Intermediates Trapped during Nitrogenase Reduction of N⋮N, CH3−NNH, and H2N−NH2

Barney, Brett M.; Yang, Tran Chin; Igarashi, Robert Y.; Santos, Patricia C. Dos; Laryukhin, Mikhail; Lee, Hong In; Hoffman, Brian M.; Dean, Dennis R.; Seefeldt, Lance C.

doi:10.1021/ja0539342

Cited by 123 publications

(136 citation statements)

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“…The trapping of substrate-derived species on FeMo-cofactor can be monitored by the change in the EPR spectrum of the FeMo-cofactor (27,36,(44)(45)(46)(47)(48)(49). In its resting state (called M N ), FeMo-cofactor is in an S = 3/2 spin state with a characteristic EPR spectrum in the perpendicular mode with g values of 4.45, 3.56, and 2.00 ( Figure 6).…”

Section: Trapping a Diazene-derived Species Bound To Femo-cofactormentioning

confidence: 99%

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

et al. 2007

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Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N 2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H 2 N-NH 2 ). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH 3 . Diazene reduction, like N 2 reduction, is inhibited by H 2 . This contrasts with the lack of H 2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N 2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with α-195 His substituted by Gln and α-70 Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g 1 = 2.09, g 2 = 2.01, and g 3 ~ 1.98. 15 N-and 1 H-ENDOR establish that this state consists of a diazene-derived [-NH x ] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NH x ] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N 2 -reduction pathway, and that the diazene-and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N 2 by nitrogenase. The implications of these findings for the mechanism of N 2 reduction by nitrogenase are discussed. KeywordsFeMo-cofactor; EPR; ENDOR; Active Site; Substrate Nitrogenase is the enzyme responsible for catalyzing biological reduction of N 2 to two NH 3 , an essential reaction in the global biogeochemical nitrogen cycle (1-3). The minimum stoichiometry for the nitrogenase catalyzed reduction of N 2 involves delivery of 8e − and 8H + (eqn 1). † This work was supported by grants from the National Institutes of Health (R01-GM59087 to LCS and DRD; HL13531 to BMH), the National Science Foundation (MCB-0316038 to BMH) and the United States Department of Agriculture Postdoctoral Fellowship program (2004-35318-14905 to BMB).*Address correspondence to these authors: LCS, phone (435) 797-3964, fax (435) email seefeldt@cc.usu.edu; DRD, phone (540) 231-5895, fax (540) email deandr@vt.edu; BMH, phone (847) (Figure 1).Relatively little is known at a molecular level about the nitrogenase N 2 -reduction mechanism beyond the fact that N 2 binds to and is reduced at one or more of the metal atoms of FeMocofactor ( Figure 1) Both the Chatt and Schrock cycles belong to one fundamental class of potential nitrogenase mechanismsin which the first three 'H-atoms' (e − /H + ) are sequentially added to a single N atom, in those instances the distal N of an end-on bound N 2 , followed by cleava...

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Section: Trapping a Diazene-derived Species Bound To Femo-cofactormentioning

confidence: 99%

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

et al. 2007

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“…13 A very recent study shows a single N environment for an apparent adduct of N 2 (or reduced form thereof) on the FeMoco, which is consistent with a symmetrical binding mode. 14 In addition to nitrogenase, there are heterogeneous iron systems that catalytically reduce dinitrogen. Some iron oxide 15 and iron sulfide 16 surfaces produce ammonia from N 2 .…”

Section: Introductionmentioning

confidence: 99%

Studies of Low-Coordinate Iron Dinitrogen Complexes

et al. 2005

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Understanding the interaction of N2 with iron is relevant to the iron catalyst used in the Haber process and to possible roles of the FeMoco active site of nitrogenase. The work reported here uses synthetic compounds to evaluate the extent of NN weakening in low-coordinate iron complexes with an FeNNFe core. The steric effects, oxidation level, presence of alkali metals, and coordination number of the iron atoms are varied, to gain insight into the factors that weaken the NN bond. Diiron complexes with a bridgingFeCl]n under a dinitrogen atmosphere, and an iron(I) precursor of an N2 complex can be observed. X-ray crystallographic and resonance Raman data for L R FeNNFeL R show a reduction in the N-N bond order, and calculations (density functional and multireference) indicate that the bond weakening arises from cooperative backbonding into the N2 π* orbitals. Increasing the coordination number of iron from three to four through binding of pyridines gives compounds with comparable N-N weakening, and both are substantially weakened relative to five-coordinate iron-N2 complexes, even those with a lower oxidation state. Treatment of L R FeNNFeL R with KC8 gives K2L R FeNNFeL R , and calculations indicate that reduction of the iron and alkali metal coordination cooperatively weaken the N-N bond. The complexes L R FeNNFeL R react as iron(I) fragments, losing N2 to yield iron(I) phosphine, CO, and benzene complexes. They also reduce ketones and aldehydes to give the products of pinacol coupling. The K2L R FeNNFeL R compounds can be alkylated at iron, with loss of N2.

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“…Another sequencing result using a primer with nifK (7822 NifK3, Table 1) confirmed the insertion of the Sp r cassette into nifK as well (data not shown). acetylene is a competitive inhibitor of N 2 fixation, whereas N 2 is a noncompetitive inhibitor of acetylene reduction (2,5,15). Consistent with this feature, air (79% N 2 ) inhibited acetylene reduction in the wild type and the ⌬nifK mutant, but to a greater extent in the ⌬nifK mutant.…”

Section: Fig 2 the Spmentioning

confidence: 54%

“…This nitrogenase phenotype is not unique, and we must consider the relationship of H 2 evolution and acetylene reduction to the nitrogenase enzyme (1,2). Hydrogen is always produced when nitrogenase reduces N 2 to NH 3 , indicating that H 2 evolution is integral to the enzyme mechanism (23).…”

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Genetic Transformation and Mutagenesis via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus Cyanothece

Huang

Sherman

2010

Appl Environ Microbiol

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Intermediates Trapped during Nitrogenase Reduction of N⋮N, CH₃−NNH, and H₂N−NH₂

Cited by 123 publications

References 19 publications

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

Studies of Low-Coordinate Iron Dinitrogen Complexes

Genetic Transformation and Mutagenesis via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus Cyanothece

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Intermediates Trapped during Nitrogenase Reduction of N⋮N, CH3−NNH, and H2N−NH2

Cited by 123 publications

References 19 publications

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N2 Reduction

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N2 Reduction

Studies of Low-Coordinate Iron Dinitrogen Complexes

Genetic Transformation and Mutagenesis via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus Cyanothece

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Intermediates Trapped during Nitrogenase Reduction of N⋮N, CH₃−NNH, and H₂N−NH₂

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N₂ Reduction