Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state

Davis, Lawrence C.; Henzl, Michael T.; Burris, R. H.; Orme‐Johnson, William H.

doi:10.1021/bi00589a014

Cited by 117 publications

(199 citation statements)

References 36 publications

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“…In the case of the ␣-Ser 69 MoFe protein, the high affinity acetylene-binding site appears to have been eliminated or severely altered without significantly altering the capacity of the altered enzyme to reduce nitrogen. Also, the remaining low affinity acetylene binding site in the ␣-Ser 69 MoFe protein does not appear to be significantly altered, maintaining a K m value similar to the value previously reported by Davis et al (15) and a V max that is only slightly less than the value derived for wild-type MoFe protein (Table II).…”

Section: Nature Of the Acetylene Resistance Phenotype Elicited By Thesupporting

confidence: 82%

“…First, in the case of the normal MoFe protein, acetylene is a noncompetitive inhibitor of nitrogen reduction, whereas nitrogen is a competitive inhibitor of acetylene reduction (12)(13)(14). Second, kinetic and spectroscopic evidence obtained using the unaltered MoFe protein (15,16) as well as a MoFe protein altered by amino acid substitution (17) indicates that there are two acetylene-binding sites located within the MoFe protein. In light of these observations we became interested in determining if it is possible to obtain a MoFe protein that is altered in its ability to reduce acetylene but remains capable of normal nitrogen reduction.…”

mentioning

confidence: 99%

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Isolation and Characterization of an Acetylene-resistant Nitrogenase

Christiansen¹,

Cash²,

Seefeldt³

et al. 2000

Journal of Biological Chemistry

111

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A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the ␣-subunit Gly 69 residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the ␣-Ser 69 substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the ␣-subunit Val 70 residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.

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Section: Nature Of the Acetylene Resistance Phenotype Elicited By Thesupporting

confidence: 82%

mentioning

confidence: 99%

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Christiansen¹,

Cash²,

Seefeldt³

et al. 2000

Journal of Biological Chemistry

111

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“…Several earlier studies have suggested two binding sites on FeMo cofactor. For example, it has been proposed that two CO molecules bind to FeMo cofactor in the high CO concentration inhibited state (29)(30)(31)(32)(33). Likewise, two acetylene binding sites have been implicated from studies combining kinetics and amino acid substitutions (31,34,35).…”

Section: Discussionmentioning

confidence: 99%

“…Several earlier studies revealed multiple inhibitor and substrate binding sites on FeMo-cofactor, including at least two binding sites for CO (29)(30)(31)(32)(33) and acetylene (31,34,35). Two adjacent binding sites can explain the earlier reports that two or three CO molecules can be reduced and coupled to form C2 and C3 hydrocarbon products (24,26).…”

Section: Co 2 Reduction To Chmentioning

confidence: 95%

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

Yang

Moure

Dean

et al. 2012

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

107

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A doubly substituted form of the nitrogenase MoFe protein (α-70 Val→Ala , α-195 His→Gln ) has the capacity to catalyze the reduction of carbon dioxide (CO 2 ) to yield methane (CH 4 ). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH 4 within 20 min. The catalytic rate depends on the partial pressure of CO 2 (or concentration of HCO 3 − ) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H 2 C = CH-CH 3 ) through the reductive coupling of CO 2 and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO 2 sequestration and formation of olefins.is an abundant and stable form of carbon that is the product of respiration and burning of fossil fuels. As a result of these activities, the atmospheric concentration of CO 2 , a greenhouse gas, has been rising over the last century and contributing to global warming (1). There is strong interest in developing methods for sequestering CO 2 either by capturing it or by chemically converting it to valuable chemicals (2-5). Of particular interest are possible routes to reduction of CO 2 by multiple electrons to yield methanol (CH 3 OH) and methane (CH 4 ), which are renewable fuels (2). The reduction of CO 2 is difficult, with a limited number of reports of metal-based compounds able to catalyze these reactions (6-13). In biology, only a few enzymes are known to reduce CO 2 (14-18), and none of these can catalyze the eight electron reduction to CH 4 .The bacterial Mo-dependent nitrogenase enzyme catalyzes the multielectron/proton reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at a metal cluster designated FeMo-cofactor [7Fe-9S-1Mo-1C-R-homocitrate] (Fig. 1) in a reaction that requires ATP hydrolysis and evolution of H 2 , with a minimal reaction stoichiometry shown in Eq. 1 (19)(20)(21)(22).Given that nitrogenase is effective at catalyzing the difficult multielectron reduction of N 2 , it was of interest to determine whether this enzyme might also catalyze the reduction of CO 2 to the level of CH 4 . Nitrogenase is known to have the capacity to reduce a variety of other small, relatively inert, doubly or triply bonded compounds, such as acetylene (HC≡CH) (19,23). It has been shown that an alternative form of nitrogenase, which contains V in place of Mo in the active site cofactor, has the remarkable capacity to reduce CO and couple multiple CO molecules, yielding short chain alkenes and alkanes such as ethylene (C 2 H 4 ), ethane (C 2 H 6 ), propylene (C 3 H 6 ), and propane (C 3 H 8 ) (24,25). In contrast, the Mo-nitrogenase is only able to reduce CO at exceedingly low rates (24). However, we have found that the MoFe protein can be remodeled by substitution of amino acid residues that provide the first shell of noncovalent interactions with the active site FeMo cofa...

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“…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%

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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Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state

Cited by 117 publications

References 36 publications

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

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

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Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state

Cited by 117 publications

References 36 publications

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Isolation and Characterization of an Acetylene-resistant Nitrogenase

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

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

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