Accumulation of 55Fe-labeled precursors of the iron-molybdenum cofactor of nitrogenase on NifH and NifX of Azotobacter vinelandii

Iron-molybdenum cofactor (FeMo-co) biosynthesis involves the participation of several proteins. We have used (55)Fe-labeled NifB-co, the specific iron and sulfur donor to FeMo-co, to investigate the accumulation of protein-bound precursors of FeMo-co. The (55)Fe label from radiolabeled NifB-co became associated with two major protein bands when the in vitro FeMo-co synthesis reaction was carried out with the extract of an Azotobacter vinelandii mutant lacking apodinitrogenase. One of the bands, termed (55)Fe-labeled upper band, was purified and shown to be NifH by immunoblot analysis. The (55)Fe-labeled lower band was identified as NifX by N-terminal sequencing. NifX purified from an A. vinelandii nifB strain showed a different electrophoretic mobility on anoxic native gels than did NifX with the FeMo-co precursor bound.     

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.     

Purification of a NifEN protein complex that contains bound molybdenum and a FeMo-Co precursor from an Azotobacter vinelandii DeltanifHDK strain

The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.     

Accumulation of 99Mo-containing iron-molybdenum cofactor precursors of nitrogenase on NifNE,NifH, and NifX of Azotobacter vinelandii

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase was investigated using the purified in vitro FeMo-co synthesis system and 99Mo. The purified system involves the addition of all components that are known to be required for FeMo-co synthesis in their purified forms. Here, we report the accumulation of a 99Mo-containing FeMo-co precursor on NifNE. Apart from NifNE, NifH and NifX also accumulate 99Mo label. We present evidence that suggests NifH may serve as the entry point for molybdenum incorporation into the FeMo-co biosynthetic pathway. We also present evidence suggesting a role for NifX in specifying the organic acid moiety of FeMo-co.     

Inhibition of iron-molybdenum cofactor biosynthesis by L127Delta NifH and evidence for a complex formation between L127Delta NifH and NifNE.

Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex.     

Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase

The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.     

Purification and characterization of NafY (apodinitrogenase gamma subunit) from Azotobacter vinelandii

The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase. In Azotobacter vinelandii, NafY (also known as gamma protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase. Here we report the purification and biochemical characterization of NafY and reach the following conclusions. First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K(d) approximately equal to 60 nm); second, the NafY-FeMo-co complex exhibits a S = 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His(121) residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein. In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.     

NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii

The major part of biological nitrogen fixation is catalysed by the molybdenum nitrogenase that carries at its active site the iron and molybdenum cofactor (FeMo-co). The nitrogen fixation (nif) genes required for the biosynthesis of FeMo-co are derepressed in the absence of a source of fixed nitrogen. The nifB gene product is remarkable because it assembles NifB-co, a complex cluster proposed to comprise a [6Fe-9S-X] cluster, from simpler [Fe-S] clusters common to other metabolic pathways. NifB-co is a common intermediate of the biosyntheses of the cofactors present in the molybdenum, vanadium and iron nitrogenases. In this work, the expression of the Azotobacter vinelandii nifB gene was uncoupled from its natural nif regulation to show that NifB protein levels are lower in cells growing diazotrophically than in cells growing at the expense of ammonium. A. vinelandii carries a duplicated copy of the ATPase component of the ubiquitous ClpXP protease (ClpX2), which is induced under nitrogen fixing conditions. Inactivation of clpX2 resulted in the accumulation of NifB and NifEN and a defect in diazotrophic growth, especially when iron was in short supply. Mutations in nifE, nifN and nifX or in nifA also affected NifB accumulation, suggesting that NifB susceptibility to degradation might vary during its catalytic cycle.     

Cloning and mutational analysis of the gamma gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization

Dinitrogenase is a heterotetrameric (alpha(2)beta(2)) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition alpha(2)beta(2)gamma(2), which can be activated in vitro by the addition of FeMo-co. The gamma protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the gamma subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now gamma. In vitro FeMo-co insertion experiments presented in this work demonstrate that gamma stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.     

Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase.

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide.     

Nucleotide and divalent cation specificity of in vitro iron-molybdenum cofactor synthesis.

