INTERNAL ELECTRON-TRANSFER IN CU-HEME OXIDASES - THERMODYNAMIC OR KINETIC CONTROL

We present novel experimental evidence that, starting with the oxidize d enzyme, the internal electron transfer in cytochrome c oxidase is ki netically controlled, The anaerobic reduction of the oxidized enzyme b y 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 hexamin e and of NO, indicating that in the oxidized enzyme cytochrome a and a s are not its very rapid redox equilibrium; on the other hand, CO prov ed to be a poor ''trapping'' ligand, We conclude that the intrinsic sa te constant for alpha --> alpha(3) electron transfer in the oxidized e nzyme 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 s ite is the rate-limiting step in the reduction of cytochrome alpha(3) in the oxidized enzyme.