Reduction of cytochrome oxidase by 5,10-dihydro-5-methylphenazine: kinetic parameters from rapid-scanning stopped-flow experiments.
The kinetics of the reduction of resting cytochrome oxidase and of its cyanide complex by 5,10-dihydro-5- methylphenazine (MPH) have been characterized by rapid-scan and fixed-wavelength stopped-flow spectrophotometry in the Soret, visible, and near-IR spectral regions. In this study, we focused on a form of the resting enzyme that is characterized by a Soret absorption maximum at 424 nm. These experiments complement earlier work on the reduction of a 418 nm absorbing form of the resting enzyme [ Halaka , F.G., Babcock , G. T., & Dye, J. L. (1981) J. Biol. Chem. 256, 1084-1087]. The reduction of cytochrome a is accomplished in a second-order reaction with a rate constant of 3 X 10(5) M-1 s-1. The reduction of the 830-nm absorber, Cua, is closely coupled to but lags the reduction of cytochrome a; we have resolved a rate constant of about 20 s-1 for the copper reduction. The reduction of cytochrome a proceeds with a rate constant that is nearly independent of the spectral properties of the resting enzyme and of the ligation state of cytochrome a3. The reduction of cytochrome a3 occurs by slow, intramolecular electron transfer. We have resolved two phases for this process that have rate constants of approximately 0.2 s-1 and approximately 0.02 s-1 for both the 418- and 424-nm forms of the resting enzyme. It appears, therefore, that spectroscopic heterogeneity at the cytochrome a3 site in the resting enzyme exerts very little influence on the kinetics of the anaerobic reduction of the oxidase metal centers. From this we conclude that the rate of electron transfer to the a3 site is probably controlled by the protein conformation and not primarily by local factors within the a3 environment.
D003576:Electron Transport Complex IV / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010619:Phenazines / Q000494:pharmacology*; D013053:Spectrophotometry
D010619:Phenazines; C041626:5,10-dihydro-5-methylphenazine; D003576:Electron Transport Complex IV