title: Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition.
abstract: Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO(-)), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO(-) to cytochrome bd in turnover with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤15%) protein fraction, due to the NO generated from ONOO(-) and not to ONOO(-) itself. Consistently, addition of ONOO(-) to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO(-) decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO(-) with an apparent turnover rate as high as ~10 mol ONOO(-) (mol enzyme)(-1) s(-1) at 25°C. To the best of our knowledge, this is the first time that the kinetics of ONOO(-) decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO(-) damage.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation.
authors.forename: Vitaliy B
authors.initials: VB
authors.lastname: Borisov
authors.affiliation: Department of Biochemical Sciences and Istituto Pasteur - Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Forte
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Siletsky
authors.affiliation: Department of Biochemical Sciences and Istituto Pasteur - Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy; CNR Institute of Molecular Biology and Pathology, Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Sarti
authors.affiliation: CNR Institute of Molecular Biology and Pathology, Rome, Italy. Electronic address: alessandro.giuffre@uniroma1.it.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Giuffrè
mesh_terms: D055162:Biocatalysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000502:physiology*; D045222:Electron Transport Chain Complex Proteins / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000502:physiology*; D010088:Oxidoreductases / Q000502:physiology*; D010101:Oxygen Consumption; D030421:Peroxynitrous Acid / Q000378:metabolism*
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D030421:Peroxynitrous Acid; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli
keywords: Cytochrome bd; Escherichia coli; Immune response; Nitrosative and oxidative stress; Peroxynitrite; Reactive nitrogen species