Product-controlled steady-state kinetics between cytochrome aa3 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).
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ABSTRACT: Subunit 5 of Saccharomyces cerevisiae CcO is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions while 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory-deficient. Previous studies reported that respiratory growth could be restored by combining Δcox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants 5B isozyme expression level was 30-50 % of wild type (5A isozyme) and exhibited a maximum catalytic activity up to 3-fold faster than that of 5A isozyme. To investigate the origin of this effect, we constructed a mutant strain in which COX5B replaced COX5A downstream of the COX5A promoter. This strain expressed wild type levels of the 5B isozyme, without the complication of additional effects caused by mutation of ROX1. When produced this way, the isozymes displayed no significant differences in their maximum catalytic activities or in their affinities for oxygen or cytochrome c. Hence, the elevated activity of the 5B isozyme in the rox1 mutant is not caused simply by exchange of isoforms and must arise from an additional effect that remains to be resolved.Biochimica et Biophysica Acta (BBA) - Bioenergetics 09/2014; DOI:10.1042/BJ20140732 · 4.83 Impact Factor