The nucleotide and divalent cation requirements of the in vitro iron-molybdenum cofactor (FeMo-co) synthesis system have been compared with those of substrate reduction by nitrogenase. The FeMo-co synthesis system specifically requires ATP, whereas both 1,N6-etheno-ATP and 2'-deoxy-ATP function in place of ATP in substrate reduction (M. F. Weston, S. Kotake, and L. C. Davis, Arch. Biochem. Biophys. 225:809-817, 1983). Mn2+, Ca2+, and Fe2+ substitute for Mg2+ to various extents in in vitro FeMo-co synthesis, whereas Ca2+ is ineffective in substrate reduction by nitrogenase. The observed differences in the nucleotide and divalent cation specificities suggest a role(s) for the nucleotide and divalent cation in in vitro FeMo-co synthesis that is distinct from their role(s) in substrate reduction.     

Biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a Mo-Fe-S cluster that has been proposed as the site of substrate reduction for the nitrogenase enzyme complex. Biosynthesis of FeMo-co in Klebsiella pneumoniae requires at least six nif (nitrogen fixation) gene products. One of the nif genes, nifV, apparently encodes a homocitrate synthase. The synthesis and accumulation of homocitrate [(R)-2-hydroxy-1,2,4-butanetricarboxylic acid] in K.pneumoniae is correlated to the presence of a functional nifV gene. K.pneumoniae strains with mutations in nifV synthesize and accumulate an aberrant form of FeMo-co. Nitrogenase from NifV- mutants is capable of reducing some of the substrates of nitrogenase effectively (e.g. acetylene), but reduces N2 poorly. With the aid of an in vitro FeMo-co synthesis system, it recently has been established that homocitrate is an endogenous component of FeMo-co. Substitution of homocitrate with other carboxylic acids results in the formation of aberrant forms of FeMo-co with altered substrate reduction capability.     

Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site

The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron-molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N₂ to NH₃, while exhibiting residual C₂H₄ and considerable H₂ production activities. Production of H₂ by NifDK/NifB-co was stimulated by N₂ and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB-co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB-co into FeMo-co, might encode an enzyme with similar catalytic properties to NifDK/NifB-co.     

A vanadium and iron cluster accumulates on VnfX during iron-vanadium-cofactor synthesis for the vanadium nitrogenase in Azotobacter vinelandii.

The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the iron-molybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe7-8Sx precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe3S4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.     

Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum cofactor

The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C2H2 reduction assays and the lack of 99Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of 99Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C2H2 or N2. Citrate also promoted the incorporation of 99Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C2H2 effectively but not N2. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV- dinitrogenase. We also observed low rates of HD formation from NifV- dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. We propose that in vivo NifV- mutants utilize citrate for FeMo-co synthesis.     

Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN–MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems.     

Role of Azotobacter vinelandii FdxN in FeMo-co biosynthesis

Biosynthesis of metal clusters for the nitrogenase component proteins NifH and NifDK involves electron donation events. Yet, electron donors specific to the biosynthetic pathways of the [4Fe–4S] cluster of NifH, or the P-cluster and the FeMo-co of NifDK, have not been identified. Here we show that an Azotobacter vinelandii mutant lacking fdxN was specifically impaired in FeMo-co biosynthesis. The ΔfdxN mutant produced 5-fold less NifB-co, an early FeMo-co biosynthetic intermediate, than wild type. As a consequence, it accumulated FeMo-co-deficient apo-NifDK and was impaired in NifDK activity. We conclude that FdxN plays a role in FeMo-co biosynthesis, presumably by donating electrons to support NifB-co synthesis by NifB. This is the first role in nitrogenase biosynthesis unequivocally assigned to any A. vinelandii ferredoxin.     

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

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

Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase

When apodinitrogenase (lacking FeMo-co) was activated with FeMo-co synthesized in vitro in the presence of 3H-labeled homocitrate, label was incorporated into dinitrogenase. The physical association of the label with FeMo-co was demonstrated by reisolation and purification of the cofactor from dinitrogenase. The presence of homocitrate in FeMo-co was established by NMR analysis of the organic acid extracted from dinitrogenase. Quantitation of homocitrate in dinitrogenase showed it to be present at a 1:1 ratio with molybdenum.     

In vitro biosynthesis of iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution for NifH with site-specifically altered forms of NifH.

NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.