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

We have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn(2+) the transition from the "peroxy" (P(R)) to the "ferryl" (F) intermediate has a time constant of approximately 100 micros and it is associated with proton transfer from the bulk solution with an intrinsic time constant of <<100 micros, but rate limited by the P(R)-->F transition. While in the presence of 100 microM Zn(2+) the P(R)-->F transition was slowed by a factor of approximately 2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn(2+)) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of approximately 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 Zn(2+) 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 Zn(2+) was observed on the F-->O transition rate when measured in D(2)O, because in D(2)O, the transition is internally slowed to approximately 10 ms, which is already slower than with bound Zn(2+). Together with earlier results showing that both the P(R)-->F and F-->O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn(2+) binds to and blocks the entrance of the D-pathway.