title: Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase.
abstract: Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-β-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a μ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2or NO permeation were found. The present data represent the first structure for a NO-selective FDP.
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Célia V
authors.initials: CV
authors.lastname: Romão
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: João B
authors.initials: JB
authors.lastname: Vicente
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Patrícia T
authors.initials: PT
authors.lastname: Borges
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Bruno L
authors.initials: BL
authors.lastname: Victor
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Lamosa
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Elísio
authors.initials: E
authors.lastname: Silva
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Luís
authors.initials: L
authors.lastname: Pereira
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal; Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
authors.forename: Tiago M
authors.initials: TM
authors.lastname: Bandeiras
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Cláudio M
authors.initials: CM
authors.lastname: Soares
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Carrondo
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: David
authors.initials: D
authors.lastname: Turner
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal. Electronic address: miguel@itqb.unl.pt.
authors.forename: Miguel
authors.initials: M
authors.lastname: Teixeira
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Av. da República, 2780-157 Oeiras, Portugal. Electronic address: frazao@itqb.unl.pt.
authors.forename: Carlos
authors.initials: C
authors.lastname: Frazão
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009569:Nitric Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D014157:Transcription Factors; C451400:YgaK protein, E coli; D009569:Nitric Oxide; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D010100:Oxygen
keywords: NorV; diiron center; flavodiiron protein; flavorubredoxin; nitric oxide