The Nitric Oxide Reductase Mechanism of a Flavo-Diiron Protein: Identification of Active-Site Intermediates and Products

[Image: see text] The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH(2)–Fe(II)Fe(II)) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mössbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (S = 0, [{FeNO}(7)](2)) species. This species has an exchange energy, J ≥ 40 cm(–1) (JS(1) ° S(2)), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl (S = (1)/(2), Fe(II){FeNO}(7)) and the S = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (Fe(III)Fe(III)) and by inference N(2)O. The proximal FMNH(2) then rapidly rereduces the diferric site to diferrous (Fe(II)Fe(II)), which can undergo a second 2NO → N(2)O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N(2)O as a product ( HayashiBiochemistry2010, 49, 704020669924). Our results do not support other proposed mechanisms, which proceed either via “super-reduction” of [{FeNO}(7)](2) by FMNH(2) or through Fe(II){FeNO}(7) directly to a diferric-hyponitrite intermediate. The results indicate that an S = 0 [{FeNO}(7)}](2) complex is a proximal precursor to N–N bond formation and N–O bond cleavage to give N(2)O and that this conversion can occur without redox participation of the FMN cofactor.