A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase in Azotobacter vinelandii*

Carmen Rüttimann-Johnson, Christopher R. Staples, Priya Rangaraj, Vinod K. Shah, and Paul W. LuddenDagger

From the Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

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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.

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Interest in vanadium as a biologically relevant element has increased in the last two decades. Although it had been known for some time that vanadium occurs in a highly enriched form in marine organisms and mushrooms (for a review, see Ref. 1), the discovery of its presence and functional role in haloperoxidases (reviewed by Butler (2)) and nitrogenases (reviewed by Eady (3)) is recent. Nitrogenase, the bacterial enzyme that catalyzes the conversion of N2 into NH4+, contains a unique iron-sulfur-heterometal cofactor, which is the site of substrate reduction. The most widely studied nitrogenases contain molybdenum as the heterometal in their active site cofactor (the iron-molybdenum cofactor (FeMo-co)1). The discovery of molybdenum-independent nitrogenases by Bishop et al. (4, 5) has led to the isolation and characterization of nitrogenases containing an iron-vanadium cofactor (FeV-co) (see Ref. 3 for a review), as well as nitrogenases in which no metal other than iron has been detected in the active site cofactor (6). These three nitrogenase systems are genetically distinct, the genes encoding them being contained in different regulons (designated nif, vnf, and anf genes for the molybdenum, vanadium, and iron-only nitrogenases, respectively). Azotobacter vinelandii harbors all three nitrogenase systems, and their expression is regulated by the metal content of the culture medium (7). Each nitrogenase consists of two easily separable protein components: dinitrogenase and dinitrogenase reductase. The reduction of substrate is catalyzed by dinitrogenase, whereas dinitrogenase reductase serves as the obligate electron donor to dintrogenase.

The nif, vnf, and anf regulons encode not only the nitrogenases but also products involved in cofactor biosynthesis and insertion in the cofactorless dinitrogenases. Among the genes known to be required for FeMo-co synthesis are nifB, nifE, nifN, nifV, and nifH. The protein encoded by nifB synthesizes NifB-co, a precursor that is the iron and sulfur donor to FeMo-co (8, 9) and most probably to FeV-co, because nifB is required for vanadium-dependent diazotrophic growth (10). The gene products of nifE and nifN form a tetrameric protein (NifEN), that is able to bind NifB-co (11). Analogs of the nifE and nifN genes exist in the vnf regulon (vnfEN (12)), probably serving a similar role during FeV-co synthesis. The gene product of nifV is homocitrate synthase (13). Homocitrate is a structural component of FeMo-co (14) and most probably FeV-co, because nifV is also required for full functionality of the vnf-dinitrogenase (15). The product of nifH, dinitrogenase reductase, plays multiple roles in the nitrogenase system. In addition to reducing dinitrogenase during nitrogenase turnover, NifH (VnfH) is required for FeMo-co (and presumably FeV-co) biosynthesis (16, 17), but its exact role in this process remains to be established. It is important to note that the structural gene products for dinitrogenase (nifDK) are not required for accumulation of FeMo-co and not involved in its synthesis (18).

Any other gene products involved in FeMo-co/FeV-co biosynthesis remain unknown, and the complete biosynthetic pathway is yet to be understood. An important unanswered question regards the gene product(s) providing the specificity for the incorporation of the heterometal (vanadium, molybdenum, or iron) into the cofactor. We report here that a V-Fe-containing cluster accumulates on the gene product of vnfX during synthesis of FeV-co. Our data suggest that this cluster is a precursor of FeV-co.

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Materials-- All materials used for growth medium preparation were analytical grade. Tris base and glycine were purchased from Fisher. DEAE-cellulose (DE-52) was from Whatman. Sephadex G-75 and phenyl-Sepharose were obtained from Amersham Pharmacia Biotech. 49V (V(VI) in 6 N HCl, 0.5-1.0 mCi/ml) was purchased from Los Alamos National Laboratories. Sodium metavanadate (99.995% purity) and all other chemicals were from Sigma. Nitrocellulose membranes and acrylamide/bisacrylamide solution were obtained from Mallinckrodt.

