Nitrogenase Proteins Research Articles

The influence of net charge on the redox potentials of Fe4S4 cubane-type clusters in aprotic solvents

The recent demonstration of the existence of the [Fe4S4]0 oxidation state in the iron protein of nitrogenase raises the issue of stabilization of reduced states of Fe4S4 clusters in proteins and synthetic compounds. In thiolate-ligated clusters, potentials of the redox couples [Fe4S4(SR)4]2−/3− have been shown to scale linearly with substituent (R) constants. In this work, the combined result of inductive and electrostatic effects on potentials has been examined by use of the previously reported clusters with R=p-C6H4NO2, C6F5 and new clusters having R=CH2CH2NMe3+, p-C6H4NR3+, and p-C6H4CH2NR3+. The crystal structure of (Bu4N)2[Fe4S4(SC6H4-p-NO2)4] reveals a tetragonally compressed [Fe4S4]2+ core. Synthesis of salts of the thiol cations p-HSC6H4NR3+ and p-HSC6H4CH2NR3+ are described. The clusters [Fe4S4(SR)4]2+ and the [1:3] site-differentiated species [Fe4S4(LS3)(SR)]1− were prepared by ligand substitution reactions. Potentials (vs. SCE) and potential shifts relative to appropriate reference clusters for 17 clusters are reported in aprotic solvents. Substantial positive potential shifts can be achieved with the use of strongly electron-withdrawing monoanionic and neutral thiolate ligands. The two least negative [Fe4S4]2+/1+ potentials are found for the couples [Fe4S4(SC6H4-p-NO2)4]2−/3− (−0.65 V) and [Fe4S4(SC6H4-p-NMe3)4]2+/1+ (−0.73 V) in DMF. Also reported are [Fe4S4]1+/0 potentials for seven clusters, all of which are ≲−1.5 V and do not correspond to strictly chemically reversible reactions. The redox behavior of the cluster in the iron protein of nitrogenase, with reported reductions at −0.31 and −0.46 V (vs. SHE), is contrasted with synthetic clusters in aqueous or non-aqueous media. None of the latter reduces at comparable potentials and the difference between first and second potentials is always much larger. Synthesis of the [Fe4S4]0 state is a remaining goal in the synthetic analogue chemistry of biological iron–sulfur clusters.

Interaction with magnesium and ADP stabilizes both components of nitrogenase from Klebsiella pneumoniae against urea denaturation.

The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from approximately 1.5 to approximately 2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to approximately 3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type.

Endophytic Expression of nif Genes of Azoarcus sp. Strain BH72 in Rice Roots

Transcriptional fusions of gusA and gfp to the nifH gene as well as immunogold labeling with antibodies against the iron protein of nitrogenase revealed high nitrogenase gene expression levels of the endophyte Azoarcus sp. strain BH72 inside infected rice roots (Oryza sativa) in the laboratory. Thus, environmental conditions inside rice roots are permissive for endophytic nitrogen fixation in bacterial microcolonies in the aerenchyma.

Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152.

In cultures of the unicellular cyanobacterium Gloeothece sp. ATCC 27152 growing under alternating 12 h light and 12 h darkness, nitrogenase activity appears as cultures enter the dark phase. Synthesis of both component proteins of nitrogenase commences immediately prior to the appearance of activity and continues until about 8 h into the period of darkness. The two components (Fe-protein and MoFe-protein) are synthesized in a molar ratio of about 3:1. Degradation of the nitrogenase proteins starts as early as 4 h into the dark period and increases markedly as cultures enter the light phase. As a result, both nitrogenase proteins are completely absent from cultures during most of the light phase. In contrast, all of the other proteins investigated appeared to be present throughout the cycle of alternating light and darkness. Degradation of nitrogenase depends upon protein synthesis during the last 6 h of darkness and is prevented by addition of protease inhibitors. Two proteins, of M(r) 47,000 and 29,000, are specifically synthesized during this period and it is possible that they have a role in nitrogenase degradation. Proteolytic activity of extracts of Gloeothece, measured as the ability to degrade azocasein, increased markedly during the early part of the light period, but this increase did not depend on protein synthesis. This activity does not therefore correspond to that specifically involved in nitrogenase catabolism, though it may act on initial breakdown products generated by a nitrogenase-specific degradative system. A phycobiliprotein appears to act as a temporary store of the degradation products of nitrogenase.

