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A model for the formation of dinitrogenase 2. The open squares represent empty FeV-co sites in the subunit of apodinitrogenase 2, and the boxed " P " represent the P clusters.
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The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an alpha2beta2delta2 hexamer. The delta subunit (the VNFG protein) has been characterized in order to further delineate its function in the nitrogenase 2 enzyme system. Two species of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is c...
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In seeking to mimic the hydrogenation of N(2) to NH(3) as effected under mild conditions by the enzyme nitrogenase, three classes of known metal sulfide clusters that resemble the NFe(7)MoS(9) core of FeMo-co, the active site of nitrogenase, have been assessed theoretically. The assessment has been made in the context of the previously proposed mec...
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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 inhibi...
The P-cluster of nitrogenase is largely known for its function to mediate electron transfer to the active cofactor site during catalysis. Here, we show that a P-cluster variant (designated P*-cluster), which consists of paired [Fe(4)S(4)]-like clusters, can catalyze ATP-independent substrate reduction in the presence of a strong reductant, europium...
The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotr...
Citations
... The G subunits are in exclusive contact with the D subunits and do not contain any metal clusters. Their assumed function is in cofactor delivery for the correct maturation of these dinitrogenases (Fig. 1b) 26 . In FeFe protein, the AnfD subunit differs from NifD of the same organism with a root-mean-squared displacement (r.m.s.d.) of 1.14 Å and from VnfD with an r.m.s.d. of 0.66 Å for all atoms. ...
Nitrogenases uniquely reduce atmospheric N2 to bioavailable ammonium. They group into three isoforms that primarily differ in the architecture of their active-site cofactors. A molybdenum or vanadium ion is introduced into a common precursor cluster to form Mo- and V-dependent nitrogenases, respectively. In contrast, the third class of the enzyme only utilizes abundant iron to reduce N2 under ambient conditions and is consequently of high interest for mechanistic studies and catalyst design. Here we report the three-dimensional structure of Fe-nitrogenase from Azotobacter vinelandii and its FeFe cofactor, a [8Fe:9S:C] cluster with an interstitial carbide and an organic homocitrate ligand at the apical iron that substitutes for Mo or V in the other isoforms. The structure reveals lability of sulfide S2B, the proposed binding site for substrate in other nitrogenases, further supporting a general mechanism of proton and electron transfer for all nitrogenases and all their substrates.
... The genes designated for V-nitrogenase are vnfH, vnfD, vnfG, and vnfK, and the genes designated for Fe-nitrogenase are anfH, anfD, anfG, and anfK, where vnfG and anfG encode additional subunits. VnfG is required for processing apodinitrogenase to functional dinitrogenase [21]. It has been shown that a significant fraction of nitrogen fixation by free-living soil diazotrophs such as a PNSB Rhodopseudomonas palustris is contributed by the alternative nitrogenases in addition to Mo-nitrogenase [16,22]. ...
Biological nitrogen fixation catalyzed by Mo-nitrogenase of symbiotic diazotrophs has attracted interest because its potential to supply plant-available nitrogen offers an alternative way of using chemical fertilizers for sustainable agriculture. Phototrophic purple nonsulfur bacteria (PNSB) diazotrophically grow under light anaerobic conditions and can be isolated from photic and microaerobic zones of rice fields. Therefore, PNSB as asymbiotic diazotrophs contribute to nitrogen fixation in rice fields. An attempt to measure nitrogen in the oxidized surface layer of paddy soil estimates that approximately 6–8 kg N/ha/year might be accumulated by phototrophic microorganisms. Species of PNSB possess one of or both alternative nitrogenases, V-nitrogenase and Fe-nitrogenase, which are found in asymbiotic diazotrophs, in addition to Mo-nitrogenase. The regulatory networks control nitrogenase activity in response to ammonium, molecular oxygen, and light irradiation. Laboratory and field studies have revealed effectiveness of PNSB inoculation to rice cultures on increases of nitrogen gain, plant growth, and/or grain yield. In this review, properties of the nitrogenase isozymes and regulation of nitrogenase activities in PNSB are described, and research challenges and potential of PNSB inoculation to rice cultures are discussed from a viewpoint of their applications as nitrogen biofertilizer.
... The G subunit does influence substrate specificity, as strains with mutations in anfG and vnfG were able to reduce acetylene but not N 2 [21]. The G subunit has also been proposed to be involved in FeV-cofactor delivery, which is supported by the crystal structure of VnfDGK where VnfG is located near the cofactor insertion site but away from the P-cluster [20,22]. ...