Bacterial Strains and Growth Conditions-- A. vinelandii strains CA12 (Delta nifHDK (19)), CA11.1 (Delta nifHDKvnfDGK::spc (20)), CA117.3 (Delta nifDKB (21)), CA48 (vnfE705::kan (12)), DJ42.48 (Delta nifENvnfE705::kan (12)), and CA11.8 (Delta nifHDKDelta vnfH (22)) have been described. All glassware used to prepare the culture medium and for cell growth was washed with 4 N HCl and rinsed thoroughly with deionized water. The strains were grown in Burk's modified medium lacking molybdenum and containing Na51VO3 (0.1 µM) spiked with radioactive 49VCl6 (0.05 µCi/ml) and 400 µg/ml nitrogen in the form of ammonium acetate at 30 °C with shaking. In order to ensure maximum incorporation of 49V into the cells, starter cultures that had been depleted of vanadium (by subculturing three times in molybdenum-free, vanadium-free medium) were used to inoculate the 49V-containing medium. Cells were grown overnight, collected by centrifugation, and resuspended in nitrogen-free medium containing Na51VO3 and 49VCl6 (same concentrations as above). The cultures were incubated then for 5 h at 30 °C for derepression of the vanadium nitrogenase system. Cells were harvested by centrifugation and frozen at -80 °C. For large scale purification of VnfX, A. vinelandii strain CA11.1 was grown in 20-liter carboys containing 15 liters of medium containing 0.1 µM NaVO3 and 40 µg/ml nitrogen in the form of ammonium acetate. The cultures were incubated at 30 °C and aerated vigorously. Cultures were monitored for the depletion of ammonium, following which, derepression of nitrogenase was allowed for 5 h. Cells were collected using a Pellicon cassette system equipped with a filtration membrane (0.45 µM, Millipore Corporation) and frozen at -80 °C. Cell-free extracts were prepared by osmotic shock, as described earlier (23).

Buffer Preparation-- All buffers were sparged with purified N2 for 20-30 min, and sodium dithionite was added to a final concentration of 1-1.7 mM. Buffers used during protein purification contained 0.5 µg/ml leupeptin and 0.2 mM phenylmethylsulfonyl fluoride.

Anoxic Native Gel Electrophoresis and Phosphorimaging-- The procedure for anoxic native gel electrophoresis and phosphorimaging has been described (8). Proteins were resolved on 7-20% acrylamide (37.5:1 acrylamide:bisacrylamide) and 0-20% sucrose gradient gels. After electrophoresis for 15 h at 100 V, the gels were dried and exposed to a phosphor screen for 1-3 days. Screens were scanned using a Molecular Dynamics model 425e PhosphorImager.

EPR Analysis-- EPR was performed at a microwave frequency of 9.23 GHz and a modulation amplitude of 12.5 Gauss using a Varian E-15 EPR spectrometer equipped with a Hewlett-Packard 5255A frequency converter, a Varian E102 microwave bridge, and an Oxford 3120 temperature controller.

Purification of VnfX-- VnfX was purified from A. vinelandii CA11.1 grown and derepressed in the absence of molybdenum and presence of vanadium. All procedures were performed anaerobically. Cell-free extract from 150 g of cells grown in the presence of NaVO3 and 6 g of cells grown in the presence of 49VCl6 plus NaVO3 were mixed and loaded on a DE-52 column (4 × 24 cm) equilibrated with 25 mM Tris-HCl, pH 7.4, containing 0.1 M NaCl. The column was washed with 300 ml of the same buffer, and protein was eluted stepwise with 0.15, 0.25, and 0.35 M NaCl in 25 mM Tris-HCl, pH 7.4, containing 20% glycerol. The presence of VnfX in the fractions was assessed by phosphorimaging of anoxic native gels. The fractions containing VnfX (0.15 M NaCl) were pooled and concentrated to 15 ml by ultrafiltration using a PM10 membrane (Amicon). The concentrated fraction was loaded on a Sephadex G-75 column (2.5 × 95 cm) equilibrated with 25 mM Tris-HCl, pH 7.4, 10% glycerol, 50 mM NaCl. The fractions from Sephadex G-75 containing VnfX were pooled, brought to 1 M (NH4)2SO4, and loaded on a phenyl-Sepharose column (1 × 15 cm) equilibrated with Tris-HCl, pH 7.4, containing 1 M ammonium sulfate. The column was washed with 1 column volume of the same buffer, and protein was eluted stepwise with two column volumes each of 0.5, 0.25, and 0.0 M ammonium sulfate in 0.025 M Tris, pH 7.4. Fractions containing VnfX (0.5 M NH4SO4) were pooled, concentrated by ultrafiltration using the same membrane as above and used for metal and EPR analyses.

