Substrate Interaction at an Iron-Sulfur Face of the FeMo-cofactor during Nitrogenase Catalysis

Barney, Brett M.; Igarashi, Robert Y.; Santos, Patricia C. Dos; Dean, Dennis R.; Seefeldt, Lance C.

doi:10.1074/jbc.m410247200

Cited by 142 publications

(167 citation statements)

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“…From the results in Figure 3B, a minimum V max for diazene reduction by nitrogenase is found to be 400 nmol NH 3 /min/ mg MoFe protein. This value compares favorably with the V max for N 2 reduction by nitrogenase determined in a parallel experiment of 600 nmol NH 3 /min/mg MoFe protein (35). Likewise, an upper limit on the K m for diazene is calculated to be 4.5 mM, compared to the K m for N 2 of 60 μM (35).…”

Section: Diazene Is a Substrate For Nitrogenasesupporting

confidence: 70%

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: Diazene Is a Substrate For Nitrogenasesupporting

confidence: 70%

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

et al. 2007

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“…Significantly, however, the nonheterocyst nitrogenase of this strain, which is expressed mainly in vegetative cells under anaerobic conditions, is incompatible with O 2 -evolving photosynthesis and thus requires continuous anaerobic conditions along with a supply of exogenous reducing sugars for H 2 production. Substitutions of selected amino acids in the vicinity of the FeMo-co active site within Azotobacter vinelandii nitrogenase were shown to eliminate or greatly diminish N 2 fixation while, in some cases, allowing for effective proton reduction (2,10,17,27,36,44,45,48). Therefore, certain amino acid exchanges near FeMo-co might produce variant MoFe proteins in heterocyst-forming Anabaena that redirect the electron flux through the enzyme preferentially to proton reduction so as to synthesize more H 2 in the presence of N 2 in an aerobic environment.…”

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confidence: 99%

“…The nitrogenase-related (nif) genes are specifically expressed in heterocysts which lack O 2 -evolving photosystem II activity and are surrounded by a thick cell envelope composed of glycolipids and polysaccharides that impede the entry of O 2 (56). Vegetative cells perform oxygenic photosynthesis and fix CO 2 . Heterocysts obtain carbohydrates from those cells and, in turn, provide them with fixed nitrogen.…”

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Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Masukawa

Inoue

Wölk

et al. 2010

Appl Environ Microbiol

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Cyanobacteria use sunlight and water to produce hydrogen gas (H 2 ), which is potentially useful as a clean and renewable biofuel. Photobiological H 2 arises primarily as an inevitable by-product of N 2 fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H 2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N 2 . Most of the 49 variants examined were deficient in N 2 -fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N 2 atmosphere significantly increased their in vivo rates of H 2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H 2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H 2 in an aerobic, nitrogen-containing environment.

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“…A number of different interaction sites can be considered upon inspection of the structure of FeMo-cofactor and calculations have been reported that support a number of different binding sites [4,12]. In our earlier studies, we began to localize the site of alkyne substrate interactions with FeMo-cofactor to one Fe-S face that includes Fe atoms numbered 2, 3, 6 and 7 [2,[13][14][15][16][17][18][19][20]. Analysis of the capacity of MoFe proteins having amino acid substitutions for residues that approach this Fe-S face to reduce a range of substrates has indicated that this face might provide the only site for substrate binding and reduction.…”

Section: Introductionmentioning

confidence: 99%

“…By using the amino acid substitution approach, conditions were also developed that allowed trapping and characterization of an intermediate that accumulates during the reduction of propargyl alcohol by the α-70 Ala -substituted MoFe protein [18]. Characterization of this intermediate by EPR and 13 C-and 1/2 H-electron nuclear double resonance (ENDOR) spectroscopies were interpreted to indicate side-on binding of a 2e − /2H + reduced allyl alcohol species (H 2 C=CH-CH 2 OH) that is bound to FeMo-cofactor through a single Fe atom [17]. The effect of pH dependence on the capacity of the α-70 Ala MoFe protein to reduce either propargyl alcohol or propargyl amine (HC≡C-CH 2 NH 2 ) further localized the proposed binding site to Fe atom 6 [16] within FeMo-cofactor (Figure 1).…”

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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Substrate Interaction at an Iron-Sulfur Face of the FeMo-cofactor during Nitrogenase Catalysis

Cited by 142 publications

References 32 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

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Alkyne substrate interaction within the nitrogenase MoFe protein

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Substrate Interaction at an Iron-Sulfur Face of the FeMo-cofactor during Nitrogenase Catalysis

Cited by 142 publications

References 32 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

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

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