The presence of ADP-ribosylated Fe protein of nitrogenase in Rhodobacter capsulatus is correlated with cellular nitrogen status.

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.

GroEL chaperonins are required for the formation of a functional nitrogenase in Bradyrhizobium japonicum

At least five highly conserved, but disparately regulated groESL operons are present in Bradyrhizobium japonicum. Expression of groESL 3 is coregulated with symbiotic nitrogen fixation genes, implying a role of GroESL chaperonins in the nitrogen fixation process. Null mutants of individual groEL genes, however, were not impaired in symbiotic nitrogen fixation activity. By contrast, the groEL 3-plus-groEL 4 double mutant strain D4, which is mutated in those groEL genes that contribute most to the GroEL pool under symbiotic conditions, exhibited less than 5% Fix activity as compared to the wild-type. Expression of lacZ fusions made to several representative nif and fix genes was not, or only marginally, reduced in mutant D4, indicating that the requirement of chaperonins for nitrogen fixation does not occur at the level of RegSR-NifA-σ54- or FixLJ-FixK2-dependent gene regulation. Instead, immunoblot analyses revealed that the level of NifH and NifDK nitrogenase proteins was drastically decreased in extracts prepared from D4 bacteroids and from free-living cells grown anaerobically. Transcriptional fusions of the anaerobically induced groESL 3 promoter (P3) to all five B. japonicum groESL operons and also to groESL from Escherichia coli were integrated into the chromosome of mutant D4. Strains harboring P3 fused to groESL 1, groESL 2, groESL 5, or E. coli groESL partially complemented the symbiotic defect of mutant D4, whereas the wild-type phenotype was completely restored in strains complemented with P3 fused to groESL 3 (control) or groESL 4. Likewise, the growth defect of an E. coli groEL mutant could be corrected at least partially by individual B. japonicum groESL operons. In conclusion, both series of complementation analyses were not indicative of a strict substrate specificity of any of the B. japonicum groESL gene products, which is in good agreement with their high degree of sequence conservation.

Mössbauer and Integer-Spin EPR Studies and Spin-Coupling Analysis of the [4Fe-4S]0Cluster of the Fe Protein fromAzotobacter vinelandiiNitrogenase

We have previously shown that the [4Fe-4S] cluster of the Fe protein of nitrogenase from Azotobacter vinelandii can be reduced to the all-ferrous state, [4Fe-4S]0. We have studied here this state with integer-spin EPR and Mössbauer spectroscopy, and analyzed the exchange couplings that give rise to a ground state with cluster spin S = 4. The results are as follows: The cluster contains four high-spin (Si = 2) ferrous sites with isomer shift δ = 0.68 mm/s. One site, FeA, has ΔEQ = 3.08 mm/s while the three other sites exhibit ΔEQ-values between 1.2 and 1.7 mm/s. This asymmetry is surprising in view of the fact that the cluster is suspended at the interface of an α2 protein dimer. A similar 3:1 symmetry is also evident in the magnetic hyperfine tensors, A(i), of the four sites; three sites have negative A-values while that of the fourth site, FeA, is positive. Analysis of the exchange couplings, assumed to be antiferromagnetic, shows that every possible S = 4 ground multiplet that can be constructed from four high-spin ferrous sites must have this property. From Mössbauer spectroscopy we obtained for the S = 4 multiplet the zero-field splitting parameter D = −0.75 ± 0.05 cm-1. Moreover, the Mössbauer studies show that the two lowest levels of the spin multiplet are split by Δground = 0.02 cm-1. Four pairs of levels from the S = 4 multiplet yield integer-spin EPR signals which can be observed, with only minor broadening, at temperatures up to 140 K. Transitions between the two lowest levels yield a sharp resonance at g = 16.4. We have simulated the EPR spectra of the all-ferrous cluster in the temperature range from 2 to 120 K and have obtained curves that fit the experimental data with high accuracy. The spin concentration obtained from these simulations is in good agreement with the [4Fe-4S] cluster concentration. Moreover, analysis of the EPR data has revealed that another spin multiplet, most likely an S = 3 manifold, becomes populated above 40 K. Exchange coupling (J) among four high-spin ferrous ions yields 85 spin multiplets, 15 of which have S = 4. To have an S = 4 ground state, two of the six J-values must differ by at least a factor 3, and two others by a factor 2.5. The computed spin projection factors, together with aiso = −22.4 MHz of ferrous rubredoxin, yield Aiso-values for three of the sites that are in good agreement with the experimental data; FeA, however, requires a much smaller aiso-value (−12 to −16 MHz).