Preparation of VnfX for N-terminal Sequencing-- One ml of partially purified VnfX was subjected to preparative anoxic native gel electrophoresis, the region of the gel containing the radioactivity was sliced, and the protein was eluted from the acrylamide. SDS gel electrophoresis of this sample showed a 20-kDa protein. The protein was transferred to a polyvinylidene difluoride membrane (Problott, Applied Biosystems), and N-terminal sequencing was done at the Department of Biochemistry, Medical College of Wisconsin (Milwaukee, WI).

Antibodies and Immunoblot Analysis-- Pure VnfX was cut out of preparative SDS-polyacrylamide gel electrophoresis gels. The protein was eluted from the acrylamide and injected into a rabbit to produce polyclonal antibodies at the Animal Care Unit of the University of Wisconsin-Madison Medical School. Immunoblot analysis was performed as described by Brandner et al. (24).

Purification of VnfX from a nifB- Strain-- VnfX was partially purified from A. vinelandii CA117.3 (Delta nifDKB) grown and derepressed in the absence of molybdenum and presence of vanadium. Extract of 40 g of cells was chromatographed on a DE-52 column (2.5 × 17 cm) equilibrated with 25 mM Tris-HCl, pH 7.4, containing 20% glycerol. The column was washed with 80 ml of the same buffer, and protein was eluted stepwise with 0.075, 0.15, and 0.30 M NaCl in 25 mM Tris, pH 7.4, containing 20% glycerol. The presence of VnfX in the fractions was assessed using immunoblot analysis of anoxic native gels. The fractions containing VnfX were pooled and concentrated to 15 ml by ultrafiltration using a PM10 membrane (Amicon). The concentrated fraction was loaded on a Sephadex G-75 column (2.5 × 95 cm) equilibrated with 25 mM Tris-HCl, pH 7.4, 10% glycerol, 50 mM NaCl. The fractions from Sephadex G-75 containing VnfX were pooled and frozen in liquid N2.

Incubations with NifB-co-- NifB-co was obtained from Klebsiella pneumoniae UN1217 (nifN4536 (25)) as has been described (9). NifB-co and 55Fe-NifB-co were in 25 mM Tris-HCl, pH 7.4, containing 2% SB12 (3-(dodecyldimethylammonio)propane-1-sulfonate, Fluka). Incubations of VnfX with NifB-co were done for 30 min at 30 °C.

Analysis of VnfX for Homocitrate-- Approximately 80 ml of 49V-VnfX partially purified from A. vinelandii CA11.1 (48 nmol of vanadium, as estimated by specific activity of 49V; 15 mg of total protein) was added dropwise to 900 ml of acetone containing 6.75 ml of 4 N HCl at 4 °C with continuous stirring over a 10-min period. The mixture was stirred for an additional 15 min, and precipitated protein was removed by filtration. The filtrate was evaporated to approximately 8 ml using a rotary evaporator, and the pH was adjusted to 8.0-9.0 before chromatography on an AG-1X8 (formate form, Bio-Rad) column (1 × 41 cm) equilibrated with water. The column was eluted with a gradient of 0-6 N formic acid (400 ml total). In order to determine in which fractions of the AG-1X8 column the homocitrate elutes, a standard of [3H]homocitrate (0.1 mmol, 36,000 dpm) was loaded previously on a comparable column and eluted under the exact same conditions. The fractions containing the 3H label were pooled, evaporated to dryness, redissolved in 5.0 ml of H2O, and brought to pH 8. The presence of homocitrate in this fraction was confirmed by in vitro FeMo-co synthesis assays (26), using extracts of A. vinelandii UW45 (Delta nifKDB) that had been desalted using Sephadex G-25 to remove endogenous homocitrate. The corresponding fractions from the AG-1X8 column on which the acidified acetone extract of VnfX had been loaded were pooled, evaporated to dryness, redissolved in 0.2 ml of double distilled H2O, and brought to pH 8.0. The presence or absence of homocitrate in this fraction was assessed using in vitro FeMo-co synthesis assays. The concentration of homocitrate expected in this fraction if the V-Fe cluster associated to VnfX contained homocitrate was calculated based on the concentration of vanadium in the sample of VnfX used for homocitrate extraction (considering 1 mol of homocitrate/mol of vanadium). The recovery of standard homocitrate that underwent these same manipulations was considered in this calculations (70%).

