Differential Effects on N2 Binding and Reduction, HD Formation, and Azide Reduction with α-195His- and α-191Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

Fisher, Karl; Dilworth, M. J.; Newton, William E.

doi:10.1021/bi0017834

Cited by 87 publications

(113 citation statements)

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“…Therefore, the corresponding residues in Anabaena NifD (C282, H449, and residues 362 to 367; VGGLRP) were left unchanged. In contrast, we carried out an extensive survey of changes to residues Q193, H197, R284, and F388 in the Anabaena enzyme, equivalent to residues (Q191, H195, R277, and F381) that were previously substituted in a limited fashion in the A. vinelandii protein (17,27,36,44,45,48). Two additional residues lying within 5 Å of FeMo-co in the A. vinelandii enzyme (Y229 and S278) were substituted in the Anabaena sequence (Y236 and S285).…”

Section: Resultsmentioning

confidence: 99%

“…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%

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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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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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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“…We considered two models for this process, both built on our characterization of the key E 4 state, and tested them against the numerous constraints imposed by turnover under N 2 plus D 2 or T 2 (5,(9)(10)(11)(12)(13)(14)(15)(16). In particular, these constraints include the key findings that during catalytic reduction of N 2 (see Scheme S1), a molecule of D 2 or T 2 will reduce two protons to form two HD or HT without D + /T + exchange with solvent, even though neither D 2 nor T 2 by themselves reacts with nitrogenase during turnover under Ar (5).…”

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confidence: 99%

On reversible H ₂ loss upon N ₂ binding to FeMo-cofactor of nitrogenase

Yang

Khadka

Lukoyanov

et al. 2013

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

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Nitrogenase is activated for N 2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E 4 , which contains two iron-bridging hydrides [Fe-H-Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H 2 per N 2 reduced, which would "waste" two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H 2 production. In this model, H 2 is produced by reductive elimination of the two bridging hydrides of E 4 during N 2 binding. This process releases H 2 , yielding N 2 bound to FeMocofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D 2 /N 2 , the reverse reaction of D 2 with the N 2 -bound product of reductive elimination would generate dideutero-E 4 [E 4 to two ammonia (NH 3 ) molecules-is primarily catalyzed by the Mo-dependent nitrogenase. This enzyme comprises an electron-delivery Fe protein and MoFe protein, which contains the active site FeMo-cofactor ( Fig. 1A) (1, 2). The nitrogenase catalyzed reaction is generally thought to have the limiting stoichiometry shown in Eq. 1 (3),This equation conveys one of the most puzzling aspects of nitrogenase function, for it incorporates an obligatory formation of one mole of H 2 per mole of N 2 reduced, which requires the waste of two reducing equivalents and four ATP (1, 2). A kinetic framework for nitrogenase function that incorporates the stoichiometry of Eq. 1 is provided by the Lowe-Thorneley model (1, 2, 4), which describes transformations among catalytic intermediates (denoted E n ), where n is the number of electrons and protons (n = 0-8) delivered to MoFe protein. N 2 reduction requires activation of the MoFe protein to the E 4 state in which FeMo-cofactor has accumulated four electrons and four protons stored as two hydrides that bridge Fe atoms [Fe-H-Fe] and two protons presumably bound to sulfides of FeMo-cofactor (Fig. 1B) (5-8). The binding of N 2 to the E 4 state is coupled to the stoichiometric evolution of one H 2 per N 2 reduced.We recently presented a draft mechanism for N 2 reduction by nitrogenase that incorporates a mechanistic explanation for obligatory, reversible H 2 loss upon N 2 binding (5). We considered two models for this process, both built on our characterization of the key E 4 state, and tested them against the numerous constraints imposed by turnover under N 2 plus D 2 or T 2 (5, 9-16). In particular, these constraints include the key findings that during catalytic reduction of N 2 (see Scheme S1), a molecule of D 2 or T 2 will reduce two protons to form two HD or HT without D + /T + exchange with solvent, even though neither D 2 nor T 2 by themselves reacts with nitrogenase during turnover under Ar (5). One model, involving protonation of one of the hydrides to form H 2 , and its replacement by N 2 , was shown to violate ...

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“…However, based on its g values, this appears to be from an N 2 -turnover state, probably formed with N 2 generated as a breakdown product of diazene (49). In contrast, conversion to an intermediate with a distinct EPR signal is observed when the α-195 Gln MoFe protein is trapped during turnover with diazene; the substitution of α-195 His by Gln has been suggested to limit proton delivery for reduction of nitrogenous substrates, and thus to arrest the reduction of these substrates (36,52,53 It was important to establish that the trapped state observed by EPR is a result of diazene binding to or reacting with nitrogenase, and not one of the diazene breakdown products. In an experiment parallel to the a kinetic studies described above, diazene was allowed to decompose for 30 min in an EPR tube prior to the addition of the nitrogenase proteins.…”

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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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Differential Effects on N₂ Binding and Reduction, HD Formation, and Azide Reduction with α-195^His- and α-191^Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

Cited by 87 publications

References 42 publications

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

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

On reversible H ₂ loss upon N ₂ binding to FeMo-cofactor of nitrogenase

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

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Differential Effects on N2 Binding and Reduction, HD Formation, and Azide Reduction with α-195His- and α-191Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

Cited by 87 publications

References 42 publications

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

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

On reversible H 2 loss upon N 2 binding to FeMo-cofactor of nitrogenase

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

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Differential Effects on N₂ Binding and Reduction, HD Formation, and Azide Reduction with α-195^His- and α-191^Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

On reversible H ₂ loss upon N ₂ binding to FeMo-cofactor of nitrogenase

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