Revealing a role for the G subunit in mediating interactions between the nitrogenase component proteins.

Azotobacter vinelandii contains three forms of nitrogenase known as the Mo-, V-, and Fe-nitrogenases. They are all two-component enzyme systems, where the catalytic component, referred to as NifDK, VnfDGK, and AnfDGK, associates with the reductase component, the Fe protein or NifH, VnfH, and AnfH respectively. AnfDGK and VnfDGK have an additional subunit compared to NifDK, termed gamma or AnfG and VnfG, whose role is unknown. The expression of each nitrogenase is tightly regulated by metal availability, however it is known that there is crosstalk between the Mo- and V­nitrogenases but the Fe­nitrogenase components cannot support substrate reduction with its Mo­nitrogenase counterparts. Here, docking models for the nitrogenase complexes were generated in ClusPro 2.0 based on the crystal structure of the Mo­nitrogenase and refined using the HADDOCK 2.2 refinement interface to identify structural determinants that enable crosstalk between the Mo- and V­nitrogenase but not the Fe­nitrogenase. Differing salt bridge interactions were identified at the binding interface of each complex. Specifically, positively charged residues of VnfG enable complementary interactions with NifH and VnfH but not AnfH. Similarly, negatively charged residues of AnfG enable interactions with AnfH but not NifH or VnfH. A role for the G subunit is revealed where VnfG could be mediating crosstalk between the Mo- and V­nitrogenases while the AnfG subunit on AnfDGK makes interactions with NifH and VnfH unfavorable, reducing competition with NifDK and funneling electrons to the most efficient nitrogenase.

Crystal structure of VnfH, the iron protein component of vanadium nitrogenase.

Nitrogenases catalyze the biological fixation of inert N2 into bioavailable ammonium. They are bipartite systems consisting of the catalytic dinitrogenase and a complementary reductase, the Fe protein that is also the site where ATP is hydrolyzed to drive the reaction forward. Three different subclasses of dinitrogenases are known, employing either molybdenum, vanadium or only iron at their active site cofactor. Although in all these classes the mode and mechanism of interaction with Fe protein is conserved, each one encodes its own orthologue of the reductase in the corresponding gene cluster. Here we present the 2.2 Å resolution structure of VnfH from Azotobacter vinelandii, the Fe protein of the alternative, vanadium-dependent nitrogenase system, in its ADP-bound state. VnfH adopts the same conformation that was observed for NifH, the Fe protein of molybdenum nitrogenase, in complex with ADP, representing a state of the functional cycle that is ready for reduction and subsequent nucleotide exchange. The overall similarity of NifH and VnfH confirms the experimentally determined cross-reactivity of both ATP-hydrolyzing reductases.

Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle.

Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation to release of Pi Because the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein-protein interactions during the Fe protein cycle.

Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

57Fe ENDOR spectroscopy and 'electron inventory' analysis of the nitrogenase E4 intermediate suggest the metal-ion core of FeMo-cofactor cycles through only one redox couple.

N(2) binds to the active-site metal cluster in the nitrogenase MoFe protein, the FeMo-cofactor ([7Fe-9S-Mo-homocitrate-X]; FeMo-co) only after the MoFe protein has accumulated three or four electrons/protons (E(3) or E(4) states), with the E(4) state being optimally activated. Here we study the FeMo-co (57)Fe atoms of E(4) trapped with the α-70(Val→Ile) MoFe protein variant through use of advanced ENDOR methods: 'random-hop' Davies pulsed 35 GHz ENDOR; difference triple resonance; the recently developed Pulse-Endor-SaTuration and REcovery (PESTRE) protocol for determining hyperfine-coupling signs; and Raw-DATA (RD)-PESTRE, a PESTRE variant that gives a continuous sign readout over a selected radiofrequency range. These methods have allowed experimental determination of the signed isotropic (57)Fe hyperfine couplings for five of the seven iron sites of the reductively activated E(4) FeMo-co, and given the magnitude of the coupling for a sixth. When supplemented by the use of sum-rules developed to describe electron-spin coupling in FeS proteins, these (57)Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E(4). In light of the previous findings that FeMo-co of E(4) binds two hydrides in the form of (Fe-(µ-H(-))-Fe) fragments, and that molybdenum has not become reduced, an 'electron inventory' analysis assigns the formal redox level of FeMo-co metal ions in E(4) to that of the resting state (M(N)), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier (57)Fe ENDOR studies and electron inventory analyses of the bio-organometallic intermediate formed during the reduction of alkynes and the CO-inhibited forms of nitrogenase (hi-CO and lo-CO) inspire the conjecture that throughout the eight-electron reduction of N(2) plus 2H(+) to two NH(3) plus H(2), the inorganic core of FeMo-co cycles through only a single redox couple connecting two formal redox levels: those associated with the resting state, M(N), and with the one-electron reduced state, M(R). We further note that this conjecture might apply to other complex FeS enzymes.

Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii.

Most biological nitrogen (N(2)) fixation results from the activity of a molybdenum-dependent nitrogenase, a complex iron-sulfur enzyme found associated with a diversity of bacteria and some methanogenic archaea. Azotobacter vinelandii, an obligate aerobe, fixes nitrogen via the oxygen-sensitive Mo nitrogenase but is also able to fix nitrogen through the activities of genetically distinct alternative forms of nitrogenase designated the Vnf and Anf systems when Mo is limiting. The Vnf system appears to replace Mo with V, and the Anf system is thought to contain Fe as the only transition metal within the respective active site metallocofactors. Prior genetic analyses suggest that a number of nif-encoded components are involved in the Vnf and Anf systems. Genome-wide transcription profiling of A. vinelandii cultured under nitrogen-fixing conditions under various metal amendments (e.g., Mo or V) revealed the discrete complement of genes associated with each nitrogenase system and the extent of cross talk between the systems. In addition, changes in transcript levels of genes not directly involved in N(2) fixation provided insight into the integration of central metabolic processes and the oxygen-sensitive process of N(2) fixation in this obligate aerobe. The results underscored significant differences between Mo-dependent and Mo-independent diazotrophic growth that highlight the significant advantages of diazotrophic growth in the presence of Mo.

A conformational mimic of the MgATP-bound "on state" of the nitrogenase iron protein.

The crystal structure of a nitrogenase Fe protein single site deletion variant reveals a distinctly new conformation of the Fe protein and indicates that, upon binding of MgATP, the Fe protein undergoes a dramatic conformational change that is largely manifested in the rigid-body reorientation of the homodimeric Fe protein subunits with respect to one another. The observed conformational state allows the rationalization of a model of structurally and chemically complementary interactions that occur upon initial complex formation with the MoFe protein component that are distinct from the protein-protein interactions that have been characterized previously for stabilized nitrogenase complexes. The crystallographic results, in combination with complementary UV-visible absorption, EPR, and resonance Raman spectroscopic data, indicate that the [4Fe-4S] cluster of both the Fe protein deletion variant and the native Fe protein in the presence of MgATP can reversibly cycle between a regular cubane-type [4Fe-4S] cluster in the reduced state and a cleaved form involving two [2Fe-2S] fragments in the oxidized state. Resonance Raman studies indicate that this novel cluster conversion is induced by glycerol, and the crystallographic data suggest that glycerol is bound as a bridging bidentate ligand to both [2Fe-2S] cluster fragments in the oxidized state.

Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.

Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure.

The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.

Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability.

Azotobacter vinelandii contains two superoxide dismutases (SODs), a cytoplasmic iron-containing enzyme (FeSOD), and a periplasmic copper/zinc-containing enzyme (CuZnSOD). In this study, the FeSOD was found to be constitutive, while the activity of CuZnSOD increased as the culture entered the stationary phase. Total SOD (units/mg protein) in stationary phase cells grown under nitrogen-fixing conditions was not significantly different from those grown under non-nitrogen-fixing conditions. The gene encoding FeSOD (sodB) was isolated from an A. vinelandii cosmid library. A 1-kb fragment containing the coding region and 400 base pairs of upstream sequence was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence had a high degree of homology with other bacterial FeSODs, particularly with P. aeruginosa. Attempts to construct a sodB mutant by recombination of a sodB::kan insertion mutation into the multicopy chromosome of A. vinelandii were unsuccessful even in the presence of SOD mimics or nutritional supplements. These results suggest that FeSOD may be essential for the growth and survival of A. vinelandii, and that the periplasmic CuZnSOD cannot replace the function of FeSOD.

Regulated expression of the nifM of Azotobacter vinelandii in response to molybdenum and vanadium supplements in Burk's nitrogen-free growth medium.

