Nitrogenase Proteins Research Articles

Intercellular localization of nitrogenase in a non-heterocystous cyanobacterium (cyanophyte), Trichodesmium sp. NIBB1067

The nitrogenase activities of cyanobacteria of the genus Trichodesmium are strictly light-dependent, although they do not develop heterocysts. Recently, the development of heterocyst-equivalent cells (diazocytes) was suggested in Trichodesmium spp. However, no cells with a similar appearance to diazocytes could be found in Trichodesmium sp. NIBB1067. An immunocytochemical analysis of nitrogenase in individual cells was performed using polyclonal antibodies generated from DNA fragments of genes encoding the Fe-protein and the α-and the β-subunit of the MoFe-protein of nitrogenase of Trichodesmium sp. NIBB1067. Visualization of the antibody binding was carried out using a horseradish peroxidase-conjugated secondary antibody and a chromogenic substrate, 3,3′-diaminobenzidine, to avoid the masking effects caused by the bright autofluorescence emitted by Trichodesmium cells. Nitrogenase proteins were detected in almost all the cells (always higher than 95% of total cells) grown under nitrogen-limited conditions. These results indicate that Trichodesmium sp. NIBB1067 does not differentiate heterocyst-equivalent cells.

ATP-driven Reduction by Dark-operative Protochlorophyllide Oxidoreductase from Chlorobium tepidum Mechanistically Resembles Nitrogenase Catalysis

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

Flux capacities and acclimation costs in Trichodesmium from the Gulf of Mexico

Phytoplankton function and acclimation are driven by catalytic protein complexes that mediate key physiological transformations, including generation of photosynthetic ATP and reductant, and carbon and nitrogen fixation. Quantitation of capacities for these processes allows estimation of rates for key ecosystem processes, and identification of factors limiting primary productivity. We herein present molar quantitations of PSI, PSII, ATP synthase, RuBisCO and the Fe protein of nitrogenase of Trichodesmium collected from the Gulf of Mexico, in comparison to determinations for a range of cyanobacteria growing in culture. Using these measurements, estimates were generated for Trichodesmium capacities for carbon fixation of 1–3.4 g C g chl a −1 h−1 and nitrogen fixation of 0.06–0.17 g N g chl a −1 h−1, with diel variations in capacities. ATP synthase levels show that ATP synthesis capacity is sufficient to support these levels of carbon and nitrogen fixation, and that ATP synthase levels change over the day in accordance with the ATP demands of nitrogenase and RuBisCO activity. Levels of measured complexes indicate that Trichodesmium manifests n-type diel light acclimation through rapid changes in RuBisCO:PSII, supported by significant investment of cellular nitrogen. The plasticity in the levels and stoichiometry of these core complexes show that changes in the abundance of core protein complexes are an important component of acclimation and regulation of metabolic function by Trichodesmium populations.

An allylic ketyl radical intermediate in clostridial amino-acid fermentation

The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO(2), 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed to mediate this reaction catalysed by an iron-sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron-sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases and the related benzoyl-CoA reductases-present in anaerobically living bacteria-employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.

Probing the MgATP-bound conformation of the nitrogenase Fe protein by solution small-angle X-ray scattering.

The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small-angle X-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single-amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits, resulting in an overall elongated structure relative to the more compact structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein largely on the basis of the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATP-bound state. In this work, SAXS studies reveal that the large-scale conformational differences between the native Fe protein and the variant observed by X-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.

Molecular insights into nitrogenase FeMoco insertion – The role of His 274 and His 451 of MoFe protein α subunit

The final step of FeMo cofactor (FeMoco) assembly involves the insertion of FeMoco into its binding site in the molybdenum–iron (MoFe) protein of nitrogenase. Here we examine the role of His α274 and His α451 of Azotobacter vinelandii MoFe protein in this process. Our results from combined metal, activity, EPR, stability and insertion analyses show that mutations of His α274 and/or His α451, two of the histidines that belong to a so-called His triad, to small uncharged Ala specifically reduce the accumulation of FeMoco in MoFe protein. This observation indicates that the enrichment of histidines at the His triad is important for FeMoco insertion and that the His triad potentially serves as an intermediate docking point for FeMoco through transitory ligand coordination and/or electrostatic interaction.