The Role of Methionine 156 in Cross-subunit Nucleotide Interactions in the Iron Protein of Nitrogenase

A variant Fe protein has been created at the completely conserved residue methionine 156 by changing it to cysteine. The Azotobacter vinelandii strain expressing M156C is unable to grow under nitrogen-fixing conditions, and the purified protein cannot support substrate reduction in vitro. This mutation has an effect on the Fe protein's ability to undergo the MgATP-induced conformational change as evidenced by the fact that M156C is chelated in the presence of MgATP with a lower observed rate than wild-type. While the electron paramagnetic resonance spectra of this protein are similar to those of the wild-type Fe protein, the circular dichroism spectrum is markedly different in the presence of MgATP, showing that the conformation adopted by M156C following nucleotide binding is different from the wild-type conformation. Although competition activity and chelation assays show that this Fe protein can still form a complex with the MoFe protein, this altered conformation only supports MgATP hydrolysis at 1% the rate of wild-type Fe protein. A model based on x-ray crystallographic information is presented to explain the importance of Met-156 in stabilization of the correct conformation of the Fe protein via critical interactions of the residue with Asp-43 and nucleotide in the other subunit.

An All-ferrous State of the Fe Protein of Nitrogenase: INTERACTION WITH NUCLEOTIDES AND ELECTRON TRANSFER TO THE MoFe PROTEIN

The MoFe protein of nitrogenase catalyzes the six-electron reduction of dinitrogen to ammonia. It has long been believed that this protein receives the multiple electrons it requires one at a time, from the [4Fe-4S]2+/+ couple of the Fe protein. Recently an all-ferrous [4Fe-4S]0 state of the Fe protein was demonstrated suggesting instead a series of two electron steps involving the [4Fe-4S]2+/0 couple. We have examined the interactions of the [4Fe-4S]0 Fe protein with nucleotides and its ability to transfer electrons to the MoFe protein. The [4Fe-4S]0 Fe protein binds both MgATP and MgADP and undergoes the MgATP induced conformational change and then binds properly to the MoFe protein, as evidenced by the fact that the behavior of the 0 and +1 oxidation states in the chelation and chelation protection assays are indistinguishable. Nucleotide binding does not effect the distinctive UV/Vis, CD, or Mössbauer spectra exhibited by the [4Fe-4S]0 Fe protein; however, because the intensity of the g = 16.4 EPR signal of the [4Fe-4S]0 Fe protein is extremely sensitive to minor variations of the rhombicity parameter E/D, the EPR signal is sensitive to the binding of nucleotides. A 50:50 mixture of [4Fe-4S]2+ and [4Fe-4S]0 Fe protein results in electron self-exchange and 100% production of [4Fe-4S]+ Fe protein, demonstrating that the +1/0 couple is fully reversible. MgATP is absolutely required for electron transfer from the [4Fe-4S]0 Fe protein to the reduced state of the MoFe protein. In that reaction both electrons are transferred and are used to reduce substrate.

Bacteroids Isolated from Ineffective Nodules of Pisum sativum Mutant E135 (syml3) Lack Nitrogenase Activity but Contain the Two Protein Components of Nitrogenase

Pea mutant E135 (syml3) forms ineffective (Fix) nodules that lack nitrogen fixing activity. To determine the developmental step blocked in E135 nodules we studied the nitrogenase activities in isolated bacteroids and in cell-free extracts of bacteroids, and measured the two components of nitrogenase protein in bacteroids. Bacteroids prepared anaerobically from E135 nodules showed no acetylene reduction activity in the presence and absence of myoglobin. Furthermore, no acetylene reduction activity by cell-free extracts of E135 bacteroids was detected in the presence of ATP-generating system and dithionite. However, immunoblotting analyses revealed the presence of nitrogenase components I and II in E135 nodule bacteroids. These results suggest that a host plant gene is involved in the expression of nitrogenase activity in symbiotic bacteria.

