Internal Electron Transfer in Cu-Heme Oxidases: Thermodynamic or Kinetic Control?

Maurizio Brunori, Alessandro Giuffre, Emilio D'Itri and Paolo Sarti J. Biol. Chem. in review 1997

SUMMARY: The internal electron transfer in beef heart cytochrome c oxidase has been re-investigated by stopped-flow spectroscpoy using a preparation of "fast" enzyme. We provide novcel experimental evidence that, starting from oxidized cytochrome c oxidase, electron transfer from cyt. a to cyt. a₃ is kinetically controlled. The anaerobic reduction of the oxidized enzyme has been followed in the presence or absence of CO or NO, using ruthenium hexammine as electron donor.In the absence of gaseous ligands the time coursrse of reduction of cyt. a₃ yields an apparent rate constant of k' = 16 +/-2 sec^-1 (20'C, pH 7). In the presence of NO acting as an "trapping" ligand the formation of the cyt. a₃²⁺-NO adduct was found to be independent of NO which indicates that in the oxidized enzyme cyt. a and cyt. a₃ are not in very rapid redox equilibrium. On the other hand, CO is a poor trapping ligand because its combination rate constant is not sufficiently high. We conclude that the intrinsic rate constant for electron transfer starting with the oxidized enzyme is 25 sec^-1. These new data are discussed with reference to the model propsoed by Verkhovsky et al (Biochemistry 34 7483-7491 (1995)) in which H⁺ diffusion and/or binding at the binuclea center is the rate limiting step in the reduction of cytochrome a₃, starting from oxidized enzyme.