Posttranslational modification of dinitrogenase reductase in Rhodospirillum rubrum treated with fluoroacetate.

Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses.

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5'-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no (15)NH4(+) assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active 'free-living' population, and are not dependent on methane or ANME activity. We investigated the possibility of N2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. (15)N2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, (15)N2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced (15)N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.

Analysis of cbbL, nifH, and pufLM in Soils from the Sør Rondane Mountains, Antarctica, Reveals a Large Diversity of Autotrophic and Phototrophic Bacteria.

Cyanobacteria are generally thought to be responsible for primary production and nitrogen fixation in the microbial communities that dominate Antarctic ecosystems. Recent studies of bacterial communities in terrestrial Antarctica, however, have shown that Cyanobacteria are sometimes only scarcely present, suggesting that other bacteria presumably take over their role as primary producers and diazotrophs. The diversity of key genes in these processes was studied in surface samples from the Sør Rondane Mountains, Dronning Maud Land, using clone libraries of the large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) genes (cbbL, cbbM) and dinitrogenase-reductase (nifH) genes. We recovered a large diversity of non-cyanobacterial cbbL type IC in addition to cyanobacterial type IB, suggesting that non-cyanobacterial autotrophs may contribute to primary production. The nifH diversity recovered was predominantly related to Cyanobacteria, particularly members of the Nostocales. We also investigated the occurrence of proteorhodopsin and anoxygenic phototrophy as mechanisms for non-Cyanobacteria to exploit solar energy. While proteorhodopsin genes were not detected, a large diversity of genes coding for the light and medium subunits of the type 2 phototrophic reaction center (pufLM) was observed, suggesting for the first time, that the aerobic photoheterotrophic lifestyle may be important in oligotrophic high-altitude ice-free terrestrial Antarctic habitats.

Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach.

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas (15)N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

Genetic differentiation of Trifolium repens microsymbionts deriving from Zn-Pb waste-heap and control area in Poland.

The aim of this work was to determine the genetic structure of Rhizobium leguminosarum bv. trifolii population isolated from root nodules of Trifolium repens growing in heavy metal contaminated Boleslaw waste-heap area and compare it with that of an unpolluted control Bolestraszyce population. The 684-bp long dinitrogenase reductase (nifH) gene fragments were amplified in a PCR reaction and then sequenced. An analysis of nifH gene amplicons of 21 rhizobial strains from each of the studied populations revealed substantially reduced genotype (h) and nucleotide (π) diversities in the metallicolous Boleslaw population in comparison to the non-metallicolous Bolestraszyce one, and showed a significant genetic differentiation between these populations (F(ST) = 0.159, p = 0.018). Among the strains under investigation, six genotypes (A-F) with 95-99% nifH gene sequence identities were distinguished. Studied T. repens nodule isolates indicated the highest nifH gene sequence similarities (95-100%) with R. leguminosarum bv. trifolii reference strains and on nifH phylogram all these strains formed monophyletic, highly supported clade (100%). The decreased genotype and nucleotide diversities of the waste-heap R. leguminosarum bv. trifolii population, compared to that from the control area and substantial genetic differentiation between populations of nifH gene, is arguably the consequence of the random genetic drift (Tajima's D = 2.042, p = 0.99).

Arsenic and cadmium are inhibitors of cyanobacterial dinitrogenase reductase (nifH1) gene.

The enzyme nitrogenase complex is a key component conferring nitrogen fixation in all known diazotrophs. This study for the first time examines the impact of As, Na, Cd, Cu and butachlor on component II (dinitrogenase reductase, nifH1) of nitrogenase from diazotrophic cyanobacterium Anabaena sp. PCC7120 using in silico and wet lab approaches. The nifH1 of Anabaena is a glycine-rich stable protein having DNA-binding properties and shows close similarity with free living compared with symbiotic diazotrophs. Phylogenetic tree revealed an adverse effect of the selected stresses on close homologs across the diazotroph community. The protein interaction network demonstrated the presence of nirA, glnA, glnB, alr4255 and alr2485 proteins besides nif proteins, suggesting their involvement in nitrogen fixation along with nifH1. Homology modelling and docking under As, Na, Cd, Cu and butachlor revealed an interaction between stressors and nifH1 protein which was further validated by a transcript of the gene through quantitative real-time PCR (qRT-PCR). Presence of binding sites for As, Na, Cd and Cu on oxyR promoter attested their adverse affects on nifH1. Maximum down-regulation of nifH1 in Cd and As followed by salt, copper and butachlor revealed that arsenic and cadmium were most potential inhibitors of nitrogenase of diazotrophic community, which might negatively affect crop yield.

Network analysis reveals ecological links between N-fixing bacteria and wood-decaying fungi.

Nitrogen availability in dead wood is highly restricted and associations with N-fixing bacteria are thought to enable wood-decaying fungi to meet their nitrogen requirements for vegetative and generative growth. We assessed the diversity of nifH (dinitrogenase reductase) genes in dead wood of the common temperate tree species Fagus sylvatica and Picea abies from differently managed forest plots in Germany using molecular tools. By incorporating these genes into a large compilation of published nifH sequences and subsequent phylogenetic analyses of deduced proteins we verified the presence of diverse pools corresponding to functional nifH, almost all of which are new to science. The distribution of nifH genes strongly correlated with tree species and decay class, but not with forest management, while higher fungal fructification was correlated with decreasing nitrogen content of the dead wood and positively correlated with nifH diversity, especially during the intermediate stage of wood decay. Network analyses based on non-random species co-occurrence patterns revealed interactions among fungi and N-fixing bacteria in the dead wood and strongly indicate the occurrence of at least commensal relationships between these taxa.

Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis.

