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 Zn2+ the transition from the "peroxy" (P-R) to the "ferryl" (F) intermediate has a time constant of similar to100 mus and it is associated with proton transfer from the bulk solution with an intrinsic time constant of << 100 mus, but rate limited by the P-R-->F transition. While in the presence of 100 muM Zn2+ the P-R-->F transition was slowed by a factor of similar to2, 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 similar to 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 similar to 10 ms, which is already slower than with bound Zn2+. 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 Zn2+ binds to and blocks the entrance of the D-pathway. (C) 2002 Elsevier Science B.V. All rights reserved.