A nifH-based Oligonucleotide Microarray for Functional Diagnostics of Nitrogen-fixing Microorganisms

Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database.

Structural Insights into HypB, a GTP-binding Protein That Regulates Metal Binding

HypB is a prokaryotic metal-binding guanine nucleotide-binding protein that is essential for nickel incorporation into hydrogenases. Here we solved the x-ray structure of HypB from Methanocaldococcus jannaschii. It shows that the G-domain has a different topology than the Ras-like proteins and belongs to the SIMIBI (after Signal Recognition Particle, MinD and BioD) class of NTP-binding proteins. We show that HypB undergoes nucleotide-dependent dimerization, which is apparently a common feature of SIMIBI class G-proteins. The nucleotides are located in the dimer interface and are contacted by both subunits. The active site features residues from both subunits arguing that hydrolysis also requires dimerization. Two metal-binding sites are found, one of which is dependent on the state of bound nucleotide. A totally conserved ENV/IGNLV/ICP motif in switch II relays the nucleotide binding with the metal ionbinding site. The homology with NifH, the Fe protein subunit of nitrogenase, suggests a mechanistic model for the switch-dependent incorporation of a metal ion into hydrogenases.

Metabolite Profiles of Nodulated Alfalfa Plants Indicate That Distinct Stages of Nodule Organogenesis Are Accompanied by Global Physiological Adaptations

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.

Identification of FX in the Heliobacterial Reaction Center as a [4Fe-4S] Cluster with an S = 3/2 Ground Spin State

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The Mössbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.

Genetic Complementation Studies of Human Pin1 in Azotobacter vinelandii Revealed that it Requires Amino Terminus of the NifM to Deliver PPIase Effect to the Fe-protein of Nitrogenase

The NifM is a peptidyl prolyl cis-trans isomerase and is required for the maturation and activation of the Fe protein of Nitrogenase. Since the carboxyl terminus of NifM is similar to the Human Pin1, we expressed the Human Pin1 in A. vinelandii BG98, a nifM mutant strain containing a kanamycin insertion and found that it could not complement the function of nifM. It was hypothesized that the amino terminus of the NifM might be required for the Pin1 to bind to NifH similar to requirement of the WW domain for the binding to pSer/Thr-Pro of Cdc25C. Therefore we expressed a NifM amino-terminal and Pin1 fusion protein chimera in A. vinelandii BG98 and this chimera was able to complement the function of NifM. This observation indicated that the amino-terminal of NifM is responsible for the specificity for NifH similar to the specificity of WW domain of Pin1 for pSer/Thr-Pro while the carboxyl terminal domain functions as peptidyl prolyl cis/trans isomerase.

Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous α and β subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353–5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length λCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal α-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the β–β interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the α-RNAP of the pTRG vector and λCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the β-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein–protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the β-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein–protein interaction between the β-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein–protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.

Symbiotic Leghemoglobins Are Crucial for Nitrogen Fixation in Legume Root Nodules but Not for General Plant Growth and Development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from Azotobacter vinelandii

