Accumulation of 55Fe-Labeled Precursors of the Iron-Molybdenum Cofactor of Nitrogenase on NifH and NifX of Azotobacter vinelandii*

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

From the Department of Biochemistry and Center for the Study of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received for publication, January 31, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Iron-molybdenum cofactor (FeMo-co) biosynthesis involves the participation of several proteins. We have used 55Fe-labeled NifB-co, the specific iron and sulfur donor to FeMo-co, to investigate the accumulation of protein-bound precursors of FeMo-co. The 55Fe 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 55Fe-labeled upper band, was purified and shown to be NifH by immunoblot analysis. The 55Fe-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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitrogenase, the enzyme responsible for the conversion of dinitrogen to ammonium, is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2). During catalysis, dinitrogenase (also called MoFe protein or NifKD)1 is specifically reduced by dinitrogenase reductase (Fe-protein or NifH), one electron at a time, until a sufficient number of electrons accumulates on dinitrogenase for the subsequent reduction of substrate (3). Dinitrogenase reductase, an alpha 2 dimer of the nifH gene product, contains one 4Fe-4S cluster which is symmetrically coordinated by the thiol groups of Cys-97 and Cys-132 from each subunit of the homodimer (4, 5). Dinitrogenase is an alpha 2beta 2 tetramer of the nifD and K gene products and is associated with two unique metal clusters: the P-cluster (6) and the iron-molybdenum cofactor (FeMo-co) (6, 7). FeMo-co is the active site in dinitrogenase where substrate reduction is believed to occur and is composed of a MoFe3-S3 cluster bridged to a Fe4-S3 cluster by three sulfur ligands. The molybdenum atom is also coordinated to the C-2 carboxyl and hydroxyl groups of the organic acid R-homocitrate.

Biochemical and genetic analyses of A. vinelandii and Klebsiella pneumoniae mutants have revealed that the biosynthesis of FeMo-co requires the participation of at least seven different nif gene products: NifQ, V, B, H, X, N, and E (8-12). Interestingly, the products of the structural genes for dinitrogenase, nifD and K, are not necessary for FeMo-co biosynthesis, suggesting that the cofactor is assembled elsewhere in the cell and then is inserted into the FeMo-co-deficient apodinitrogenase (13, 14). In the absence of apodinitrogenase (NifKD), it is known that FeMo-co accumulates on a non-nif protein, gamma (gamma ), that serves as a chaperone-insertase during FeMo-co biosynthesis and its insertion into NifKD (15). An in vitro reaction for the biosynthesis of FeMo-co has been described that requires the presence of an ATP-regenerating system, MoO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, homocitrate, NifB-cofactor (NifB-co), and the gene products of nifH, N, and E (11). However, the in vitro FeMo-co synthesis system involving only purified proteins and components that are known for FeMo-co synthesis has not yielded significant levels of FeMo-co synthesis, thereby suggesting the requirement for other factors in this process (12).

Because FeMo-co is unstable in aqueous solutions, intermediates in the FeMo-co biosynthetic pathway are believed to be present as protein-bound species. The accumulation of molybdenum in the FeMo-co biosynthetic pathway has been studied in various nif mutants of K. pneumoniae (13) and A. vinelandii (16) as an approach to identify the sequence of steps in the FeMo-co biosynthetic pathway. Previous studies have identified the metabolic product of NifB, NifB-co, as a potential iron and sulfur source for FeMo-co biosynthesis (17). Studies with purified 55Fe-labeled NifB-co have conclusively shown that NifB-co serves as the specific iron and sulfur donor to FeMo-co (18). Furthermore, nifB is one of the nif genes that is required for the full activity of the Mo-independent nitrogen fixation systems (19). Thus, NifB-co is proposed to function as the fundamental iron-sulfur cluster for the synthesis of FeMo-co of Mo-nitrogenase, FeV-co of V-nitrogenase, and FeFe-co of Fe-only-nitrogenase.

