title: An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III).
abstract: As part of the machinery to acquire, internalize and utilize heme as a source of iron from the host, some bacteria possess a canonical heme oxygenase, where heme plays the dual role of substrate and cofactor, the later catalyzing the cleavage of the heme moiety using O2 and electrons, and resulting in biliverdin, carbon monoxide and ferrous non-heme iron. We have previously reported that the Escherichia coli O157:H7 ChuS protein, which is not homologous to heme oxygenases, can bind and degrade heme in a reaction that releases carbon monoxide. Here, we have pursued a detailed characterization of such heme degradation reaction using stopped-flow UV-visible absorption spectrometry, the characterization of the intermediate species formed in such reaction by EPR spectroscopy and the identification of reaction products by NMR spectroscopy and Mass spectrometry. We show that hydrogen peroxide (in molar equivalent) is the key player in the degradation reaction, at variance to canonical heme oxygenases. While the initial intermediates of the reaction of ChuS with hydrogen peroxide (a ferrous keto π neutral radical and ferric verdoheme, both identified by EPR spectroscopy) are in common with heme oxygenases, a further and unprecedented reaction step, involving the cleavage of the porphyrin ring at adjacent meso-carbons, results in the release of hematinic acid (a monopyrrole moiety identified by NMR spectroscopy), a tripyrrole product (identified by Mass spectrometry) and non-heme iron in the ferric oxidation state (identified by EPR spectroscopy). Overall, the unprecedented reaction of E. coli O157:H7 ChuS provides evidence for a novel heme degradation activity in a Gram-negative bacterium.
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS and PROTEO, Université Laval, Québec G1V 0A6, Canada.
authors.forename: Yannick H
authors.initials: YH
authors.lastname: Ouellet
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS and PROTEO, Université Laval, Québec G1V 0A6, Canada.
authors.forename: Cheikh Tidiane
authors.initials: CT
authors.lastname: Ndiaye
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS and PROTEO, Université Laval, Québec G1V 0A6, Canada.
authors.forename: Stéphane M
authors.initials: SM
authors.lastname: Gagné
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS and PROTEO, Université Laval, Québec G1V 0A6, Canada.
authors.forename: Anne
authors.initials: A
authors.lastname: Sebilo
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Michael D L
authors.initials: MD
authors.lastname: Suits
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS and PROTEO, Université Laval, Québec G1V 0A6, Canada.
authors.forename: Éric
authors.initials: É
authors.lastname: Jubinville
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
authors.affiliation: CNRS, Unité de Recherche Mixte CNRS/Aix-Marseille Université (UMR 7281), Laboratoire de Bioénergétique et Ingénierie des Protéines, 13009 Marseille, France. Electronic address: aivancich@imm.cnrs.fr.
authors.forename: Anabella
authors.initials: A
authors.lastname: Ivancich
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, IBIS and PROTEO, Université Laval, Québec G1V 0A6, Canada. Electronic address: Manon.Couture@bcm.ulaval.ca.
authors.forename: Manon
authors.initials: M
authors.lastname: Couture
mesh_terms: D019453:Escherichia coli O157 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D006418:Heme / Q000737:chemistry*; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry* / Q000502:physiology; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D011422:Propionates / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry
chemical_list: C000610974:ChuS protein, E coli; D029968:Escherichia coli Proteins; D008301:Maleimides; D011422:Propionates; D011725:Pyridines; D011758:Pyrroles; D006418:Heme; C090385:hematinic acid; D006861:Hydrogen Peroxide; D007501:Iron; D006419:Heme Oxygenase (Decyclizing)
keywords: ChuS; EPR spectroscopy; Hematinic acid; Heme degradation; Iron; Verdoheme