    RESULTS AND DISCUSSION
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In order to study which proteins play a role in FeV-co biosynthesis, mutant strains of A. vinelandii were grown in a medium depleted of molybdenum and containing 51V spiked with 49V (radioactive). The labeled proteins present in cell extracts were analyzed through phosphorimaging of anoxic native gels (Fig. 1). In an extract of a strain that is a wild-type for the vnf-nitrogenase system (CA12 (Delta nifHDK), the major protein band containing 49V was the vnf-dinitrogenase, as expected (Fig. 1, lane 1). In extracts of a mutant strain unable to synthesize the structural proteins of either the molybdenum nitrogenase or the vanadium nitrogenase (CA11.1 Delta nifHDKDelta vnfDGK1::spc), one prominent radiolabeled protein band was observed (Fig. 1, lane 2, VnfX). 49V did not accumulate on this protein in a strain that lacks NifB-co, the iron and sulfur donor to FeMo-co (and presumably FeV-co) (CA117.3 Delta nifDKDelta nifB, Fig. 1, lane 3). Furthermore a strain lacking both NifH and VnfH (CA11.8 Delta nifHDKDelta vnfH) also failed to accumulate 49V (Fig. 1, lane 4) on this protein. NifH is known to be required for FeMo-co synthesis, and its vnf-encoded homolog is proposed to play a similar role in FeV-co synthesis. These observations suggest that a 49V-labeled precursor of FeV-co is associated with the protein labeled VnfX. The same radiolabeled protein was also present, although at a much lower intensity in the extract of the wild-type strain (Fig. 1, lane 1). A 49V-labeled band was seen in an extract of cells grown in the presence of NH4+ (Fig. 1, lane 5, E). The same band was also seen in all other extracts used. This band did not stain for protein by Coomassie Blue or silver stain. Work is in progress to determine the identity of this band. This band does not correspond to H49VO42- (the vanadium species expected to be in solution at this pH), because this species migrated as a diffuse band at a different position in the gel (Fig. 1, lane 6).


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Fig. 1.   Phosphorimage of a native anoxic gel of extracts of A. vinelandii mutant strains grown and derepressed in the presence of 49VCl6. Lane 1, strain CA12 (Delta nifHDK, 81,000 cpm). Lane 2, strain CA11.1 (Delta nifHDKvnfDGK::spc, 32,000 cpm). Lane 3, strain CA117.3 (Delta nifDKB, 80,000 cpm). Lane 4, strain CA11.8 (Delta nifHDKDelta vnfH, 80,000 cpm). Lane 5, strain CA12 (Delta nifHDK, 81,000 cpm) grown in the presence of ammonium. Lane 6, H249VO4- in 0.1 M Mops, pH 7.5. Lane 7, strain DJ42.48 (Delta nifENXDelta vnfE, 81,000 cpm). Lane 8, strain CA48 (vnfE705::kan, 80,000 cpm).

Purification of VnfX-- The radiolabeled band present in extracts of A. vinelandii CA11.1 (Fig. 1, VnfX) was purified by following the radioactivity associated with it. Purification steps included DEAE-cellulose, Sephadex G-75, and phenyl-Sepharose chromatography, as shown in Table I. Purified VnfX showed a specific activity of 133,948 cpm/mg. A purification fold of 165.5 and a final yield of 26% were obtained. After this purification procedure, the protein was not homogenously pure. In order to obtain pure protein, 1 ml of the fraction obtained after phenyl-Sepharose chromatography was electrophoresed in an anoxic native gel, the region of the gel containing the radioactivity was sliced, and the protein was eluted from the acrylamide. This step is not shown in the purification table, because VnfX lost the 49V label upon exposure to air, when eluting from the gel. The purified protein was identified as VnfX, based on its N-terminal sequence identity to the amino acid sequence predicted from the vnfX gene (12) (MIKVAFASN). In A. vinelandii, the vnfX gene is located downstream of the vnfEN genes, which are also proposed to be involved in FeV-co synthesis (12).

