An Organometallic Intermediate during Alkyne Reduction by Nitrogenase

Lee, Hong‐In; Igarashi, Robert Y.; Laryukhin, Mikhail; Doan, Peter E.; Santos, Patricia C. Dos; Dean, Dennis R.; Seefeldt, Lance C.; Hoffman, Brian M.

doi:10.1021/ja048714n

Cited by 123 publications

(202 citation statements)

References 47 publications

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“…4). Given the similarity in lineshape of the EPR spectra elicited under freeze-quench conditions when either propargyl-OH or propargyl-NH 2 is used as substrate, it is presumed that an allyl-NH 2 adduct is bound to an iron atom of the FeMo-cofactor in a way similar to that proposed for propargyl-OH (17). One difference in behavior between these two substrates is the lower intensity of the propargyl-NH 2 (15 mM)-elicited EPR signal (approximately one-fourth) when compared with propargyl-OH (3 mM)-dependent EPR signal intensity.…”

Section: Appearance Of a Propargyl-nh 2 Reductionmentioning

confidence: 98%

“…This new EPRactive state originates from an intermediate derived from propargyl-OH that is bound to the FeMo-cofactor. Electron nuclear double resonance studies coupled with the use of isotopically labeled propargyl-OH have established that allyl alcohol, CH 2 ϭCH-CH 2 OH, is the probable species bound (Scheme 1) (17).…”

Section: Relevant Features Of the ␣-70 Ala -Substituted Mofe Proteinsmentioning

confidence: 99%

“…Using 13 C-and 1/2 H-labeled propargyl-OH and electron nuclear double resonance spectroscopic methods, we have recently deduced that the trapped intermediate has two hydrogen atoms added (i.e. allyl alcohol, H 2 CϭCHCH 2 OH) and is bound to iron such that the two terminal hydrogen atoms are spectroscopically indis-tinguishable, suggesting the bio-organometallic complex shown in Scheme 1 (17).…”

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confidence: 99%

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Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase

Igarashi

Santos

Niehaus

et al. 2004

Journal of Biological Chemistry

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104

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Nitrogenase catalyzes the biological reduction of N 2 to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HCϵCH) to ethylene (H 2 C‫؍‬CH 2 ). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the ␣-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; ␣-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HCϵCCH 2 OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S ‫؍‬ 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HCϵCCH 2 NH 2 , propargyl-NH 2 )-derived intermediates is shown to be dependent on pH and the presence of histidine at position ␣-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the -OH group or the -NH 3 ؉ group and the imidazole ⑀-NH of ␣-195 His . Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an 2 coordination at Fe-6 of the FeMo-cofactor.Nitrogenase is comprised of two component proteins, called the iron protein and the MoFe protein, which together catalyze the nucleotide-dependent reduction of N 2 to ammonia (Equation 1). N 2 ϩ 8e Ϫ ϩ 16MgATP ϩ 8H ϩ 3 2NH 3 ϩ H 2 ϩ 16MgADP ϩ 16P i (Eq. 1) During catalysis, electrons are delivered one at a time from the iron protein to the MoFe protein in a reaction coupled to the hydrolysis of 2 eq of MgATP for each equivalent of electrons transferred (1, 2). The MoFe protein contains two metalloclusters called the P-cluster [8Fe-7S] and the FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], where X is proposed to be nitrogen, carbon, or oxygen (3). The P-clusters are thought to mediate electron transfer from the iron protein to the FeMo-cofactor, which in turn provides the site for substrate binding and reduction. The structure of the FeMo-cofactor has been elucidated from the solution of x-ray structures of MoFe proteins (3-7), yet where and how substrates interact with the FeMocofactor is still unknown. Different models for where substrates bind to the FeMo-cofactor have been developed; they were built on evidence from model compounds, theoretical calculations, and kinetic and biophysical studies on the wildtype (WT) 1 and genetically altered MoFe proteins (8). Some models propose binding and reduction of substrates at th...

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Section: Appearance Of a Propargyl-nh 2 Reductionmentioning

confidence: 98%

Section: Relevant Features Of the ␣-70 Ala -Substituted Mofe Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase

Igarashi

Santos

Niehaus

et al. 2004

Journal of Biological Chemistry

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104

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“…This observation can be explained in one of two different ways. According to our model, alkyne binding within the MoFe protein occurs through exclusive coordination with Fe6 of FeMo-cofactor in a side-on fashion [15,16]. Thus, steric constraints imposed by side-chains other than α-70 could prevent the productive interaction with nitrogenase by alkynes that have an internal triple bond.…”

Section: A Genetic Screen For a Vinelandii Strains That Can Reduce Imentioning

confidence: 96%

“…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%

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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Nitrogenase: Recent Advances

Andrade

Ribbe

et al. 2004

Handbook of Metalloproteins

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Nitrogenase catalyzes the reductive fixation of nitrogen from atmospheric N 2 for biological syntheses, and as such it constitutes the only enzyme known to be able to cleave the highly stable triple bond of the dinitrogen molecule. Structures are known of both component proteins of nitrogenase, the enzymatically active MoFe protein and its electron donor, the Fe protein. In spite of a wealth of biochemical and biophysical data on nitrogenase, the mechanistic details of its action remain poorly understood. This article provides an update on recent results in nitrogenase research, including the discovery of a central, light atom in the center of the FeMo cofactor, studies on substrate binding to the cofactor in mutant proteins, and novel aspects of complex formation between both component proteins. Particular emphasis is given to the significant progress made in understanding the biosynthesis of the complex metal clusters of the MoFe protein, the P‐cluster and the FeMo cofactor.

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An Organometallic Intermediate during Alkyne Reduction by Nitrogenase

Cited by 123 publications

References 47 publications

Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase

Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase: Recent Advances

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