Proton transfer from glutamate 286 determines the transition rates betweenoxygen 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 f rom Rhodobacter sphaeroides. The kinetics of these reactions were investiga ted 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 ha s a shorter side chain than that of the glutamate and, (2) ML(II-263), in w hich the redox potential of CUA is increased by similar to 100 mV, which sl ows electron transfer to the binuclear centre during the F-->O transition b y a factor of similar to 200. In ED(I-286) proton uptake during P-R --> F w as 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 wi th 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 i n which the proton donor and acceptor must approach each other (M. Karpefor s, P. Angstrom delroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the sm aller 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-26 3) the electron-transfer to the binuclear centre is slower than the intrins ic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope e ffect of similar to 8, i.e., about the same as in the wild-type enzyme, whi ch shows that these reactions are intimately coupled. (C) 2000 Elsevier Sci ence B.V. All rights reserved.