To identify proteins that play a role in FeMo-co biosynthesis, we have used 55Fe-labeled NifB-co in the in vitro FeMo-co synthesis reaction. We have followed the incorporation of the radiolabel into FeMo-co and its precursors by electrophorescing the reaction mixtures on non-denaturing polyacrylamide gels and detecting the radiolabeled protein bands by phosphorimaging analysis. Here, we report the accumulation of a radiolabeled species on NifH under conditions in which FeMo-co synthesis is allowed to take place. Another protein, NifX, also accumulates the radiolabel from 55Fe-labeled NifB-co under similar conditions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All materials used for growth medium preparation were of analytical grade. Sodium dithionite (DTH) was from Fluka. Leupeptin, phenylmethylsulfonyl fluoride, phosphocreatine, creatine phosphokinase, and homocitrate lactone were from Sigma Chemical Co. ATP was purchased as a disodium salt from Sigma. Tris base and glycine were from Fisher Scientific Co. Nitrocellulose membrane was from Millipore, and the polyvinylidene difluoride membrane (Problott) was from Applied Biosystems. Acrylamide/bis acrylamide solution (37.5%:1%) and the equipment for SDS-PAGE were from Bio-Rad. Sephacryl S-200, Sephadex G-75, Q-Sepharose, and phenyl-Sepharose were from Amersham Pharmacia Biotech Inc. DE52 was a Whatman product. 55FeCl3 was from PerkinElmer Life Sciences.

Strains and Growth Conditions-- All strains were grown in the presence of molybdenum and were nif-derepressed. A list of the A. vinelandii mutant strains used in this study is presented in Table I. The procedure for the preparation of cell-free extracts by osmotic shock has been described previously (20).

                              
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Table I
A. vinelandii strains

A. vinelandii strain CA11.3 (Delta nifHDKDelta nifB::kan) was constructed in the laboratory of P. E. Bishop by transforming A. vinelandii strain CA11 with genomic DNA from CA30 (19) and selecting for kanamycin-resistant transformants. A. vinelandii strain CA142 (Delta nifDKYDelta nifENX::kan) was also constructed in P. E. Bishop's laboratory by transforming A. vinelandii strain CA117 (21) with genomic DNA from strain DJ678 (18); transformants were selected for kanamycin resistance.

Conditions for the Growth of A. vinelandii Strain CA11.1 in the Presence of 55FeCl3-- All glassware was rinsed with 4 N HCl overnight to remove traces of contaminating iron and then rinsed thoroughly with deionized water. A 25-ml starter culture of strain CA11.1 was grown in Burk's modified medium (22) in the presence of 18 µM unlabeled iron and 400 µg of nitrogen/ml in the form of ammonium acetate at 30 °C for 20-24 h in a rotary shaker. One milliliter of this culture was used as the inoculum for each of six 250-ml cultures, each containing 9 µM iron and 400 µg fixed nitrogen/ml. The cultures were grown for 20-24 h, and the cells were then collected by centrifugation. The supernatant was discarded, and the cell pellets were resuspended in fresh medium containing 9 µM iron, 2 mCi of 55FeCl3 (18.5-20 mCi/ml) and with no source of fixed nitrogen. 55Fe was added to the cultures as ferric citrate, prepared according to the procedure described previously (18). The cultures were derepressed for 4 h after which the cells were harvested by centrifugation. The cell pellets were stored at -80 °C. Typically about 60-75% of the radioactivity was incorporated into the cells.

Buffer Preparation-- All buffers were sparged with nitrogen for 20-30 min and degassed on a gassing manifold three times, with alternating cycles of vacuum and flushing with argon gas. Sodium dithionite (DTH) was added to a final concentration of 1.7 mM. Buffers used for protein purification contained 0.5 µg/ml leupeptin and 0.2 mM phenylmethylsulfonyl fluoride. 25 mM Tris-HCl (pH 7.5) was used throughout as the buffer unless otherwise stated.

NifB-co Purification-- NifB-co was purified from the cell-free extracts of unlabeled and 55Fe-grown A. vinelandii mutant CA11.1 (Delta nifHDKDelta vnfDGK::spc) according to published procedures (17, 18).

