Product-controlled steady-state kinetics between cytochrome aa₃ from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c^{2 +}) as the electron donor. In this study, the oxidation of horse cyt c^{2 +} by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c^{2 +} and cyt c^{3 +}. This allowed an analysis of the effects of cyt c^{3 +} on the steady-state kinetics between CcO and cyt c^{2 +}. The results show that cyt c^{3 +} exhibits product inhibition by two mechanisms: competition with cyt c^{2 +} at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c ^{2 +} at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics.