X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-.ANG. resolution

Kim, Jongsun; Woo, D.; Rees, Douglas C.

doi:10.1021/bi00079a006

Cited by 232 publications

(156 citation statements)

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“…The nitrogenase component proteins are structurally conserved to such an extent that heterologous pairs are usually active (Emerich et al, 1981). However, the Fe and MoFe proteins of C. pasteurianum have unique structures (Chen et al, 1986;Wang et al, 1988;Kim & Rees, 1993;Schlessman et al, 1998;Chen, 2004), which could explain the lack of activity in heterologous pairings involving the nitrogenase of C. pasteurianum. Whether or not the distinctive nitrogen-fixing system of C. pasteurianum contributes to the robustness of this organism is not known.…”

Section: Introductionmentioning

confidence: 99%

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Kasap

Chen

2005

Microbiology

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Previous studies identified five nifH-like genes (nifH2 through nifH6) in Clostridium pasteurianum (strain W5), where the nifH1 gene encodes the nitrogenase iron protein. Transcripts of these nifH genes, with the exception of nifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by the nifH-like genes were not reported. The nifH2 and nifH6 genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region of nifH2 and nifH6 was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of the nif genes of single-colony isolates of C. pasteurianum suggest that the nifH6 designation should be eliminated. Here the size of mRNA from nifH2 and the detection of the NifH2 polypeptide in nitrogen-fixing cells of C. pasteurianum are reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that the nifH1 and nifH2 mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH of Azotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixing C. pasteurianum cells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixing C. pasteurianum cells.

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Section: Introductionmentioning

confidence: 99%

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Kasap

Chen

2005

Microbiology

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“…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)(4)(5)(6)(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).…”

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confidence: 99%

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

Igarashi

Santos

Niehaus

et al. 2004

Journal of Biological Chemistry

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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“…The M and P nomenclature for these clusters derives from early Mössbauer spectroscopy studies that assigned iron sites to either a "magnetic" (M) or "protein" (P) cluster within the MoFe-protein (32). The nitrogenase proteins and associated metalloclusters have been extensively characterized crystallographically, with structures available for the MoFe-protein from Azotobacter vinelandii (33)(34)(35)(36), Clostridium pasteurianum (37,38), and Klebsiella pasteurianum (39), and for the Fe-protein from A. vinelandii (17, 40 -43) and C. pasteurianum (41). Complexes of the nitrogenase proteins have been characterized crystallographically (44,45) and by small-angle scattering (46,47).…”

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confidence: 99%

Great Metalloclusters in Enzymology

Rees

2002

Annu. Rev. Biochem.

204

134

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Key Words metalloproteins, iron-sulfur clusters, nitrogenase, hydrogenase, carbon monoxide dehydrogenase, nitrous oxide reductase f Abstract Metallocluster-containing enzymes catalyze some of the most basic redox transformations in the biosphere. The reactions catalyzed by these enzymes typically involve small molecules such as N 2 , CO, and H 2 that are used to generate both chemical building blocks and energy for metabolic purposes. During the past decade, structures have been established for the iron-sulfur-based metalloclusters present in the molybdenum nitrogenase, the iron-only hydrogenase, and the nickel-carbon monoxide dehydrogenase, and for the coppersulfide-based cluster in nitrous oxide reductase. Although these clusters are built from interactions observed in simpler metalloproteins, they contain novel features that may be relevant for their catalytic function. The mechanisms of metallocluster-containing enzymes are still poorly defined and represent substantial and continuing challenges to biochemists, biophysicists, and synthetic chemists. These proteins also provide a window into the union of the biological and inorganic worlds that may have been relevant to the early evolution of biochemical catalysis. Annu. Rev. Biochem. 2002. 71:221-46 DOI: 10.1146 Copyright © 2002 by Annual Reviews. All rights reserved 221 0066-4154/02/0707-0221$14.00 Annu. Rev. Biochem. 2002.71:221-246 CONTENTS INTRODUCTIONAmong the most remarkable chemical transformations in biological systems are the oxidation-reduction reactions of some of the smallest molecules, including N 2 , CO, and H 2 . Redox processes involving these species not only generate basic chemical building blocks required by living organisms but can also generate the energy needed to fuel their metabolisms. Although binding sites of exquisite specificity for molecules ranging from small organic metabolites to enormous macromolecules can be constructed from the standard amino acids and nucleotides, these groups are poorly suited for binding and catalyzing the redox reactions of diatomic and similar molecules. Consequently, it is necessary to incorporate specialized metallocofactors into enzymes to provide the binding interactions for these small substrates, as well as to confer the catalytic capabilities necessary to achieve the desired transformations. Metalloclusters also provide a fascinating window into the union of the biological and inorganic worlds that may have arisen early in evolution to effect the metabolism of small molecules required to drive the energetic and growth needs of primitive organisms. Indeed, it has been proposed that before an RNA world could even exist, the central biochemical processes arose from a surface-based metabolism on the iron-sulfur-containing mineral pyrite (1). What may be the vestiges of this metabolism can perhaps still be perceived in these contemporary clusters.This review surveys the metalloclusters that have been structurally characterized in enzymes (Figure 1). Although the choice of clusters covered is partl...

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X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-.ANG. resolution

Cited by 232 publications

References 52 publications

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

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

Great Metalloclusters in Enzymology

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