Azotobacter vinelandii contains three forms of nitrogenase known as the Mo-, V-, and Fe-nitrogenases. They are all two-component enzyme systems, where the catalytic component, referred to as NifDK, VnfDGK, and AnfDGK, associates with the reductase component, the Fe protein or NifH, VnfH, and AnfH respectively. AnfDGK and VnfDGK have an additional subunit compared to NifDK, termed gamma or AnfG and VnfG, whose role is unknown. The expression of each nitrogenase is tightly regulated by metal availability, however it is known that there is crosstalk between the Mo- and V‑nitrogenases but the Fe‑nitrogenase components cannot support substrate reduction with its Mo‑nitrogenase counterparts. Here, docking models for the nitrogenase complexes were generated in ClusPro 2.0 based on the crystal structure of the Mo‑nitrogenase and refined using the HADDOCK 2.2 refinement interface to identify structural determinants that enable crosstalk between the Mo- and V‑nitrogenase but not the Fe‑nitrogenase. Differing salt bridge interactions were identified at the binding interface of each complex. Specifically, positively charged residues of VnfG enable complementary interactions with NifH and VnfH but not AnfH. Similarly, negatively charged residues of AnfG enable interactions with AnfH but not NifH or VnfH. A role for the G subunit is revealed where VnfG could be mediating crosstalk between the Mo- and V‑nitrogenases while the AnfG subunit on AnfDGK makes interactions with NifH and VnfH unfavorable, reducing competition with NifDK and funneling electrons to the most efficient nitrogenase.
... Azotobacter vinelandii has the peculiarity of having genes to encode the Mo-nitrogenase (nif ) and the alternative V (vnf ) and Fe-only (anf ) nitrogenases . The dinitrogenase components of the alternative nitrogenases contain additional subunits (VnfG or AnfG) essential for N 2 reduction (Chatterjee et al., 1997;Krahn et al., 2002) and present subtle differences in cofactor structure (Sippel and Einsle, 2017). However, amino acid sequence comparisons of NifD/VnfD/AnfD and NifK/VnfK/AnfK indicate that residues that serve as ligands to the metal cofactors are conserved in all three nitrogenases . ...
The N2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybdenum cofactor (FeMo-co). The in vitro FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an A. vinelandii strain lacking the MoSto subunit genes (ΔmosAB) was analyzed. Consistent with its role as storage protein, the ΔmosAB strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased in vivo nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The ΔmosAB strain was found to readily derepress vnfDGK upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The ΔmosAB mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δvnf Δanf) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the ΔmosAB and mosAB+ strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to A. vinelandii in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.
... They each have two main components: dinitrogenase reductase (DNR) and dinitrogenase [75,76]. The former is a single subunit encoded by one gene and the latter comprises at least two subunits (a and b) that form an a 2 b 2 tetramer in the Mo nitrogenase [76,77], while the V and Fe nitrogenases also contain a d subunit [78,79]. Some studies have investigated the specific amino acid residues essential for the catalysis, especially in the Mo nitrogenase [80,81]. ...
Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of many different biological systems, including enzyme kinetics and the genetic code. It has been especially useful in working out the structures and mechanisms of different nitrogenase enzymes, how they can function in oxic environments and the interactions of nitrogen fixation with other aspects of metabolism. Interest in studying A. vinelandii has waned in recent decades, but this bacterium still possesses great potential for new discoveries in many fields and commercial applications. The species is of interest for research because of its genetic pliability and natural competence. Its features of particular interest to industry are its ability to produce multiple valuable polymers - bioplastic and alginate in particular; its nitrogen-fixing prowess, which could reduce the need for synthetic fertilizer in agriculture and industrial fermentations, via coculture; its production of potentially useful enzymes and metabolic pathways; and even its biofuel production abilities. This review summarizes the history and potential for future research using this versatile microbe.
... In the last few decades, an immense attention has been paid to the chemistry of vanadium due to the discovery of the vital role of vanadium in the biological system [1]. Vanadium complexes have numerous applications in analytical, clinical, biological and pharmacological fields [2]. ...
... Then methanolic solution of VOSO 4 ÁxH 2 O/VCl 3 (THF) 3 in different M/L ratios was added drop wise in above solution (Scheme 1). The reaction mixture was stirred for 6 h at room temperature for complexes (1) and (2) and refluxed for 4 h in case of complex (3). The pH of reaction mixture was adjusted from 3.5 to 4.5. ...
... Partial charges of atoms of geometry optimized structure of complexes(1) and(3)in gas phase. ...