Azotobacter is a diazotrophic bacterium that harbors three genetically distinct nitrogenases referred to as nif, vnf, and anf systems. The nifM is an accessory gene located in the nif gene cluster and is transcriptionally regulated by the NifA. However, Azotobacter mutants that lack NifA are known to synthesize functional NifM and this accessory protein is known to be needed for the activity of nitrogenase-2 and nitrogenase-3. To determine how the transcription of nifM is regulated when Azotobacter is grown under conditions in which nitrogenase-2 or nitrogenase-3 is expressed, we generated an Azotobacter vinelandii strain that carries a nifM:lacZ-kanamycin resistance gene cassette in its chromosome. In this strain the nifM open reading frame was disrupted by the presence of a lacZ-kanamycin resistance gene cassette so that it could not produce active NifM. Moreover, the lacZ gene was placed under the transcriptional control elements of the nifM gene so that the lacZ expression could be used as a marker to determine the extent of expression of the nifM gene under different growth conditions. Our results show that this strain was unable to grow in Burk's nitrogen-free medium supplemented with either molybdenum or vanadium or lacking both metals suggesting that in the absence of functional NifM none of the nitrogenases were active. It was also found that the nifM expression was differentially regulated when the A. vinelandii cells were grown under conditions that activate nitrogenase-2 and nitrogenase-3, as determined by liquid beta-galactosidase activity measurements. These results suggest that the transcriptional activators, VnfA and AnfA, may regulate the nifM expression.

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum.

A spontaneous mutant derivative of Azotobacter vinelandii CA12 (delta nif HDK), which vanadium-dependent nitrogen fixation is not inhibited by molybdenum (A. vinelandii CARR), grows profusely on BNF-agar containing 1 microM Na2MoO4, alone or supplemented with 1 microM V2O5. The expression of A. vinelandii vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CARR was not inhibited by 1 mM Na2MoO4, whereas molybdenum at much lower concentration inhibited the expression of vnfH::lacZ and vnfA::lacZ fusions in A. vinlandii CA12. The mutant also exhibited normal acetylene reduction activity in the presence of 1 microM Na2MoO4. The expression of A. vinelandii nifH::lacZ fusion in A. vinelandii CARR was low even though the cells were cultured under non-repressing conditions with urea as nitrogen source in the presence of Na2MoO4. The molybdenum content of A. vinelandii CARR cells was found to be about one-fourth that of A. vinelandii CA12. No nitrate reductase activity could be detected in A. vinelandii CARR when the cells were cultured in the presence of 10 microM Na2MoO4, whereas A. vinelandii CA12 exhibited some activity even with 100 pM Na2MoO4.

Hydrodynamic properties of nitrogenase--the MoFe protein from Azotobacter vinelandii studied by dynamic light scattering and hydrodynamic modelling.

Experimental results for the nitrogenase MoFe protein from Azotobacter vinelandii obtained by dynamic light scattering (DLS) are presented. The translational diffusion coefficient was determined to D = (4.0 +/- 0.2) x 10(-7) cm2/s. Complementary, we have performed hydrodynamic model calculations based on the X-ray crystallographic data of the MoFe protein. The calculated transport coefficient suggests that the size and shape of the protein in solution is consistent with that in the crystal structure.

Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii.

1. A soluble reduced Methyl Viologen-dependent assimilatory nitrate reductase from Azotobacter vinelandii strain UW136 grown aerobically on nitrate was purified to homogeneity by the criteria of nitrate reductase activity staining, and coincidence of a Coomassie Blue-staining protein band on polyacrylamide gels run under non-denaturing conditions. The specific activity was 3 mumol of NO2- formed/min per mg of protein. 2. Gel filtration on Superose-12 and SDS/PAGE showed that the enzyme had an M(r) of 105,000 and was monomeric. The enzyme contained 1 Mo atom, 4 Fe atoms and 4 acid-labile sulphide atoms per molecule; no evidence for the presence of cytochrome or FAD was found. 3. Mo was present in a molybdenum cofactor, which on extraction was capable of activating apo-(nit-1) nitrate reductase present in crude extracts of nit-1 mutants of Neurospora crassa. 4. As isolated, the enzyme had e.p.r. signals assigned to Mo(V) with g-values g1 = 2.023; g2 = 1.998; g3 = 1.993 and with gav. = 2.004 indicating an unusual environment of Mo in this enzyme. 5. Reduction with S2O4(2-) bleached the e.p.r. signals which, on reoxidation after the addition of NO3(2-) to initiate enzyme turnover, exhibited at short times Mo(V) signals similar to those of dissimilatory nitrate reductases, with g1 = 1.998; g2 = 1.989; g3 = 1.981 and gav. = 1.989. Prolonged incubation subsequently gave a mixture of both e.p.r. species. 6. Neither NADH nor NADPH was effective as an electron donor, but reduced Methyl Viologen (apparent Km 998 microM) and reduced Bromophenol Blue (apparent Km 158 microM) were effective. With these donors the apparent Km values for nitrate were 70 microM and 217 microM respectively.