title: Product-controlled steady-state kinetics between cytochrome aa(3) from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach.
abstract: 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. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Myat T
authors.initials: MT
authors.lastname: Lin
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D045304:Cytochromes c / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006736:Horses; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D045304:Cytochromes c; D003576:Electron Transport Complex IV