title: A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP.
abstract: MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein fromEscherichia coliWe optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify theE. coliflavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Céline
authors.initials: C
authors.lastname: Juillan-Binard
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Antoine
authors.initials: A
authors.lastname: Picciocchi
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Andrieu
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Jerome
authors.identifier: 0000-0002-2721-5365
authors.initials: J
authors.lastname: Dupuy
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Petit-Hartlein
authors.affiliation: the Université Grenoble Alpes, Grenoble.|CNRS LCBM UMR 5249, Grenoble, and.|CEA-DRF-BIG-Laboratoire de Chimie et Biologie des Métaux, 17 Rue des Martyrs, 38054 Grenoble, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Caux-Thang
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Corinne
authors.initials: C
authors.lastname: Vivès
authors.affiliation: the Université Grenoble Alpes, Grenoble.|CNRS LCBM UMR 5249, Grenoble, and.|CEA-DRF-BIG-Laboratoire de Chimie et Biologie des Métaux, 17 Rue des Martyrs, 38054 Grenoble, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Nivière
authors.affiliation: From the Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 38044 Grenoble, fieschi@ibs.fr.|the IBS, Commissariat à l'Energie Atomique (CEA), 38027 Grenoble.|the IBS, CNRS, 38027 Grenoble.
authors.forename: Franck
authors.identifier: 0000-0003-1194-8107
authors.initials: F
authors.lastname: Fieschi
mesh_terms: D000595:Amino Acid Sequence; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D020349:Surface Plasmon Resonance
chemical_list: D049452:Green Fluorescent Proteins; D019255:NADPH Oxidases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase
keywords: Escherichia coli (E. coli); NADPH oxidase; YedZ; electron transfer; flavin mononucleotide (FMN); flavin reductase Fre; heme; membrane protein; methionine sulfide reductase MsrPQ