Role of Nucleotides in Nitrogenase Catalysis

Seefeldt, Lance C.; Dean, Dennis R.

doi:10.1021/ar960260e

Cited by 110 publications

(105 citation statements)

References 69 publications

(135 reference statements)

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“…15 N 2 H 2 was prepared by first synthesizing biurea (3 in Figure 2) from 15 N-hydrazine (1 in Figure 2). The biurea (3) was oxidized to azocarboamide (4), which in turn could be readily converted to azodiformate (5 Selected 15 N-ENDOR spectra for the diazene-dependent state and for the hydrazine-dependent state are shown at fields near to the common principal g-values ( Figure 9A). Spectra collected over a wider frequency range (not shown) revealed no additional signals from 15 N with larger couplings for either sample; neither were additional signals detected with smaller hyperfine couplings through lengthening of the interpulse spacing to τ = 800 ns, which accentuates smaller couplings (see Materials and Methods).…”

Section: N 1 H-endormentioning

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: N 1 H-endormentioning

confidence: 99%

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

et al. 2007

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“…ϩ1 Fe protein induces a conformational change that involves the contraction of the protein (44,(55)(56)(57). Binding of two molecules of MgADP to the state of the Fe protein causes a different, less dramatic conformational change that does not involve a global change in the protein radius of gyration (57).…”

Section: The Fe Protein Forms a Normal Complex With The ⌬Nifh Mofe Prmentioning

confidence: 99%

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

Ribbe¹,

Hu²,

Guo³

et al. 2002

Journal of Biological Chemistry

146

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The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (⌬nifH MoFe protein) was purified in large quantity. The which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.The metalloenzyme nitrogenase complex catalyzes the biological reduction of dinitrogen to ammonia (for recent reviews, see Refs. 1-6). The enzyme is composed of two separately purifiable proteins, the iron (Fe) protein and the molybdenumiron (MoFe) 1 protein. The Fe Protein is a 60-kDa dimer of two identical subunits encoded by the nifH gene. The two subunits are bridged by a [4Fe-4S] cluster, and each subunit has a binding site for MgATP. The more complicated MoFe protein is a 230-kDa ␣ 2 ␤ 2 tetramer with the ␣ and ␤ subunits encoded by the nifD and nifK genes, respectively. The MoFe protein contains two different types of metal clusters, the [8Fe-7S] cluster (P-cluster) bridged between each ␣␤ subunit pair and the [Mo7Fe-9S-homocitrate] cluster (FeMoco) located within each ␣ subunits. Substrate reduction by the enzyme requires both component proteins, with the Fe protein serving as a specific reductant of the MoFe protein, which in turn provides the site of substrate reduction. To carry out the catalytic function of nitrogenase, the reduced Fe protein first binds two molecules of MgATP and undergoes a conformational change before forming a complex with the MoFe protein. Then, coupled with MgATP hydrolysis, electrons are transferred from the Fe protein to the P-clusters of the MoFe protein within the complex. This process is followed by the dissociation and re-reduction of the oxidized Fe protein and the dissociation of MgADP from the MoFe protein. Finally, the electrons are believed to be transferred from the P-cluster to the FeMoco, where substrate reduction occurs.FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7-18) and the features of the P-cluster (

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“…The Fe protein is a homodimer that contains a [4Fe-4S] cluster bridging the two subunits [5], with each subunit having a site for MgATP binding. During turnover, the reduced Fe protein transfers one electron at a time from its [4Fe-4S] cluster to the MoFe protein in a reaction coupled to the hydrolysis of two MgATP molecules [3,6]. The MoFe protein is an α 2 β 2 heterotetramer that contains two unique types of [Fe-S] clusters [7,8].…”

Section: Introductionmentioning

confidence: 99%

Alkyne substrate interaction within the nitrogenase MoFe protein

Santos

Mayer

Barney

et al. 2007

Journal of Inorganic Biochemistry

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Nitrogenase catalyzes the biological reduction of N 2 to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC≡CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein α-70 Val residue, whose side-chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of α-70 Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡C-CH 3 ), whereas substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the α-70 Val residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC≡C-CH 2 -CH 3 ). This protein shows no detectable reduction of the internal alkyne 2-butyne (H 3 C-C≡C-CH 3 ). In contrast, substitution of the nearby α-191 Gln residue by alanine, in combination with the α-70 Ala substitution, does result in significant reduction 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves sideon binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.

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Role of Nucleotides in Nitrogenase Catalysis

Cited by 110 publications

References 69 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

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

Alkyne substrate interaction within the nitrogenase MoFe protein

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Role of Nucleotides in Nitrogenase Catalysis

Cited by 110 publications

References 69 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

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

Alkyne substrate interaction within the nitrogenase MoFe protein

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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