In Vitro FeMo-co Synthesis Assay-- FeMo-co synthesis reactions were carried out as described by Shah et al. (11). To 9-ml serum vials flushed with purified argon and rinsed with anaerobic Tris-HCl buffer were added the following: 100 µl of anaerobic Tris-HCl, 10 nmol of sodium molybdate, 100 nmol of homocitrate, 200 µl of an ATP-regenerating mixture (containing 3.6 mM ATP, 6.3 mM MgCl2, 51 mM creatine phosphate, 20 units/ml creatine phosphokinase, and 6.3 mM DTH), 200 µl of the appropriate A. vinelandii extract, and 10 µl of a solution containing purified 55Fe-labeled NifB-co (60,000 cpm). The total volume of the reaction mixture was 555 µl. The reaction mixtures were incubated for 30 min at 30 °C. After incubation, the samples to be applied to native polyacrylamide gels were placed on ice. Purified apodinitrogenase (the alpha 2beta 2gamma 2 form, 50 µg of total protein) was added to certain reaction mixtures. Acetylene reduction was initiated in the remainder of the vials by the addition of excess MgATP, purified NifH (0.1 mg of protein), and 0.5 ml of acetylene. Nitrogenase activity was then quantitated by acetylene reduction as described elsewhere (11).

Purification of Upper and Lower 55Fe-Labeled Bands-- A 1200-fold scale-up of the in vitro FeMo-co synthesis reaction containing the extract of strain CA117.3 and 55Fe-NifB-co was performed as follows. Thirty 100-ml vials were stoppered and degassed thoroughly on a gassing manifold four times and rinsed with anoxic Tris-HCl buffer. To each vial were added the following: 2 ml of anoxic Tris-HCl containing 1 mM DTH, 0.4 ml of 5 mM homocitrate, 0.2 ml of 1 mM sodium molybdate, 8 ml of an ATP-regenerating mixture (as described earlier), 8 ml of extract of strain CA117.3 (Delta nifDKDelta nifB), 0.4 ml of purified unlabeled NifB-co solution (equivalent to 200 nmol of Fe), and 0.1 ml of purified 55Fe-NifB-co solution (6 × 106 cpm/ml). The vials were placed in a rotary shaker and were gently shaken at 30 °C for 30 min. The reaction mixtures were pooled after the incubation period and applied to a 93-ml DEAE-cellulose column (2.5 × 19 cm) equilibrated with anoxic Tris-HCl containing 0.1 M NaCl. The column was washed with 100 ml of the same buffer, and proteins were eluted from the column using a step gradient of 0.15, 0.25, 0.35, and 0.5 M NaCl in the above buffer containing 20% glycerol. The presence of upper and lower bands was determined by subjecting aliquots of the DEAE-cellulose column fractions to anoxic, native gel electrophoresis, and phosphorimaging analyses. The upper band was found to elute with buffer containing 0.25 M NaCl, whereas the lower band eluted with buffer containing 0.35 M NaCl.

The DEAE-cellulose fractions containing the upper band were separated and pooled from those containing the lower band. The upper band- and lower band-containing pools were separately concentrated down to 12 ml using an Amicon system fitted with a PM-10 membrane and subjected to Sephadex G-75 column (2.5 × 95 cm) chromatography. The column was equilibrated with anoxic Tris-HCl containing 0.05 M NaCl and 10% glycerol. Ten-milliliter fractions were collected, and those containing the upper band, as determined by anoxic gel electrophoresis and phosphorimaging analysis, were pooled and brought to 1 M (NH4)2SO4. This was applied to a 1- × 10-cm phenyl-Sepharose column equilibrated with 1 M (NH4)2SO4 in Tris-HCl, washed with one column volume of the same buffer, and protein was eluted stepwise with two column volumes each of 0.75, 0.5, 0.25, 0.025, and 0.0 M (NH4)2SO4. The upper band-containing fractions (0.25 M (NH4)2SO4)) were pooled, diluted 2-fold with anoxic Tris-HCl, and concentrated by ultrafiltration using the same membrane as stated above. The sample produced in this manner was not homogeneously pure, so ~1 ml of this sample was subjected to preparative anoxic native gel electrophoresis; the region of the gel containing radioactivity was sliced, and the protein was then eluted from the acrylamide, as described below.

