title: Insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme.
abstract: Flavodiiron proteins (FDPs) catalyze reductive scavenging of dioxygen and nitric oxide in air-sensitive microorganisms. FDPs contain a distinctive non-heme diiron/flavin mononucleotide (FMN) active site. Alternative mechanisms for the nitric oxide reductase (NOR) activity consisting of either protonation of a diiron-bridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal FMNH(2) in the rate-determining step have been proposed. To test these alternative mechanisms, we examined a deflavinated FDP (deflavo-FDP) from Thermotoga maritima. The deflavo-FDP retains an intact diiron site but does not exhibit multiturnover NOR or O(2) reductase (O(2)R) activity. Reactions of the reduced (diferrous) deflavo-FDP with nitric oxide were examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. Anaerobic addition of nitric oxide up to one NO per diferrous deflavo-FDP results in formation of a diiron-mononitrosyl complex characterized by a broad S = (1)/(2 )EPR signal arising from antiferromagnetic coupling of an S = (3)/(2) {FeNO}(7) with an S = 2 Fe(II). Further addition of NO results in two reaction pathways, one of which produces N(2)O and the diferric site and the other of which produces a stable diiron-dinitrosyl complex. Both NO-treated and as-isolated deflavo-FDPs regain full NOR and O(2)R activities upon simple addition of FMN. The production of N(2)O upon addition of NO to the mononitrosyl deflavo-FDP supports the hyponitrite mechanism, but the concomitant formation of a stable diiron-dinitrosyl complex in the deflavo-FDP is consistent with a super-reduction pathway in the flavinated enzyme. We conclude that a diiron-mononitrosyl complex is an intermediate in the NOR catalytic cycle of FDPs.
authors.affiliation: Department of Science and Engineering, School of Medicine, Oregon Health and Science University, 20000 Northwest Walker Road, Beaverton, Oregon 97006, USA.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hayashi
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Caranto
authors.forename: David A
authors.initials: DA
authors.lastname: Wampler
authors.forename: Donald M
authors.initials: DM
authors.lastname: Kurtz
authors.forename: Pierre
authors.initials: P
authors.lastname: Moënne-Loccoz
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000378:metabolism; D015870:Gene Expression; D007501:Iron / Q000378:metabolism*; D008667:Metalloproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D005415:Flavins; D008667:Metalloproteins; D009569:Nitric Oxide; D007501:Iron; D010088:Oxidoreductases; C034747:nitric-oxide reductase