Reduction of cytochrome oxidase by 5,10-dihydro-5-methylphenazine: kinetic parameters from rapid-scanning stopped-flow experiments

Abstract

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.

The use of principal component analysis to resolve the spectra and kinetics of cytochrome c oxidase reduction by 5,10-dihydro-5-methyl phenazine

“…The results, shown in Fig. 4, provide a striking confirmation of the observations described previously (Halaka et al, 1981(Halaka et al, , 1984, that the decay of the oxidized cytochrome a3 lags that of the oxidized a. Also, the growth of the reduced heme (a + a3) band results from an averaging of the two profiles.…”

“…Cytochrome oxidase was prepared as described by Hartzell and Beinert (1974); the details of the handling of the enzyme and of the apparatus used in recording the stopped flow, rapid scan data are given elsewhere (Halaka et al, 1981(Halaka et al, , 1984. Principal component analysis was carried out in detail on data obtained in two separate studies with two different forms of the oxidized protein (for biochemical details [Halaka et al, 1984]): the resting enzyme with a Soret absorption maximum as isolated at 418 nm (designated the 418-nm species) and the resting enzyme with a Soret absorption maximum as isolated at 424 nm (designated as the 424-nm species). Applying PCA to these forms complements our previous work on the kinetics of the anaerobic four-electron reduction of the oxidase (Halaka et al, 1981).…”

“…For the past few years, we have been using rapid scanning stopped-flow spectrophotometry (RSSFS) for the study of the electron transfer kinetics of cytochrome oxidase (Halaka et al, 1981(Halaka et al, , 1984. In RSSFS, a particular region of the absorption spectrum of the compounds under study is rapidly and repeatedly scanned.…”

“…The results indicated that MPH interacts with the a site of the oxidase (Halaka et al, 1981(Halaka et al, , 1984. Other reductants, for example, hexaaquochromium(II) (Greenwood et al, 1977) and hexaammineruthenium(II) (Scott and Gray, 1980), have also been used in reduction studies of the enzyme.…”

A modified stopped-flow apparatus for time-resolved protein phosphorescence

“…In recent years, the time resolution of many important spectroscopic methods, such as nuclear magnetic resonance ͑NMR͒, 1-3 fluorescence emission, 4,5 circular dichroism in the near-and far-ultraviolet ͑UV͒, 6-8 infrared absorption, 9,10 electron paramagnetic resonance ͑EPR͒, 11 and Raman scattering, 12 has improved considerably, down to the millisecond range, with their implementation in the stoppedflow technique.…”