The structures of nitrogenase Fe proteins with defined amino acid substitutions in the previously implicated nucleotide-dependent signal transduction pathways termed switch I and switch II have been determined by X-ray diffraction methods. In the Fe protein of nitrogenase the nucleotide-dependent switch regions are responsible for communication between the sites responsible for nucleotide binding and hydrolysis and the [4Fe-4S] cluster of the Fe protein and the docking interface that interacts with the MoFe protein upon macromolecular complex formation. In this study the structural characterization of the Azotobacter vinelandii nitrogenase Fe protein with Asp at position 39 substituted by Asn in MgADP-bound and nucleotide-free states provides an explanation for the experimental observation that the altered Fe proteins form a trapped complex subsequent to a single electron transfer event. The structures reveal that the substitution allows the formation of a hydrogen bond between the switch I Asn39 and the switch II Asp125. In the structure of the native enzyme the analogous interaction between the side chains of Asp39 and Asp125 is precluded due to electrostatic repulsion. These results suggest that the electrostatic repulsion between Asp39 and Asp125 is important for dissociation of the Fe protein:MoFe protein complex during catalysis. In a separate study, the structural characterization of the Fe protein with Asp129 substituted by Glu provides the structural basis for the observation that the Glu129-substituted variant in the absence of bound nucleotides has biochemical properties in common with the native Fe protein with bound MgADP. Interactions of the longer Glu side chain with the phosphate binding loop (P-loop) results in a similar conformation of the switch II region as the conformation that results from the binding of the phosphate of ADP to the P-loop.

Differentiation of acetylene-reduction sites by stereoselective proton addition during Azotobacter vinelandii nitrogenase-catalyzed C2D2 reduction.

The interactions of acetylene with its binding site(s) on the FeMo cofactor of the MoFe protein of Azotobacter vinelandii nitrogenase were probed using C(2)D(2). Specifically, the effects of changing the C(2)D(2) concentration, electron flux, pH, or the individual presence of N(2), ethylene, or CO on the formation of both cis- and trans-1,2-ethylene-d(2) from C(2)D(2) were measured. A hypothesis, involving two acetylene-reduction sites, was developed to explain the changes observed in the stereoselective protonation during both substrate-concentration-dependent and electron-flux-dependent C(2)D(2) reduction. One of these sites is a higher-affinity acetylene-binding site that produces only cis-1,2-ethylene-d(2) from C(2)D(2). The other is a lower-affinity acetylene-binding site, which produces both cis- and trans-1,2-ethylene-d(2). Added N(2) specifically inhibited the production of cis-1,2-ethylene-d(2) from C(2)D(2), which indicates that N(2) binds to (and is reduced at) the higher-affinity acetylene-binding site. High concentrations of added ethylene behaved like very high concentrations of acetylene and inhibited both the electron flux flowing through the enzyme and cis-isomer formation. Added CO, at very low concentrations, did not affect the relative distribution of cis- and trans-isomers, indicating a separate CO-binding site. The results of pH-dependence experiments showed that substrate inhibition at high C(2)D(2) concentrations is enhanced under acidic conditions but is absent under basic conditions and suggest that a low proton flux has a similar impact to that of a low electron flux; both inhibit cis-1,2-ethylene-d(2) formation selectively. Apparently, the factors affecting stereoselective protonation during C(2)D(2) reduction could be the same as those that perturb protonation of the FeMo cofactor when acetylene is reduced. The observed nitrogenase-catalyzed production of ethylene-d(1) from C(2)D(2) implicates a reversible protonation step in the mechanistic pathway.

Density functional theory calculations and exploration of a possible mechanism of N2 reduction by nitrogenase.

