn2+ the transition from the “peroxy” (PR) to the “ferryl” (F) intermediate has a time constant of ∼100 μs and it is associated with proton transfer from the bulk solution with an intrinsic time constant of ≪100 μs, but rate limited by the PR→F transition. While in the presence of 100 μM Zn2+ the PR→F transition was slowed by a factor of ∼2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn2+) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of ∼2.5 ms. Thus, the results show that there is one proton available within the enzyme that can be used for oxygen reduction and confirm our previous observation that F can be formed without proton uptake from the bulk solution. No effect of Zn2+ was observed with a mutant enzyme in which Asp(I-132), at the entry point of the D-pathway, was replaced by its non-protonatable analogue Asn. In addition, no effect of Zn2+ was observed on the F→O transition rate when measured in D2O, because in D2O, the transition is internally slowed to ∼10 ms, which is already slower than with bound Zn2+. Together with earlier results showing that both the PR→F and F→O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn2+ binds to and blocks the entrance of the D-pathway