                              
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Table I
Purification of VnfX from A. vinelandii CA11.1

Characterization of VnfX-- The temperature dependence at 10.0 mW of the X-band EPR spectrum of dithionite-treated (as isolated) VnfX is shown in Fig. 2A. The spectral properties can be readily interpreted as arising from a majority S = 3/2 species that is of maximum observed intensity at 4.8 K (Fig. 2B) and a minority S = 1/2 species that increases in intensity with increasing temperature to 50 K. The g values of the majority S = 3/2 species can be analyzed using a spin Hamiltonian, He, with D and E as the axial and rhombic splitting parameters, and an isotropic g-tensor, g0, as given by Equation 1.
H<SUB>e</SUB>=<UP>g</UP><SUB>0</SUB>&bgr;<UP><B>H · S</B></UP>+D(<UP><B>S</B></UP><SUB>z</SUB><SUP>2</SUP>−S(S+1)/3+(E/D)(<UP><B>S</B></UP><SUB>x</SUB><SUP>2</SUP>−<UP><B>S</B></UP><SUB>y</SUB><SUP>2</SUP>)) (Eq. 1)
On the basis of their temperature dependence behavior, the features at g = 4.82, g = 3.36, and g ~1.90 are attributed to resonances from the lower doublet of the S = 3/2 system with a rhombicity parameter (E/D) of 0.15 and D > 0, using g0 = 2.00. An accurate assessment of the high field g value is hindered by the diffuse nature of the resonance and the presence of the minority species. As Fig. 2B demonstrates, power-saturation of the majority species (determined by following the intensity of the g = 4.82 resonance) only becomes evident at temperatures below 10.0 K when using a 10.0 mW microwave power. A plot of the normalized intensity of the g = 4.82 resonance divided by the square root of the microwave power versus the log of microwave power at a temperature of 4.8 K, according to Equation 2, is shown in Fig. 3A (27).
S ∝ P<SUP>0.5</SUP>/(1+P/P<SUB>1/2</SUB>)<SUP>b/2</SUP> (Eq. 2)
P1/2 is the power for half-saturation, and b is the inhomogeneity parameter. Analysis of the data reveals a P1/2 of 4.3 mW using b = 1, which provided the best fit. Fig. 3A shows that at 4.8 K and 1.0 mW, the S = 3/2 species is largely not power-saturated. Because the minority S = 1/2 species was not observed at 4.8 K and 1.0 mW (Fig. 3B), it can be assumed that the g = 2 region of the spectrum is arising from only the majority species. Magnetic hyperfine structure due to 51V (I = 7/2; the majority isotopic species of vanadium present) is apparent in the g = 2 region under these conditions (Fig. 3B, inset). Because the observation of this hyperfine structure is maximized at the same conditions as is the intensity of the S = 3/2 species, it is probable that the hyperfine structure is due to 51V incorporated into the species that produces the S = 3/2 resonances, rather than due to a protein-bound 51V4+ (S = 1/2) species. The ground state spin and spectral line shapes of the majority species resemble those of isolated FeV-co (28), the reduced vnf-dinitrogenase of A. chroococcum (29), and a synthetic [VFe3S4] cubane cluster (30). The minority S = 1/2 component (g = 2.05, 1.96, 1.89) was most clearly observable at higher temperatures (Fig. 2A), and its relative abundance varied from sample to sample. The minority species may represent a damaged version of the majority species. Analogous signals have been seen in the vnf-dinitrogenases of A. chroococcum (31) and A. vinelandii (32) and have been suggested to represent damaged FeV-co in those proteins. Therefore, the spectroscopic properties of the S = 3/2 species are consistent with an [Fe-V-S] cluster with similar properties to both FeV-co (proposed to be [VFe7S9]) and a [VFe3S4] cluster. Metal analysis revealed that there are 8 ± 1 mol of Fe and 0.5 mol of vanadium bound per mol of VnfX. The levels of vanadium and Fe are more consistent with a model in which a [VFe7Sx] cluster is bound to VnfX, as opposed to a [VFe3S4] cluster. However, it does not appear that FeV-co itself accumulates on VnfX, as vanadium- and iron-containing VnfX (as purified) is not able to donate 49V label to FeV-co-deficient dinitrogenase (VnfDGK) (see below). The low level of vanadium (0.5 mol/mol of VnfX) may indicate that some vanadium was lost during purification or that a fraction of the cluster on the isolated VnfX had not yet acquired an atom of vanadium. It is possible that the minority species arises from a damaged [Fe-S] cluster in which vanadium has been lost. As discussed below, NifB-co is able to bind to VnfX in the absence of vanadium. Isolated NifB-co contains only Fe and S, and is diamagnetic in the presence of dithionite. Whereas it is possible that VnfX-bound NifB-co is paramagnetic, it would not exhibit the magnetic hyperfine that the S = 3/2 species described above does. Experiments are in progress to determine the spectral properties of VnfX-bound NifB-co.


