Factors determining electron-transfer rates in cytochrome c oxidase:: investigation of the oxygen reaction in the R-sphaeroides enzyme

We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa(3)) from Rhodobacter sphaeroides with dioxygen using the flow-flash methodology and compared the results to those obtained with the well-characterised bovine mitochondrial enzyme. The overall reaction sequence was the same in the two enzymes, but the extents and rates of the electron-transfer reactions differed, implying differences in redox potentials, and/or interaction energies between electrons and protons during oxygen reduction. As with the bovine enzyme, the R. sphaeroides enzyme displayed two major kinetic phases of proton uptake with rate constants of similar to 5000 s(-1) and similar to 500 s(-1) at pH 7.9, concomitant with the peroxy to oxoferryl and oxoferryl to oxidised states. The net number of protons taken up in the R sphaeroides enzyme was about similar to 1.9, which implies that upon reduction, the enzyme has to pick up similar to 2.1 H+ from the medium. On the basis of the comparison of electron-transfer reactions in the two enzymes, we conclude that the transfer rate of the fourth electron to the binuclear centre is not only determined by the electron-transfer rate from haem a to the binuclear centre, but also by the electron equilibrium between Cu-A and haem a. In addition, in contrast to the bovine enzyme, where the electron- and proton-transfer rates during oxidation of the fully reduced enzyme by O-2 are all faster than the overall turnover rate, in the R sphaeroides enzyme, the slowest kinetic phase was rate limiting for the overall turnover. Moreover, the comparison of the reactions in the two systems shows that in the R. sphaeroides enzyme, the electrons are more evenly distributed among the redox centres during oxygen reduction. This enables investigations of effects also of minor perturbations on, e.g., the electron-transfer characteristics in mutant enzymes, for which this study forms the basis. (C) 1998 Elsevier Science B.V. All rights reserved.