N(2) fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N(2) and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N(2) at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of (15)N(2)-incubated samples revealed significant incorporation of (15)N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining (15)N(2) tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N(2) fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.

Diversity of nitrogen-fixing bacteria in cyanobacterial mats.

The structure of the microbial community and the diversity of the functional gene for dinitrogenase reductase and its transcripts were investigated by analyzing >1400 16S rRNA gene and nifH sequences from two microbial mats situated in the intertidal zone of the Dutch barrier island Schiermonnikoog. Although both microbial mat communities were dominated by Cyanobacteria, they differed with respect to the composition of the total bacterial community. Proteobacteria-related sequences were retrieved as the second most abundant group higher up in the littoral (Station I), whereas Bacteroidetes were the second most abundant group at the low water mark (Station II). The diazotrophic (nitrogen-fixing) communities at both stations were also different, but had more operational taxonomic units in common than the total bacterial community. Denaturing gradient gel electrophoresis also revealed differences in the total bacterial and diazotrophic community in two consecutive years. Analysis of the expression of nifH at Station I showed a discrepancy between the present and the active diazotrophic community. Transcript abundances of the different diazotrophs changed over a 24-h cycle and were dominated by cyanobacterial lineages in the daytime, while Gammaproteobacteria peaked at night. These variations might be responsible for the pattern in nitrogenase activity observed in these mats.

Diversity of nifH genotypes in floating periphyton mats along a nutrient gradient in the Florida Everglades.

Periphyton mats are an important component of many wetland ecosystems, performing a range of vital ecosystem functions, including nitrogen fixation. The composition and integrity of these mats are affected by nutrient additions, which might result in changes in their function. The overall objective of this study was to investigate the distribution of nifH sequences in floating periphyton mats collected along a nutrient gradient in the Florida Everglades. Distribution of nifH clone libraries indicated nutrient enrichment selected primarily for sequences branching deeply within the heterocystous cyanobacteria and within a novel group of cyanobacteria; sequences from low-nutrient sites were broadly distributed, with no clear dominance of sequences associated with heterocystous and nonheterocystous cyanobacteria and alpha-, gamma-, and delta-proteobacteria. The dominance of heterocystous cyanobacteria in nutrient-enriched sites and the lack of clear dominance by heterocystous cyanobacteria is consistent with previously reported diurnal cycles of nitrogen fixation rates in these systems. Sequences clustering with those harbored by methanotrophs were also identified; sequences from nutrient-impacted and transition regions clustered with those characteristic of type II methanotrophs, and sequences from oligotrophic regions clustered with type I methanotrophs.

A novel potassium deficiency-induced stimulon in Anabaena torulosa.

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacterium Anabaena torulosa and of nine proteins in Escherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K+ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase of A. torulosa and the glycine betaine-binding protein of E. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria

VnfY is required for full activity of the vanadium-containing dinitrogenase in Azotobacter vinelandii.

A gene from Azotobacter vinelandii whose product exhibits primary sequence similarity to the NifY, NafY, NifX, and VnfX family of proteins, and which is required for effective V-dependent diazotrophic growth, was identified. Because this gene is located downstream from vnfK in an arrangement similar to the relative organization of the nifK and nifY genes, it was designated vnfY. A mutant strain having an insertion mutation in vnfY has 10-fold less vnf dinitrogenase activity and exhibits a greatly diminished level of (49)V label incorporation into the V-dependent dinitrogenase when compared to the wild type. These results indicate that VnfY has a role in the maturation of the V-dependent dinitrogenase, with a specific role in the formation of the V-containing cofactor and/or its insertion into apodinitrogenase.

Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage.

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-(32)P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-(14)C]NAD individually upon UV irradiation, but most (14)C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-(14)C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes.

The pairs of nitrogen fixation genes nifDK and nifEN encode for the alpha and beta subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed.

ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase.

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.

Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L.

The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.

Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites.

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.

Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii.

BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, whereas addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.

The Azotobacter vinelandii NifEN complex contains two identical [4Fe-4S] clusters.

The nifE and nifN gene products from Azotobacter vinelandii form an alpha2beta2 tetramer (NifEN complex) that is required for the biosynthesis of the nitrogenase FeMo cofactor. In the current model for NifEN complex organization and function, the complex is structurally analogous to the nitrogenase MoFe protein and provides an assembly site for a portion of FeMo cofactor biosynthesis. In this work, gene fusion and immobilized metal-affinity chromatography strategies were used to elevate the in vivo production of the NifEN complex and to facilitate its rapid and efficient purification. The NifEN complex produced and purified in this way exhibits an FeMo cofactor biosynthetic activity similar to that previously described for the NifEN complex purified by traditional chromatography methods. UV-visible, EPR, variable-temperature magnetic circular dichroism, and resonance Raman spectroscopies were used to show that the NifEN complex contains two identical [4Fe-4S]2+ clusters. These clusters have a predominantly S = 1/2 ground state in the reduced form, exhibit a reduction potential of -350 mV, and are likely to be coordinated entirely by cysteinyl residues on the basis of spectroscopic properties and sequence comparisons. A model is proposed where each NifEN complex [4Fe-4S] cluster is bridged between a NifE-NifN subunit interface at a position analogous to that occupied by the P clusters in the nitrogenase MoFe protein. In contrast to the MoFe protein P clusters, the NifEN complex [4Fe-4S] clusters are proposed to be asymmetrically coordinated to the NifEN complex where NifE cysteines-37, -62, and -124 and NifN cysteine-44 are the coordinating ligands. On the basis of a homology model of the three-dimensional structure of the NifEN complex, the [4Fe-4S] cluster sites are likely to be remote from the proposed FeMo cofactor assembly site and are unlikely to become incorporated into the FeMo cofactor during its assembly.