title: The roles of the hybrid cluster protein, Hcp and its reductase, Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress.
abstract: The hybrid cluster protein, Hcp, contains a 4Fe-2S-2O iron-sulfur-oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N2 O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron-sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp-hcr operon encodes an NADH-dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co-purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO.
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Vine
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Basema K
authors.initials: BK
authors.lastname: Balasiny
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: John
authors.initials: J
authors.lastname: Rizk
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Charlene L
authors.initials: CL
authors.lastname: Bradley
authors.affiliation: Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
authors.forename: Mariana
authors.initials: M
authors.lastname: Tinajero-Trejo
authors.affiliation: Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
authors.affiliation: Norwegian University of Life Science, PO box 5003, N-1432, Ås, Norway.
authors.forename: Linda L
authors.initials: LL
authors.lastname: Bergaust
authors.affiliation: Norwegian University of Life Science, PO box 5003, N-1432, Ås, Norway.
authors.forename: Lars R
authors.initials: LR
authors.lastname: Bakken
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Cole
mesh_terms: D000693:Anaerobiosis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D015538:Nitrosation; D009876:Operon; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009566:Nitrates; C000621362:hybrid cluster protein, E coli; D009569:Nitric Oxide; D010088:Oxidoreductases; C000621361:hybrid cluster reductase, E coli; C034747:nitric-oxide reductase