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PubMed2002

Structure of a cofactor-deficient nitrogenase MoFe protein.

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.

Amino Acid Sequence↗
PubMed2002

Nitrogenase MoFe-protein at 1.16 A resolution: a central ligand in the FeMo-cofactor.

A high-resolution crystallographic analysis of the nitrogenase MoFe-protein reveals a previously unrecognized ligand coordinated to six iron atoms in the center of the catalytically essential FeMo-cofactor. The electron density for this ligand is masked in structures with resolutions lower than 1.55 angstroms, owing to Fourier series termination ripples from the surrounding iron and sulfur atoms in the cofactor. The central atom completes an approximate tetrahedral coordination for the six iron atoms, instead of the trigonal coordination proposed on the basis of lower resolution structures. The crystallographic refinement at 1.16 angstrom resolution is consistent with this newly detected component being a light element, most plausibly nitrogen. The presence of a nitrogen atom in the cofactor would have important implications for the mechanism of dinitrogen reduction by nitrogenase.

Azotobacter vinelandii↗
PubMed2005

Nitrogenase complexes: multiple docking sites for a nucleotide switch protein.

Adenosine triphosphate (ATP) hydrolysis in the nitrogenase complex controls the cycle of association and dissociation between the electron donor adenosine triphosphatase (ATPase) (Fe-protein) and its target catalytic protein (MoFe-protein), driving the reduction of dinitrogen into ammonia. Crystal structures in different nucleotide states have been determined that identify conformational changes in the nitrogenase complex during ATP turnover. These structures reveal distinct and mutually exclusive interaction sites on the MoFe-protein surface that are selectively populated, depending on the Fe-protein nucleotide state. A consequence of these different docking geometries is that the distance between redox cofactors, a critical determinant of the intermolecular electron transfer rate, is coupled to the nucleotide state. More generally, stabilization of distinct docking geometries by different nucleotide states, as seen for nitrogenase, could enable nucleotide hydrolysis to drive the relative motion of protein partners in molecular motors and other systems.

Adenosine Diphosphate↗
PubMed1992

Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii.

The nitrogenase enzyme system catalyzes the ATP (adenosine triphosphate)-dependent reduction of dinitrogen to ammonia during the process of nitrogen fixation. Nitrogenase consists of two proteins: the iron (Fe)-protein, which couples hydrolysis of ATP to electron transfer, and the molybdenum-iron (MoFe)-protein, which contains the dinitrogen binding site. In order to address the role of ATP in nitrogen fixation, the crystal structure of the nitrogenase Fe-protein from Azotobacter vinelandii has been determined at 2.9 angstrom (A) resolution. Fe-protein is a dimer of two identical subunits that coordinate a single 4Fe:4S cluster. Each subunit folds as a single alpha/beta type domain, which together symmetrically ligate the surface exposed 4Fe:4S cluster through two cysteines from each subunit. A single bound ADP (adenosine diphosphate) molecule is located in the interface region between the two subunits. Because the phosphate groups of this nucleotide are approximately 20 A from the 4Fe:4S cluster, it is unlikely that ATP hydrolysis and electron transfer are directly coupled. Instead, it appears that interactions between the nucleotide and cluster sites must be indirectly coupled by allosteric changes occurring at the subunit interface. The coupling between protein conformation and nucleotide hydrolysis in Fe-protein exhibits general similarities to the H-Ras p21 and recA proteins that have been recently characterized structurally. The Fe-protein structure may be relevant to the functioning of other biochemical energy-transducing systems containing two nucleotide-binding sites, including membrane transport proteins.

Adenosine Diphosphate↗
PubMed1992

Structural models for the metal centers in the nitrogenase molybdenum-iron protein.