Oxovanadium (IV) complexes (1)-(3) have been synthesized by treating 2-thiophene carboxylic acid hydrazide with VOSO4⋅xH2O and VCl3(THF)3 in different M/L ratios. These complexes have been characterized by elemental analysis, UV-vis, FT-IR and mass spectrometry. The FT-IR data predicts the bidentate nature of the ligand which is also confirmed by semi-empirical study. Mass spectrometric data shows that molecular ion peak is only observed for 2-thiophene carboxylic acid hydrazide. The ESP map and thermodynamic parameters shows the presence of partial charge on atoms and stability of synthesized oxovanadium complexes, respectively. DNA binding study of complexes (1)-(3) was carried out by UV-vis and cyclic voltammetric methods which suggests the intercalative binding mode of the complexes with DNA. Cytotoxicity was checked by brine shrimp lethality assay and complex (1) showed greater cytotoxicity towards Artemia salina as compared to free ligand. Immuno-modulatory activity data shows that hydrazide ligand was more active as compared to oxovanadium complexes and standard drug. Complex (2) shows significant urease inhibition activity. The ligand and synthesized complexes were found inactive against all tested bacterial and fungal strains.
... In contrast uncharacterized nitrogenase genes occur as the only nitrogenase homolog in their respective genomes. Furthermore these genomes lack genes which encode a third structural subunit (anfG, vnfG) found to be associated with known alternative nitrogenases (Chatterjee et al., 1997;Lee et al., 2009), making it unlikely that they bind either FeV-co or FeFe-co. ...
Nitrogenase enzymes have evolved complex iron–sulfur (Fe–S) containing cofactors that most commonly contain molybdenum (MoFe, Nif) as a heterometal but also exist as vanadium (VFe, Vnf) and heterometal-independent (Fe-only, Anf) forms. All three varieties are capable of the reduction of dinitrogen (N2) to ammonia (NH3) but exhibit differences in catalytic rates and substrate specificity unique to metal type. Recently, N2 reduction activity was observed in archaeal methanotrophs and methanogens that encode for nitrogenase homologs which do not cluster phylogenetically with previously characterized nitrogenases. To gain insight into the metal cofactors of these uncharacterized nitrogenase homologs, predicted three-dimensional structures of the nitrogenase active site metal-cofactor binding subunits NifD, VnfD, and AnfD were generated and compared. Dendrograms based on structural similarity indicate nitrogenase homologs cluster based on heterometal content and that uncharacterized nitrogenase D homologs cluster with NifD, providing evidence that the structure of the enzyme has evolved in response to metal utilization. Characterization of the structural environment of the nitrogenase active site revealed amino acid variations that are unique to each class of nitrogenase as defined by heterometal cofactor content; uncharacterized nitrogenases contain amino acids near the active site most similar to NifD. Together, these results suggest that uncharacterized nitrogenase homologs present in numerous anaerobic methanogens, archaeal methanotrophs, and firmicutes bind FeMo-co in their active site, and add to growing evidence that diversification of metal utilization likely occurred in an anoxic habitat.
... Its function has not been finally resolved, but it is apparently required for processing the apoprotein of the alternative nitrogenases to the functional enzyme complex by assisting in the insertion of the cofactor, as has been specifically shown for the V nitrogenase (45,46). Remarkably, although the proteins VnfG and AnfG are required for N 2 fixation by A. vinelandii, they are not required for C 2 H 2 reduction (45,46,228). ...
... Its function has not been finally resolved, but it is apparently required for processing the apoprotein of the alternative nitrogenases to the functional enzyme complex by assisting in the insertion of the cofactor, as has been specifically shown for the V nitrogenase (45,46). Remarkably, although the proteins VnfG and AnfG are required for N 2 fixation by A. vinelandii, they are not required for C 2 H 2 reduction (45,46,228). ...
This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N(2) fixation and/or H(2) formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H(2) as a source of combustible energy. To enhance the rates of H(2) production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H(2) formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.
... The VnfG subunit has been shown to be loosely associated with apo-VFe protein in extracts of a strain incapable of synthesizing FeV-co. When subjected to electrophoresis, the three subunits comigrate on anoxic native gels, but the VnfG subunit separates from the complex by gel filtration chromatography (12). The ␣ 2  2 ␦ 2 apo-VFe protein (lacking FeV-co but containing the P-clusters) present in these cell extracts is competent to activation by partially purified FeV-co. ...
... Tiemann, S. Fuchs, K. Schneider & A. Mu¨ller, unpublished results). Dissociation of the d subunit from the apodinitrogenase under certain conditions (e.g. during gel filtration) has also been reported in the case of the vanadium nitrogenase (VFe protein) from A. vinelandii [42]. The tetrameric FeFe apoprotein from R. capsulatus did not show any EPR signal typical of a P-cluster signal. ...
The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of −195 mV (appearance) and −30 mV (disappearance), (2) an axial signal (g|| = 2.00, g⊥ = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the ‘low-potential’ rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the ‘high-potential’ rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: −80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this ‘turnover signal’ nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.