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Fig. 2.   Temperature dependence of the X-band EPR spectrum of VnfX. A, temperature dependence of the EPR spectrum at 10 mW of microwave power. Spectra are the sums of six scans, with a six-scan cavity spectrum performed at the same experimental conditions subtracted. VnfX purified from A. vinelandii CA11.1 (Delta nifHDKvnfDGK::spc) was at a final concentration of 23 µM. B, plot of g = 4.82 resonance intensity versus reciprocal temperature.


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Fig. 3.   Microwave power dependence of the X-band EPR spectrum of VnfX. A, power study of the S = 3/2 signal. The intensity of the g = 4.82 resonance was divided by the square root of the microwave power in mW (PmW), normalized, and plotted versus the log of PmW. The data was fit using Equation 2, affording a P1/2 = 4.3 mW. Best fits were obtained with b = 1. Microwave power levels used were 0.10, 0.20, 0.50, 1.0, 2.0, 3.5, 5.0, 10, 15, and 20 mW. B, EPR spectrum of VnfX obtained at 4.8 K and 1.0 mW. The inset expands the region of the spectrum containing the nuclear hyperfine resonances (2600-3800 Gauss). The spectrum shown is the sum of six scans, with a six-scan cavity spectrum performed at the same experimental conditions subtracted. VnfX purified from A. vinelandii CA11.1 (Delta nifHDKvnfDGK::spc) was at a final concentration of 23 µM.

As indicated above, vanadium- and iron-containing VnfX is not able to donate the V-Fe cluster to FeV-co-deficient dinitrogenase (VnfDGK) under the conditions tested to date. Assay mixtures used for this purpose contained 25 mM Tris-HCl, pH 7.4, extract of A. vinelandii CA117.3 (Delta nifKDB) grown in the presence of vanadium (as a source of FeV-co-deficient vnf-dinitrogenase, VnfH, and other vnf proteins), an ATP generating system, MgCl2 (1.8 mM), and partially purified VnfX with the 49V-Fe cluster bound (122,000 cpm/assay). Assays were performed in the absence or presence of homocitrate (104 µM). Purified NifH and partially purified NifEN were added to some assays, in case the vnf counterparts of these proteins were limiting in the extract of A. vinelandii CA117.3. Under none of the conditions used was the 49V-Fe cluster donated to apo-dinitrogenase, as estimated by phosphorimaging of anoxic native gels of the assay mixtures, nor could nitrogenase activity be detected.

The V-Fe Cluster Accumulating on VnfX Does Not Contain Homocitrate-- Experiments were conducted to determine whether the V-Fe cluster associated to VnfX contains homocitrate. Partially purified VnfX was extracted with acidified acetone, and the extract was purified by AG-1X8 chromatography. As shown in Table II, no homocitrate could be detected in the extract of VnfX, as assessed by in vitro FeMo-co synthesis assays. Table II also shows the activities obtained in vitro FeMo-co synthesis assays when adding amounts of homocitrate equivalent to the amounts expected to be in the VnfX extract if this organic acid was a part of the cluster associated to VnfX. Homocitrate is probably added to FeV-co at a later step than the one occurring on VnfX.

                              
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Table II
Detection of homocitrate by in vitro FeMo-co synthesis assay

VnfX Is Not Absolutely Required for FeV-co Synthesis-- Surprisingly, A. vinelandii CA48 (vnfE705::kan), a strain that does not express VnfX (as shown by immunoblot analysis, apparently due to polarity of the mutation in vnfE on vnfX) is able to grow diazotrophically in the presence of vanadium, although after a prolonged lag period (12). This strain does produce vnf-encoded dinitrogenase, as shown in Fig. 1, lane 8. As expected, no label was seen in this extract at the position of VnfX. It is possible that NifX or some other protein replaces VnfX in the synthesis of FeV-co. Wolfinger and Bishop (12) have suggested that NifEN replaces VnfEN in this strain during vanadium-dependent diazotrophic growth. Immunoblot analysis shows that both NifX and NifEN are expressed in vanadium-grown CA48 (data not shown). Nevertheless, no 49V was associated to NifX in extract of this strain (Fig. 1, lane 8). Association of 49V to NifX may be weaker than to VnfX and not withstand the electrophoresis conditions. No 49V-labeled proteins were seen in extracts of strain DJ42.48 (Delta nifENvnfE705::kan) (Fig. 1, lane 7). The mutations in this strain are polar on nifX and vnfX.