Structural models for the nitrogenase FeMo-cofactor and P-clusters are proposed based on crystallographic analysis of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii at 2.7 angstrom resolution. Each center consists of two bridged clusters; the FeMo-cofactor has 4Fe:3S and 1Mo:3Fe:3S clusters bridged by three non-protein ligands, and the P-clusters contain two 4Fe:4S clusters bridged by two cysteine thiol ligands. Six of the seven Fe sites in the FeMo-cofactor appear to have trigonal coordination geometry, including one ligand provided by a bridging group. The remaining Fe site has tetrahedral geometry and is liganded to the side chain of Cys alpha 275. The Mo site exhibits approximate octahedral coordination geometry and is liganded by three sulfurs in the cofactor, two oxygens from homocitrate, and the imidazole side chain of His alpha 442. The P-clusters are liganded by six cysteine thiol groups, two which bridge the two clusters, alpha 88 and beta 95, and four which singly coordinate the remaining Fe sites, alpha 62, alpha 154, beta 70, and beta 153. The side chain of Ser beta 188 may also coordinate one iron. The polypeptide folds of the homologous alpha and beta subunits surrounding the P-clusters are approximately related by a twofold rotation that may be utilized in the binding interactions between the MoFe-protein and the nitrogenase Fe-protein. Neither the FeMo-cofactor nor the P-clusters are exposed to the surface, suggesting that substrate entry, electron transfer, and product release must involve a carefully regulated sequence of interactions between the MoFe-protein and Fe-protein of nitrogenase.

Azotobacter vinelandii↗
PubMed1993

The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 A resolution structures.

Structures recently proposed for the FeMo-cofactor and P-cluster pair of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii have been crystallographically verified at 2.2 angstrom resolution. Significantly, no hexacoordinate sulfur atoms are observed in either type of metal center. Consequently, the six bridged iron atoms in the FeMo-cofactor are trigonally coordinated by nonprotein ligands, although there may be some iron-iron bonding interactions that could provide a fourth coordination interaction for these sites. Two of the cluster sulfurs in the P-cluster pair are very close together (approximately 2.1 angstroms), indicating that they form a disulfide bond. These findings indicate that a cavity exists in the interior of the FeMo-cofactor that could be involved in substrate binding and suggest that redox reactions at the P-cluster pair may be linked to transitions of two cluster-bound sulfurs between disulfide and sulfide oxidation states.

Azotobacter vinelandii↗
PubMed1979

Two forms of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

Acetylene reduction by nitrogenase from Rhodospirillum rubrum, unlike that by other nitrogenases, was recently found by other investigators to require an activation of the iron protein of nitrogenase by an activating system comprising a chromatophore membrane component, adenosine 5'-triphosphate (ATP), and divalent metal ions. In an extension of this work, we observed that the same activating system was also required for nitrogenase-linked H(2) evolution. However, we found that, depending on their nitrogen nutrition regime, R. rubrum cells produced two forms of nitrogenase that differed in their Fe protein components. Cells whose nitrogen supply was totally exhausted before harvest yielded predominantly a form of nitrogenase (A) whose enzymatic activity was not governed by the activating system, whereas cells supplied up to harvest time with N(2) or glutamate yielded predominantly a form of nitrogenase (R) whose enzymatic activity was regulated by the activating system. An unexpected finding was the rapid (less than 10 min in some cases) intracellular conversion of nitrogenase A to nitrogenase R brought about by the addition to nitrogen-starved cells of glutamine, asparagine, or, particularly, ammonia. This finding suggests that mechanisms other than de novo protein synthesis were involved in the conversion of nitrogenase A to the R form. The molecular weights of the Fe protein and Mo-Fe protein components from nitrogenases A and R were the same. However, nitrogenase A appeared to be larger in size, because it had more Fe protein units per Mo-Fe protein than did nitrogenase R. A distinguishing property of the Fe protein from nitrogenase R was its ATP requirement. When combined with the Mo-Fe protein (from either nitrogenase A or nitrogenase R), the R form of Fe protein required a lower ATP concentration but bound or utilized more ATP molecules during acetylene reduction than did the A form of Fe protein. No differences between the Fe proteins from the two forms of nitrogenase were found in the electron paramagnetic resonance spectrum, midpoint oxidation-reduction potential, or sensitivity to iron chelators.

