Studies on the hydrogenation steps of the nitrogen molecule at the Azotobacter vinelandii nitrogenase site

Stavrev, Krassimir K.; Zerner, Michael C.

doi:10.1002/(sici)1097-461x(1998)70:6<1159::aid-qua5>3.3.co;2-1

Cited by 17 publications

(47 citation statements)

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“…Overlaying the X-and Q-band EPR spectra for the hydrazine-and diazene-dependent intermediates suggests that they represent the same intermediate, with slightly different populations of two conformational substates. More definitively, 15 N and 1 H-ENDOR spectra of the diazene-and hydrazine-dependent states show that they contain substrate-derived [-NH x ] species bound to FeMo-cofactor whose characteristics are identical. Each shows a 15 N-ENDOR signal from a single (type of) 15 The equivalence of the EPR and 15 N, 1 H-ENDOR spectra for the diazene-and hydrazinedependent states can be explained in three different ways: (i) Diazene could first decompose to hydrazine, and then the resulting hydrazine could react with the MoFe protein, being trapped in the same state as accumulates when hydrazine is used as a substrate; (ii) diazene could be reduced by nitrogenase to the same intermediate state that is trapped when hydrazine is presented as the substrate; and (iii) the diazene-and hydrazine-dependent states could represent different species bound to FeMo-cofactor, but with such similar properties for the bound [-NH x ] fragment that these differences are not reflected in the EPR or 15 N-ENDOR spectra.…”

Section: Trapping a Diazene-derived Statementioning

confidence: 93%

“…The single-crystal-like spectra for the two states, collected at a field corresponding to g || = 2.09, show a doublet from a single (type of) 15 N, centered at the 15 N Larmor frequency and split by the 15 N hyperfine coupling, A = 1.80(4) MHz. The individual peaks broaden and show additional resolved features as the field is increased, a consequence of an anisotropic contribution to the hyperfine tensor.…”

Section: N 1 H-endormentioning

confidence: 99%

“…It is generally accepted that the sequential addition of electrons and protons to the N 2 bound on FeMo-cofactor results in a series of semi-reduced and semiprotonated intermediates (11)(12)(13)(14)(15)(16)(17)(18)(19), ultimately yielding two ammonia molecules. In contrast to this situation, much more is known about how N 2 is activated and reduced by metal complexes (20).…”

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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: 93%

Section: N 1 H-endormentioning

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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“…It is therefore not surprising that agreement concerning the mechanism of dinitrogen activation and the evolution of dihydrogen (both general and obligatory) has not yet been reached. Significant insight into this problem may be gained by carrying out ab initio density functional calculations [18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33] on a quantum model that faithfully represents the active site of the protein. The caveat is, however, that the active site must not be so large that the calculations become intractable at the high level of theory required to afford reliable energetics.…”

Section: And H 2 Under Ambient Conditions and At Biological Redox Potmentioning

confidence: 99%

FeMo cofactor of nitrogenase: energetics and local interactions in the protein environment

Lovell

Case

et al. 2002

J Biol Inorg Chem

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A combined broken-symmetry density functional and continuum electrostatics approach has been applied to the iron-molybdenum center (FeMoco) of nitrogenase to evaluate the energetic effects of the local amino acid environment for several spin alignments of FeMoco. The protein environment preferentially stabilizes certain spin coupling patterns. The lowest energy spin alignment pattern in the protein displays calculated properties that match the experimental data better than any of the alternative possibilities. The total interaction energy of the protein with FeMoco has been evaluated and the contribution of each amino acid residue has been broken down into sidechain and backbone components. Arginine, lysine, aspartate and glutamate sidechains exert the largest electrostatic influence on FeMoco; specific residues are highlighted and their interaction with FeMoco discussed in the context of the available X-ray data from Azotobacter vinelandii (Av). Observed data for the M(N)(resting state)-->M(OX)(one-electron oxidized state) and M(N)-->M(R)(one-electron reduced state) or M(I)(alternative one-electron reduced state) redox couples are compared with those calculated for Av. The calculated redox potentials are fairly insensitive to the spin state of the oxidized or reduced states and the predicted qualitative trend of a more negative redox potential for the more reduced M(N)-->M(R) or M(I) couple is in accord with the available redox data. These calculations represent a first step towards the development of a microscopic model of electron and proton transfer events at the nitrogenase active site.

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“…Many computational studies appeared purporting to shed light on the site of N 2 coordination and the mechanism of its protonation and reduction to ammonia [74][75][76][77][78][79][80][81][82]. However, the calculations are extremely difficult since it is hard to know what the ionisation state of the cluster is and what is its overall spin state.…”

Section: Nitrogenasementioning

confidence: 99%

Recent Developments in Computational Bioinorganic Chemistry

Deeth

Structure and Bonding

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Selected examples of density functional theory applied to modelling the active site structures and mechanisms of metalloenzymes are discussed. Factors influencing the design of suitable structural models and the theoretical strategies for computing their structures, energies and properties are presented before describing the calculations on a number of redox-active enzymes containing one or more of Cu, Co, Ni, Fe and Mo.

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Studies on the hydrogenation steps of the nitrogen molecule at the Azotobacter vinelandii nitrogenase site

Cited by 17 publications

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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

FeMo cofactor of nitrogenase: energetics and local interactions in the protein environment

Recent Developments in Computational Bioinorganic Chemistry

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Studies on the hydrogenation steps of the nitrogen molecule at the Azotobacter vinelandii nitrogenase site

Cited by 17 publications

References 0 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

FeMo cofactor of nitrogenase: energetics and local interactions in the protein environment

Recent Developments in Computational Bioinorganic Chemistry

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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