Density functional theory (DFT) calculations have been performed on the nitrogenase cofactor, FeMoco. Issues that have been addressed concern the nature of M-M interactions and the identity and origin of the central light atom, revealed in a recent crystallographic study of the FeMo protein of nitrogenase (Einsle, O.; et al. Science 2002, 297, 871). Introduction of Se in place of the S atoms in the cofactor and energy minimization results in an optimized structure very similar to that in the native enzyme. The nearly identical, short, lengths of the Fe-Fe distances in the Se and S analogues are interpreted in terms of M-M weak bonding interactions. DFT calculations with O or N as the central atoms in the FeMoco marginally support the assignment of the central atom as N rather than O. The assumption was made that the central atom is the N atom, and steps of a catalytic cycle were calculated starting with either of two possible states for the cofactor and maintaining the same charge throughout (by addition of equal numbers of H(+) and e(-)) between steps. The states were [(Cl)Fe(II)(6)Fe(III)Mo(IV)S(9)(H(+))(3)N(3-)(Gl)(Im)](2-), [I-N-3H](2-), and [(Cl)Fe(II)(4)Fe(III)(3)Mo(IV)S(9)(H(+))(3)N(3-)(Gl)(Im)], [I-N-3H](0) (Gl = deprotonated glycol; Im = imidazole). These are the triply protonated ENDOR/ESEEM [I-N](5-) and Mössbauer [I-N](3-) models, respectively. The proposed mechanism explores the possibilities that (a) redox-induced distortions facilitate insertion of N(2) and derivative substrates into the Fe(6) central unit of the cofactor, (b) the central atom in the cofactor is an exchangeable nitrogen, and (c) the individual steps are related by H(+)/e(-) additions (and reduction of substrate) or aquation/dehydration (and distortion of the Fe(6) center). The Delta E's associated with the individual steps of the proposed mechanism are small and either positive or negative. The largest positive Delta E is +121 kJ/mol. The largest negative Delta E of -333 kJ/mol is for the FeMoco with a N(3-) in the center (the isolated form) and an intermediate in the proposed mechanism.

Characterization of Bacteroid Proteins in Soybean Nodules Formed with Bradyrhizobium japonicum USDA110

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins of Bradyrhizobium japonicum USDA110 in the symbiotic and non-symbiotic state. When proteome maps were compared and the characterization of bacteroid-specific proteins was performed by N-terminal amino acid sequencing and matrix-assisted desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, putative identity was assigned to 61 bacteroid protein spots, including many metabolic proteins and ABC transporters as well as nitrogenase proteins like NifH. This study shows that proteome analysis will be a useful tool for surveying genes contributing to rhizobial functions in symbiosis.

Density functional study of the electric hyperfine interactions and the redox-structural correlations in the cofactor of nitrogenase. Analysis of general trends in (57)Fe isomer shifts.

The influence of the interstitial atom, X, discovered in a recent crystallographic study of the MoFe protein of nitrogenase, on the electric hyperfine interactions of (57)Fe has been investigated with density functional theory. A semiempirical theory for the isomer shift, delta, is formulated and applied to the cofactor. The values of delta for the relevant redox states of the cofactor are predicted to be higher in the presence of X than in its absence. The analysis strongly suggests a [Mo(4+)4Fe(2+)3Fe(3+)] oxidation state for the S = 3/2 state M(N). Among C(4-), N(3-), and O(2-), oxide is found to be the least likely candidate for X. The analysis suggests that X should be present in the cofactor states M(OX) and M(R) as well as in the alternative nitrogenases. The calculations of the electric field gradients (EFGs) indicate that the small values for DeltaE(Q) in M(N) result from an extensive cancellation between valence and ligand contributions. X emerges from the analysis of the hyperfine interactions as an ionically bonded species. Its major effect is on the asymmetry parameters for the EFGs at the six equatorial sites, Fe(Eq). A spin-coupling scheme is proposed for the state [Mo(4+)4Fe(2+)3Fe(3+)] that is consistent with the measured (57)Fe A-tensors and DeltaE(Q) values for M(N) and identifies the unique site exhibiting the small A value with the terminal Fe site, Fe(T). The optimized structure of a cofactor model has been calculated for several oxidation states. The study reveals a contraction in the average Fe-Fe distance upon increasing the number of electrons stored in the cluster, in accord with extended X-ray absorption fine structure studies. The reliability of the adopted methodology for predicting redox-structural correlations is tested for cuboidal [4Fe-4S] clusters. The calculations reveal a systematic increase in the S...S sulfide distances, in quantitative agreement with the available data. These trends are rationalized by a simple electrostatic model.

The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules.

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.

Functional Expression of a Fusion-dimeric MoFe Protein of Nitrogenase in Azotobacter vinelandii

The MoFe protein component of the complex metalloenzyme nitrogenase is an alpha2beta2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the alpha and beta subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an alpha2beta2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.