A methyldiazene (HNNCH
            <sub>3</sub>
            )-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

Barney, Brett M.; Lukoyanov, Dmitriy; Yang, Tran Chin; Dean, Dennis R.; Hoffman, Brian M.; Seefeldt, Lance C.

doi:10.1073/pnas.0602130103

Cited by 87 publications

(104 citation statements)

References 39 publications

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“…In other words, the enzyme has reduced the diazene substrate enough that it has 'caught up' to the hydrazine-derived species trapped during turnover with hydrazine. As hydrazine, and now diazene, are bona fide substrates that are reduced to NH 3 , this would then mean that both of these substrates in fact access the same, productive, mechanistic pathway to NH 3 that is used by N 2 , thereby validating the initial hypothesis about reaction intermediates (eqn 2), which implies that nitrogenase functions through an alternating mechanism, rather than a distal mechanism (27).…”

Section: Trapping a Diazene-derived Statementioning

confidence: 84%

“…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%

“…More recently, Schrock and coworkers (24)(25)(26) have demonstrated the catalytic reduction of N 2 to two NH 3 by a different mononuclear Mo complex, and have been able to capture and characterize many of the intermediate states along the reaction pathway. 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 cleavage of the N-N bond and release of the first NH 3 (this class of mechanisms thus has been termed 'distal') (27). The resulting metal (M)-bound nitride (M≡N) is reduced by three additional H-atoms to yield the second NH 3 .…”

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confidence: 99%

“…The resulting metal (M)-bound nitride (M≡N) is reduced by three additional H-atoms to yield the second NH 3 . In contrast to this mechanism are potential nitrogenase mechanisms in which the first five H-atoms are added alternately to the two N atoms of N 2 , followed by cleavage of the N-N bond and release of the first NH 3 (27). Reduction of the remaining metal bound amido (-NH 2 ) by a sixth H-atom results in release of the second NH 3 (this class of mechanisms has been termed 'alternating').…”

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confidence: 99%

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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 Statementioning

confidence: 84%

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

confidence: 99%

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confidence: 99%

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

et al. 2007

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“…Sample preparation followed freeze-quench procedures previously described (22,24,25); the 35 GHz CW (26) and pulsed (27) EPR spectrometers also have been described. Fourier transforms were performed with Bruker software, Win-EPR.…”

Section: Methodsmentioning

confidence: 99%

Unification of reaction pathway and kinetic scheme for N ₂ reduction catalyzed by nitrogenase

Lukoyanov

Yang

Barney

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Nitrogenase catalyzes the reduction of N 2 and protons to yield two NH 3 and one H 2 . Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH 3 ), or hydrazine as substrate recently was shown to trap a common intermediate, designated I . It was further concluded that the two N-atoms of N 2 are hydrogenated alternately (“Alternating” ( A ) pathway). In the present work, Q-band CW EPR and 95 Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H , has been characterized by ESEEM spectroscopy using a combination of 14,15 N isotopologs plus 1,2 H isotopologs of methyldiazene. It is concluded that: H has NH 2 bound to FeMo-co and corresponds to the penultimate intermediate of N 2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH 3 ; I corresponds to the final intermediate in N 2 reduction, the state formed after accumulation of eight electrons/protons, with NH 3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N 2 reduction by nitrogenase.

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Nitrogenase: Recent Advances

Andrade

Ribbe

et al. 2004

Handbook of Metalloproteins

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Nitrogenase catalyzes the reductive fixation of nitrogen from atmospheric N 2 for biological syntheses, and as such it constitutes the only enzyme known to be able to cleave the highly stable triple bond of the dinitrogen molecule. Structures are known of both component proteins of nitrogenase, the enzymatically active MoFe protein and its electron donor, the Fe protein. In spite of a wealth of biochemical and biophysical data on nitrogenase, the mechanistic details of its action remain poorly understood. This article provides an update on recent results in nitrogenase research, including the discovery of a central, light atom in the center of the FeMo cofactor, studies on substrate binding to the cofactor in mutant proteins, and novel aspects of complex formation between both component proteins. Particular emphasis is given to the significant progress made in understanding the biosynthesis of the complex metal clusters of the MoFe protein, the P‐cluster and the FeMo cofactor.

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A methyldiazene (HNNCH ₃ )-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

Abstract: Methyldiazene (HN‫؍‬N-CH3

Cited by 87 publications

References 39 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

Unification of reaction pathway and kinetic scheme for N ₂ reduction catalyzed by nitrogenase

Nitrogenase: Recent Advances

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A methyldiazene (HNNCH 3 )-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

Abstract: Methyldiazene (HN‫؍‬N-CH3

Cited by 87 publications

References 39 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

Unification of reaction pathway and kinetic scheme for N 2 reduction catalyzed by nitrogenase

Nitrogenase: Recent Advances

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A methyldiazene (HNNCH ₃ )-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

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

Unification of reaction pathway and kinetic scheme for N ₂ reduction catalyzed by nitrogenase