EXAFS studies on the PN and POX states of the P-clusters in nitrogenase

The MoFe protein of nitrogenase is an α2β2 tetramer that contains two each of two different types of metal centers, the FeMo-cofactor and the P-clusters. The function of the P-clusters is believed to be to accept electrons from the Fe protein of nitrogenase and to donate them to the FeMo-cofactor. We have studied the P-clusters of Azotobacter vinelandii nitrogenase in both the PN and POX states utilizing Fe K-edge X-ray absorption spectroscopy. Since the MoFe holoprotein contains the seven iron FeMo-cofactor centers in addition to P-clusters, we have utilized a FeMo-cofactor-deficient MoFe protein synthesized by the Δnif H strain DJ54. That MoFe protein is an α2β2 tetramer that contains P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the POX state. Several important results have emerged from our XAS studies. The first shell Fe-S coordination shows the same average Fe-S distance (2.26 A) in both states. The second coordination shells could only be well fit using two different Fe-Fe contributions. In both states, short Fe-Fe components with distances of 2.57 A and 2.42 A for the PN and POX states, respectively, were required to complement longer 2.75 A and 2.70 A distances. Understanding of the P-cluster structure is essential if we are to make advances in understanding the role of the P-clusters and their participation in electron transfer through the nitrogenase system.

Redox properties and electron paramagnetic resonance spectroscopy of the transition state complex of Azotobacter vinelandii nitrogenase

Nitrogenase is a two-component metalloenzyme that catalyzes a MgATP hydrolysis driven reduction of substrates. Aluminum fluoride plus MgADP inhibits nitrogenase by stabilizing an intermediate of the on-enzyme MgATP hydrolysis reaction. We report here the redox properties and electron paramagnetic resonance (EPR) signals of the aluminum fluoride-MgADP stabilized nitrogenase complex of Azotobacter vinelandii. Complex formation lowers the midpoint potential of the [4Fe-4S] cluster in the Fe protein. Also, the two-electron reaction of the unique [8Fe-7S] cluster in the MoFe protein is split in two one-electron reactions both with lower midpoint potentials. Furthermore, a change in spin-state of the two-electron oxidized [8Fe-7S] cluster is observed. The implications of these findings for the mechanism of MgATP hydrolysis driven electron transport within the nitrogenase protein complex are discussed.

Characterization of a Variant Iron Protein of Nitrogenase That Is Impaired in Its Ability to Adopt the MgATP-induced Conformational Change

An Azotobacter vinelandii nitrogenase iron protein mutant has been created which contains an alanine to glycine substitution at amino acid 157. The strain expressing this mutant Fe protein is able to grow under nitrogen-fixing conditions. This contrasts with an A. vinelandii strain described previously which is unable to grow under nitrogen-fixing conditions and which expresses an Fe protein variant that has an alanine to serine mutation at position 157. The A157S Fe protein was unable to support substrate reduction by nitrogenase because of an inability to undergo a required MgATP-induced conformational change. Although the A157G strain grows at 55% of the rate of the wild-type strain, purified A157G Fe protein is only able to support substrate reduction in in vitro assays at a rate that is approximately 20% of the rate supported by the wild-type Fe protein. Electron paramagnetic resonance, circular dichroism spectroscopies, and enzymatic activity data indicate that the A157G Fe protein adopts the correct conformation upon the binding of MgATP. However, kinetic studies using chelation show that this protein undergoes the conformational change more slowly than the wild-type protein. Thus, this mutant has lower activity because of an impaired ability to undergo this conformational change. Comparison of two available x-ray crystal structures of the native Fe protein alone and complexed with the MoFe protein has provided us with a model to explain the change in activity in alanine 157 mutants. Steric interactions with the side chain of residue 157 influence the protein's ability to undergo the initial MgATP-induced conformational change. In the case of the A157G mutant, however, once the correct conformation is attained, the protein can participate in all subsequent reactions including complex formation, electron transfer, and MgATP hydrolysis. Thus, the role of alanine 157 is to stabilize the proper initial conformation upon MgATP binding.