Two Electrophoretically Distinct Species of VnfX-- Polyclonal antibodies to VnfX were generated using pure VnfX, which was extracted from an SDS gel. Immunoblot analysis of anoxic native gels of the partially purified VnfX (from A. vinelandii CA11.1) showed a major band cross-reacting with the antibody, which comigrated with the 49V-labeled VnfX (Fig. 4, lanes 2 and 5). A minor band of slower electrophoretic mobility also cross-reacted with the VnfX antibody (labeled V-deficient VnfX in Fig. 4, lane 5). After treatment of VnfX with air, the band corresponding to 49V-VnfX could no longer be seen, but the band migrating in the higher position remained (Fig. 4, lane 6). Treatment of VnfX with air resulted in loss of vanadium and iron (data not shown). The slower migrating band in lane 5 probably arose from dissociation of the V-Fe cluster from VnfX during purification (see below). In extracts of a mutant strain incapable of NifB-co production (A. vinelandii CA117.3 (Delta nifDKDelta nifB)), there was no vanadium or iron associated with VnfX (as estimated by phosphorimaging (Fig. 1, lane 3) and iron staining of anoxic native gels, not shown). The vanadium-deficient species cross-reacting with the anti-VnfX antibody in extracts of this strain had a slower mobility in anoxic native gel electrophoresis (Fig. 4, lane 7) than the species that has the V-Fe cluster bound. Vanadium-deficient VnfX was partially purified from an extract of A. vinelandii CA117.3. When subjected to gel filtration electrophoresis using Sephadex G-75, this form of VnfX eluted at a volume that corresponds to an apparent molecular mass of 33 kDa, compared with an elution volume corresponding to an apparent molecular mass of 20 kDa for the species that has the V-Fe cluster bound (the molecular mass predicted from the DNA sequence of vnfX is 19 kDa (12)). The subunit molecular mass of both vanadium-deficient VnfX and V-Fe-containing VnfX was determined to be 20 kDa by SDS-polyacrylamide gel electrophoresis. We hypothesize that a dimeric form of VnfX monomerizes upon binding a V-Fe precursor of FeV-co. This would be analogous to the monomerization of the FeMo-co-binding protein, gamma , which is involved in insertion of FeMo-co into the cofactorless nif-apodinitrogenase (33). Other interpretations are also consistent with our observations, such as a dramatic conformational change in a monomeric VnfX upon metal cluster binding.


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Fig. 4.   Analysis of VnfX by anoxic, native gel electrophoresis. Phosphorimage of an anoxic native gel. Lane 1, extract of A. vinelandii CA12 (Delta nifHDK) grown in the presence of 49V (1.5 mg of total protein; 81,000 cpm). Lane 2, VnfX partially purified from A. vinelandii CA 11.1 (Delta nifHDKDelta vnfDGK) grown in the presence of 49V (263 µg of total protein; 28,500 cpm). Lane 3, VnfX partially purified from A. vinelandii CA117.3 (Delta nifDKDelta nifB) grown in the presence of vanadium incubated with 55Fe-NifB-co (425 µg of total protein; 5,000 cpm). Lane 4, 55Fe-NifB-co (5,000 cpm). Immunoblot of an anoxic native gel developed with antibody to VnfX. Lane 5, VnfX partially purified from A. vinelandii CA 11.1 (Delta nifHDKDelta vnfDGK) grown in the presence of 49V (1.0 µg of total protein). Lane 6, VnfX partially purified from A. vinelandii strain CA 11.1 exposed to air for 5 min before electrophoresis (1.0 µg of total protein). Lane 7, VnfX partially purified from NifB-co-deficient A. vinelandii strain CA117.3 (43.0 µg of total protein). Lane 8, VnfX partially purified from NifB-co-deficient A. vinelandii strain CA117.3 (43.0 µg of total protein) incubated with NifB-co (72.5 pmol of Fe). Lane 9, VnfX partially purified from NifB-co-deficient A. vinelandii strain CA117.3 (43.0 µg of total protein) incubated with air-inactivated NifB-co (72.5 pmol of Fe). Lane 10, VnfX partially purified from NifB-co-deficient A. vinelandii strain CA117.3 (43.0 µg of total protein) incubated with SB12 (final concentration, 0.03%) in 0.025 M Tris-HCl, pH 7.4. Lane 11, extract of A. vinelandii strain CA11.1 grown and derepressed in the presence of molybdenum (150 µg of total protein). Lane 12, extract of A. vinelandii CA11.1 grown in the presence of vanadium and ammonium acetate (150 µg of total protein).

