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Product-controlled Steady-State Kinetics Between Cytochrome aa(3) From Rhodobacter Sphaeroides and Equine Ferrocytochrome C Analyzed by a Novel Spectrophotometric Approach

Product-controlled Steady-State Kinetics Between Cytochrome aa(3) From Rhodobacter Sphaeroides and Equine Ferrocytochrome C Analyzed by a Novel Spectrophotometric Approach

Myat T Lin, Robert B Gennis

Biochim Biophys Acta. 2012 Oct;1817(10):1894-900.

PMID: 22516686

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).

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42412104	Cytochrome c equine			Price

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