Acetylene↗
PubMed1980

Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae.

Crude extracts of the wild-type Klebsiella pneumoniae reduced C2H2 with either pyruvate or formate as reductant (specific activity, 3 nmol min-1 mg of protein-1), whereas crude extracts of nifF mutant were almost inactive (specific activity, 0.05). However, activity in the latter extracts was stimulated by adding Azotobacter chroococcum flavodoxin (specific activity, 10). Thus, nifF mutants may lack an electron transport factor. Crude extracts of nifJ mutants had about 20% of the wild-type level of active MoFe protein, and thus nifJ has a presumptive role in maintaining active MoFe protein. Studies on pyruvate or formate as reductants for nitrogenase in extracts of the nifJ mutants suggest in addition a role in electron input to nitrogenase for the following reasons. (i) Nitrogenase activity with these reductants was very low (specific activity, 0.06) and was not stimulated by extra MoFe protein or the flavodoxin. (ii) Activity was increased by adding a crude extract of a mutant lacking the structural nif genes (specific activity, 1) or a crude extract of the nifF mutant (specific activity, 4).

Acetylene↗
PubMed1981

In vitro activation of inactive nitrogenase component I with molybdate.

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in the presence of WO42- rather than MoO42-, it synthesized active component II and inactive component I of nitrogenase. This inactive component I could be activated in vitro with the iron-molybdenum cofactor or with MoO42-. The latter reaction required adenosine 5'-triphosphate and was inhibited by adenosine 5'-diphosphate. FeMo cofactor and MoO42- produced different levels of activation, but there was no evidence that they acted upon different species of demolybdo component I. Rather, it may be that an additional factor necessary for MoO42-mediated activation but not for FeMo cofactor-mediated activation was limiting. Mo was inserted into component I during both FeMo cofactor- and MoO42- mediated activations.

Adenosine Triphosphate↗
PubMed1984

Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase.

Klebsiella pneumoniae accumulates molybdenum during nitrogenase derepression. The molybdenum is primarily in nitrogenase component I in the form of iron-molybdenum cofactor (FeMo-co). Mutations in any of three genes (nifB, nifN, and nifE) involved in the biosynthesis of FeMo-co resulted in very low molybdenum accumulation and in a molybdenum-free nitrogenase component I. A mutant lacking both subunits of nitrogenase component I accumulated 60% of the amount of molybdenum present in the wild type. The molybdenum was in protein-bound form and behaved differently than that in the wild type with respect to electrophoretic mobility, size, and extractability by organic solvents. Two forms of molybdenum could be extracted from the protein fraction of the mutant; one of them was not detected in the wild type, and the other behaved like FeMo-co in nonaqueous gel filtration chromatography. Crude extracts of this mutant were able to complement in vitro K. pneumoniae or Azotobacter vinelandii mutants unable to produce FeMo-co. These data show that biosynthesis of FeMo-co does not require the presence of nitrogenase component I. In its absence, FeMo-co is accumulated on a different protein, presumably an intermediate in the normal FeMo-co biosynthetic pathway.

Azotobacter↗
PubMed1985

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.

Cysteine↗
PubMed1986

nifV-dependent, low-molecular-weight factor required for in vitro synthesis of iron-molybdenum cofactor of nitrogenase.

The molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase requires a low-molecular-weight factor. The factor is present in extracts of nitrogen fixation-derepressed cultures of Klebsiella pneumoniae and Azotobacter vinelandii, but not in extracts of repressed cultures of these bacteria. Analysis of K. pneumoniae Nif mutants has indicated that the nifV gene product is the only nif protein (besides nifA) necessary for the synthesis and accumulation of the factor. The factor is stable to oxygen, temperatures below 120 degrees C, and extremely acidic and basic conditions. The activity of the factor was completely destroyed by dry ashing or digestion with sulfuric acid. The factor has been partially purified by filtration through an Amicon PM-10 DIAFLO membrane and chromatography on DEAE-cellulose, hydroxylapatite, silica gel, and Sephadex G-25.

