Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase

We have investigated the electron-proton coupling during the peroxy (PR) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of CUA is increased by similar to 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of similar to 200. In ED(I-286) proton uptake during P-R --> F was slowed by a factor of similar to 5, which indicates that E(I-286) is the proton donor to PR. In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of similar to 2.5 as compared with similar to 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Angstrom delroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of similar to 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled. (C) 2000 Elsevier Science B.V. All rights reserved.