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

Abstract

We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa3) 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 approximately 5000 s-1 and approximately 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 approximately 1.9, which implies that upon reduction, the enzyme has to pick up approximately 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 CuA 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 O2 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.

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

“…4A). As observed earlier (25), with the wild-type CytcO, two kinetic phases are seen, with time constants corresponding to those of the P R → F (∼45% of the total amplitude) and F → O reactions (∼55%). With the N139T/D132N double-mutant CytcO, the absorbance changes at pH 7.8 displayed two components with rate constants of 8,000 ± 250 s −1 (τ ≅ 130 μs; ∼70% of the total amplitude) and 1,200 ± 100 s −1 (τ ≅ 800 μs; ∼30% of the total amplitude).…”

Vectorial proton transfer coupled to reduction of O 2 and NO by a heme-copper oxidase

“…3), we estimate that Ϸ2 H ϩ are taken up when O 2 is the oxidant (similar to the scenario in aa 3 oxidases where Ϸ2 H ϩ are taken up during oxidation and Ϸ2 H ϩ are ''preloaded'' on reduction (see, e.g., ref. 21). For simplicity, we will assume that the active site is located halfway through the membrane dielectric.…”

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

“…all four redox centers reduced) with dioxygen (see Fig. 2), binding of O 2 to reduced heme a 3 is followed by electron transfer from heme a to the catalytic site with a time constant of ϳ50 s (24), forming an intermediate, P R , which presumably has the same chemical structure as P M , with the only difference being an additional electron at the catalytic site in P R (25,26). As with P M , formation of P R is not associated with proton uptake from solution (22,23).…”