Azotobacter↗
PubMed1987

Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK.

The genes from Azotobacter vinelandii, which are homologous to the iron-molybdenum cofactor biosynthetic genes, nifE and nifN, from Klebsiella pneumoniae, have been cloned and sequenced. These genes comprise a single transcription unit and are located immediately downstream from the nitrogenase structural gene cluster (nifHDK). DNA sequence analysis has revealed that the products of the nifE and nifN genes contain considerable homology when compared with the nifD (MoFe protein alpha subunit) and the nifK (MoFe protein beta subunit) gene products, respectively. These striking sequence homologies indicate a structural and functional relationship between a proposed nifEN product complex and the nitrogenase MoFe protein as well as imply an ancestral relationship between these gene clusters. The isolation and characterization of strains which contain deletions within the nifEN gene cluster demonstrate a role for these products in iron-molybdenum cofactor biosynthesis in A. vinelandii.

Amino Acid Sequence↗
PubMed1987

Iron-molybdenum cofactor synthesis in Azotobacter vinelandii Nif- mutants.

Nif- mutants of Azotobacter vinelandii defective in dinitrogenase activity synthesized iron-molybdenum cofactor (FeMo-co) and accumulated it in two protein-bound forms: inactive dinitrogenase and a possible intermediate involved in the FeMo-co biosynthetic pathway. FeMo-co from both these proteins could activate apo-dinitrogenase from FeMo-co-deficient mutants.

Azotobacter↗
PubMed1990

Analysis of Azotobacter vinelandii strains containing defined deletions in the nifD and nifK genes.

Strains of Azotobacter vinelandii which contain defined deletions within the nifD and nifK genes which encode, respectively, the alpha and beta subunits of the MoFe protein of nitrogenase were analyzed. When synthesized without its partner, the beta subunit accumulated as a soluble beta 4 tetramer. In contrast, when the alpha subunit was present without its partner, it accumulated primarily as an insoluble aggregate. The solubility of this protein was increased by the presence of a form of the beta subunit which contained a large internal deletion, such that the alpha subunit could participate in the assembly of small amounts of an alpha 2 beta 2 holoprotein. When synthesized alone, the beta subunit was remarkably stable, even when the protein contained a large internal deletion. The alpha subunit, however, was much more rapidly degraded than the beta subunit, both when it was synthesized alone in its native background and when it was synthesized with its beta subunit partner in a foreign background. Antibodies raised against purified alpha 2 beta 2 MoFe protein recognized epitopes only on the nondenatured beta subunit and not on the nondenatured alpha subunit. Our findings that all epitopes for the alpha2beta2 tetramer appeared to be on the beta subunit, that the beta subunit assembled into beta4 tetramers, and that the alpha subunit alone was very insoluble, combined with the previous finding that the Fe protein binds to the beta subunit (A. H. Willing, M. M. Georgiadis, D. C. Rees, and J. B. Howard, J. Biol. Chem. 264:8499-8503, 1989) all suggest that the beta subunit has a more surface location than the alpha subunit in the alpha2beta2 tetramer.

Antibodies↗
PubMed1990

Synthesis of the iron-molybdenum cofactor of nitrogenase is inhibited by a low-molecular-weight metabolite of Klebsiella pneumoniae.

The in vitro synthesis of the iron-molybdenum cofactor nitrogenase was inhibited by a low-molecular-weight factor. This inhibitory factor was present in the membrane extracts of wild-type and nif mutant strains of Klebsiella pneumoniae that were grown under conditions that either repressed or derepressed nitrogenase expression. In vitro, the inhibition was specific for the NifB protein. Addition of this factor to K. pneumoniae cells at various times during nif derepression decreased nitrogenase activity, presumably through inhibition of iron-molybdenum cofactor synthesis. The inhibitor was purified by solvent extraction and chromatography on DEAE-cellulose, silica gel, and aluminum oxide columns.

Bacterial Proteins↗