Vanadium-deficient VnfX (as Purified from the nifB- Strain) Is Able to Bind NifB-co-- Treatment of vanadium-deficient VnfX with purified NifB-co caused a shift in the migration of VnfX in anoxic native gels (Fig. 4, lane 8). The VnfX-NifB-co complex migrated at the same position as does VnfX that has the V-Fe cluster bound to it (e.g. the VnfX purified from A. vinelandii strain CA11.1) (Fig. 4, lane 2). Oxygen-inactivated NifB-co did not produce the shift of VnfX (Fig. 4, lane 9), nor did a solution of SB12 (Fig. 4, lane 10), a detergent that was present at that concentration in the NifB-co solution. The binding of NifB-co to VnfX was confirmed by incubating the partially purified protein with 55Fe-NifB-co. When this incubation mixture was subjected to anoxic native gel electrophoresis and the gel was analyzed by phosphorimaging, the 55Fe label was found associated with VnfX (Fig. 4, lane 3).

Immunoblot analysis was also used to confirm that VnfX was not expressed in A. vinelandii grown in the presence of molybdenum (Fig. 4, lane 11) or in the presence of ammonium acetate (Fig. 4, lane 12), which represses synthesis of all three nitrogenase systems. Furthermore, anti-VnfX antibody did not cross-react with NifX (data not shown). No association that is stable to anoxic native gel electrophoresis was observed between VnfX and dinitrogenase (VnfDGK) or VnfH (data not shown).

A homolog of vnfX exists in the nif regulon (nifX). The product of nifX is required for in vitro FeMo-co synthesis with purified components (34). Nevertheless, nifX is not absolutely required in vivo for FeMo-co synthesis, because strains of A. vinelandii and K. pneumoniae with mutations in nifX are Nif+ (35, 36). The same seems to be true for VnfX. A. vinelandii CA48 (vnfE705::kan) is able to grow diazotrophically in the presence of vanadium, although after a prolonged lag period. It is possible that some other gene product replaces VnfX (or NifX) in vivo, although with lower efficiency.

As stated before, in extracts of strains with mutations in nifB or a double mutation in vnfH and nifH, no V-Fe cluster accumulates on VnfX. Thus, the involvement of NifB and VnfH in FeV-co biosynthesis must occur earlier in the biosynthetic pathway than the step occurring on VnfX. We speculate that the gene products believed to play a role in FeV-co synthesis would do so in the following order (Scheme I).
<UP>NifB → NifB-co → VnfNE</UP>(<UP>NifB-co</UP>) <LIM><OP><UP>→→</UP></OP><UL><UP>VnfH</UP></UL></LIM> 
<UP>+ V →→ VnfX-V-Fe </UP><LIM><OP><UP>→→</UP></OP><UL><UP>HC↘</UP></UL></LIM><UP> FeV-co</UP>
<UP><SC>Scheme</SC> 1</UP>

Conclusions-- We have identified and characterized what appears to be a precursor of FeV-co that contains the heterometal (vanadium). This intermediate is associated with VnfX, which suggests that this protein is involved in FeV-co synthesis. The step occurring on VnfX could be the addition of vanadium to the partially formed cluster, because both a vanadium-containing cluster and NifB-co, its Fe-S precursor, can accumulate on VnfX. Alternatively, a cluster already containing vanadium could be transferred to VnfX, and a further processing step could occur on this protein.

    ACKNOWLEDGEMENTS

We thank Dr. George Reed for the use of his EPR facility, Drs. Paul Bishop and Dennis Dean for generously providing the mutant strains used in this study, Dr. Gary Roberts for helpful suggestions, and Dr. L. Mende-Müller for N-terminal sequencing of VnfX.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM35332.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-6859; Fax: 608-262-3453; E-mail: ludden{at}biochem.wisc.edu.

    ABBREVIATIONS

The abbreviations used are: co, cofactor; Mops, 4-morpholinepropanesulfonic acid; EPR, electron paramagnetic resonance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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