Internal Electron Transfer in Cu-Heme Oxidases THERMODYNAMIC OR KINETIC CONTROL?

Abstract 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 a 3. In the presence of NO, the rate of formation of the cytochromea 3 2+-NO adduct is independent of the concentration of ruthenium hexamine and of NO, indicating that in the oxidized enzyme cytochrome a anda 3 are not in very rapid redox equilibrium; on the other hand, CO proved to be a poor "trapping" ligand. We conclude that the intrinsic rate constant for a →a 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 a 3 in the oxidized enzyme