All-Ferrous Titanium(III) Citrate Reduced Fe Protein of Nitrogenase: An XAS Study of Electronic and Metrical Structure

All-Ferrous Titanium(III) Citrate Reduced Fe Protein of Nitrogenase: An XAS Study of Electronic and Metrical Structure

Specific interaction of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with the nitrogenase MoFe protein.

Putative physiological partners of the [2Fe-2S] ferredoxin from Clostridium pasteurianum have been searched by running crude soluble extracts of this bacterium through an affinity column to which the [2Fe-2S] ferredoxin had been covalently bound. Subsequent washing of the column with buffers of increasing ionic strength revealed a strong and specific interaction of the ferredoxin with the MoFe protein of nitrogenase. This interaction was further investigated by performing cross-linking experiments with mixtures of the two purified proteins in solution. Analysis of the reactions by SDS-polyacrylamide gel electrophoresis evidenced only two covalently linked products. These were identified by N-terminal sequencing as the alpha and beta subunits of the MoFe protein, each cross-linked to a single polypeptide chain of the ferredoxin. This result, taking into account the dimeric structure of the ferredoxin in solution, strongly suggests an interaction of the ferredoxin with the MoFe protein at a site contributed to by both subunits of the MoFe protein. The ionic strength dependence of the interaction evidenced by affinity chromatography was confirmed in the cross-linking reactions, and its specificity was assessed by showing that no cross-linking occurred when the [2Fe-2S] C. pasteurianum ferredoxin was denatured or replaced by spinach ferredoxin or by clostridial rubredoxin, or when the MoFe protein from C. pasteurianum was either inactivated or replaced by its counterpart from Azotobacter vinelandii. It has also been observed that the ferredoxin inhibits cross-linking between the nitrogenase Fe protein and the MoFe protein, which suggests overlapping binding sites of the ferredoxin and of the Fe protein on the MoFe protein. Cross-linking experiments implementing a number of molecular variants of the [2Fe-2S] C. pasteurianum ferredoxin demonstrated that glutamate residues 31, 34, and 38 are important contributors to the interaction with the MoFe protein.

Mössbauer and EPR Evidence for an All-Ferrous Fe4S4 Cluster with S = 4 in the Fe Protein of Nitrogenase

Mössbauer and EPR Evidence for an All-Ferrous Fe₄S₄ Cluster with <i>S</i> = 4 in the Fe Protein of Nitrogenase

Actinorhizal symbioses and their N2 fixation.

More than 200 angiosperms, distributed in 25 genera, develop root nodule symbioses (actinorhizas) with soil bacteria of the actinomycetous genus Frankia. Although most soils studied contain infective Frankia, cultured strains are available only after isolation from root nodules. Frankia infects roots via root hairs in some hosts or via intercellular penetration in others. The nodule originates in the pericycle. The number of nodules in Alnus is determined by the plant in an autoregulated process that, in turn, is modulated by nutrients such as nitrogen and phosphate. Except in the genera Allocausarina and Casuarina, Frankia in nodules develops so-called vesicles where nitrogenase is localized. Sporulation of Frankia occurs in some symbioses. As a group, actinorhizal plants show a large range of anatomical and biochemical adaptations in order to balance the oxygen tension near nitrogenase. In symbioses with well aerated nodule tissue like Alnus, the vesicles have a multilayered envelope composed mainly of lipids, bacterio-hopanetetrol and their derivatives. This envelope is assumed to retard the diffusion of oxygen into the nitrogenase-containing vesicle. In symbioses like Casuarina, the infected plant cells themselves, rather than Frankia, appear to retard oxygen diffusion, and high concentrations of haemoglobin indicate an infected region with a low oxygen tension. At least in Alnus spp., ammonia resulting from N2 fixation is assimilated by glutamine synthetase in the plant. The carbon compound(s) used by Frankia in nodules is not yet known. Nitrogenase activity decreases in response to a number of environmental factors but recovers upon return to normal conditions. This dynamism in nitrogenase activity is often explained by loss and recovery of active nitrogenase and has been traced to loss and recovery of the nitrogenase proteins themselves. Recovery is partly due to growth of Frankia and to development of new vesicles in the Alnus nodules. In the field, varying conditions continuously affect the plants and the measured rate of N2 fixation is a result not only of the conditions prevailing at the moment but also of the conditions experienced over preceding days. N2 fixed by actinorhizal plants is substantial and actinorhizal plants have great potential in soil reclamation and in various types of forestry. Several species are also useful in horticulture. CONTENTS Summary 375 I. Introduction 376 II. The partners of actinorhizal symbioses 377 III. Root nodules 380 IV. Nitrogen fixation and related processes 385 V. Environmental effects on nitrogen fixation 389 VI. Ecological role 397 VII. Concluding remarks 398 Acknowledgements 398 References 398.

Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the beta subclass

Nitrogenase is a functionally constant protein catalyzing N2 reduction, which is found in many phylogenetic lineages of Archaea and Bacteria. A phylogenetic analysis of nif genes may provide insights into the evolution of the bacterial genomes. Moreover, it may be used to study diazotrophic communities, when classical isolation techniques may fail to detect all contributing populations. Among six species of the genus Azoarcus, diazotrophic Proteobacteria of the beta subclass, the deduced amino acid sequences of nifH genes of two species were unusually divergent from each other. Nitrogenases of the "authentic" Azoarcus branch formed a monophyletic unit with those of gamma Proteobacteria, thus being in accordance with 16S ribosomal DNA phylogeny. The nitrogenase proteins of the two aberrant strains clustered within the alpha proteobacterial clade with rhizobial nitrogenases. This relationship was supported by bootstrap values of 87 to 98% obtained by various distance and maximum parsimony methods. Phylogenetic distances of NifH proteins indicate a possible lateral gene transfer of nif genes to Azoarcus from a common donor of the alpha subclass at the time of species diversification or several more recent, independent transfers. Application of the phylogenetic analysis to DNA isolated from environmental samples demonstrated novel habitats for Azoarcus: in guts of termites and rice grown in Japan, nifH genes belonging to the authentic Azoarcus branch were detected. This is the first evidence suggesting the occurrence of Azoarcus spp. in a plant other than its originally described host, Kallar grass. Moreover, evidence for expression of nif genes inside grass roots was obtained by in situ hybridization studies with antisense nifH probes.

Evidence for multiple steps in the pre-steady-state electron transfer reaction of nitrogenase from Azotobacter vinelandii

The effect of the NaCl concentration and the reaction temperature on the MgATP-dependent pre-steady-state electron transfer reaction (from the Fe protein to the MoFe protein) of nitrogenase from Azotobacter vinelandii was studied by stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy. Besides lowering the reaction temperature, also the addition of NaCl decreased the observed rate constant and the amplitude of the absorbance increase (at 430 nm) which accompanies pre-steady-state electron transfer. The diminished absorbance increase observed at 5°C (without NaCl) can be explained by assuming reversible electron transfer, which was revealed by rapid-freeze EPR experiments that indicated an incomplete reduction of the FeMo cofactor. This was not the case with the salt-induced decrease of the amplitude of the stopped-flow signal: the observed absorbance amplitude of the electron transfer reaction predicted only 35% reduction of the MoFe protein, whereas rapid-freeze EPR showed 80% reduction of the FeMo cofactor. In the presence of salt, the kinetics of the reduction of the FeMo cofactor showed a lag period which was not observed in the absorbance changes. It is proposed that the pre-steady-state electron transfer reaction is not a single reaction but consists of two steps: electron transfer from the Fe protein to a still unidentified site on the MoFe protein, followed by the reduction of the FeMo cofactor. The consequences of our finding that the pre-steady-state FeMo cofactor reduction does not correlate with the amplitude and kinetics of the pre-steady-state absorbance increase will be discussed with respect to the present model of the kinetic cycle of nitrogenase.

Reconstitution activity of partial metallocluster-deficient molybdenum-iron protein of nitrogenase with a series of molybdenum-sulfur compounds

with a homocitrate ligand coordinated to Mo atom,bridged by three nonprotein lig-ands,two of which are inorganic sulfur atoms and the other is nitrogen- or oxygen-containing