Internal electron transfer in Cu-heme oxidases - Thermodynamic or kinetic control?

We present novel experimental evidence that, starting with the oxidized enzyme, the internal electron transfer in cytochrome c oxidase is kinetically controlled, The anaerobic reduction of the oxidized enzyme by ruthenium hexamine has been followed in the absence and presence of CO or NO, used as trapping ligands for reduced cytochrome alpha(3). In the presence of NO, the rate of formation of the cytochrome alpha(3)(2+)-NO adduct is independent of the concentration of ruthenium hexamine and of NO, indicating that in the oxidized enzyme cytochrome a and as are not its very rapid redox equilibrium; on the other hand, CO proved to be a poor ''trapping'' ligand, We conclude that the intrinsic sate constant for alpha --> alpha(3) electron transfer in the oxidized enzyme is 25 s(-1). These data are discussed with reference to a model (Verkhovsky, M.I., Morgan, J.E., and Wikstrom, M. (1995) Biochemistry 34, 7483-7491) in which H+ diffusion and/or binding at the binuclear site is the rate-limiting step in the reduction of cytochrome alpha(3) in the oxidized enzyme.