The Sephadex G-75 fractions containing the 55Fe-labeled lower band were pooled and subjected to phenyl-Sepharose column chromatography, as described above. The lower band containing fractions (0.5-0.25 M (NH4)2SO4) were then pooled and concentrated using an Amicon system fitted with a PM-10 membrane. One milliliter of this concentrate was applied to a preparative anoxic native gel, and the region of the gel containing radioactivity was sliced. The protein from the gel was eluted as described below. A SDS gel of the elute showed an 18-kDa protein. The protein was transferred to a polyvinylidene difluoride membrane, and N-terminal sequencing was performed at the Protein/Peptide Micro Analytical Laboratory at the California Institute of Technology (Pasadena, CA).

Purification of NifX from A. vinelandii Strain CA11.3-- NifX was partially purified from an A. vinelandii nifB strain, CA11.3 (Delta nifHDKDelta nifB::kan). The cell-free extract of 100 g of cells was applied to a DEAE-cellulose column (4 × 15 cm), equilibrated with anoxic Tris-HCl containing 0.1 M NaCl and 20% glycerol. The column was washed with 1 column volume of the same buffer, and proteins were eluted stepwise with 0.15, 0.25, 0.35, and 0.5 M NaCl contained in the buffer described above. The presence of NifX was determined by SDS-PAGE electrophoresis followed by immunoblot analysis of the fractions using anti-NifX antibody. The fractions containing NifX (0.15 M NaCl elute) were pooled and concentrated using an Amicon system fitted with a XM100A membrane. The concentrate (15 ml) was then chromatographed on a Sephacryl S-100 column (2.5 × 96 cm), equilibrated with buffer containing anoxic Tris-HCl, 0.05 M NaCl, and 10% glycerol. The fractions containing NifX, as determined by immunoblot analysis, were pooled and applied to a Q-Sepharose column (1.5 × 12.5 cm), equilibrated with buffer containing anoxic Tris-HCl and 0.1 M NaCl. The column was washed with 25 ml of the above buffer, and protein was eluted stepwise with one column volume each of 0.15, 0.35, and 0.5 M of NaCl in Tris-HCl. The NifX-containing fractions (0.35 M elute) were pooled, brought to 1 M in (NH4)2SO4, and applied to a phenyl-Sepharose column (1 × 10 cm) equilibrated with 1 M (NH4)2SO4 in Tris-HCl. The column was washed with 12 ml of the same buffer, and protein was eluted stepwise with 0.5, 0.25, 0.125, and 0.0 M (NH4)2SO4 in Tris-HCl. The fractions containing NifX (0.5 and 0.25 M (NH4)2SO4) were pooled and frozen as pellets in liquid nitrogen.

Assaying Activity of Partially Purified NifX-- The activity of the partially purified NifX was followed by use of the in vitro FeMo-co synthesis assay using the extract of the strain DJ42.48 (Delta nifENXDelta vnfX), as described previously (12).

Purification of Other Components-- FeMo-co was purified as described by Shah and Brill (7). Dinitrogenase and NifH were purified as previously described (23). Apodinitrogenase was purified from strain UW45 (nifB) as described by Paustian et al. (24), and NifNE was purified from strain CA117.3 (Delta nifDKDelta nifB) as described by Roll et al. (25).

O2 Denaturation of Components-- O2 treatment of cell-free extracts and various purified components was performed by exposure of the samples to air for 10 min at room temperature with gentle shaking. Following this, the samples were made anaerobic by thoroughly degassing the stoppered vials on a gassing manifold with alternating cycles of vacuum and flushing with Ar gas. DTH was added to a final concentration of 1.7 mM.

Incubation of 55Fe-NifX with Apodinitrogenase-- Apodinitrogenase (25 µg of total protein) purified from A. vinelandii strain UW45 (nifB-) was incubated with the partially purified 55Fe-NifX (5000 cpm) and purified NifH (4.5 µg of protein) in an anoxic, stoppered 9-ml vial. The total volume of the reaction mixture was 170 µl. The reaction mixture was incubated for 15 min at 30 °C, after which 100 µl was subjected to anoxic, native gel electrophoresis. For the C2H2 reduction assay, 800 µl of ATP-regenerating mixture and 0.5 ml of C2H2 were added to the reaction mixture (as described above), and nitrogenase activity was then quantitated as described elsewhere (11).

Elution of Proteins from Acrylamide Gels-- The gel slices containing either 55Fe-labeled upper or lower bands were placed in clean 10-ml culture tubes to which 2 ml of the elution buffer (0.05 M Tris-HCl, pH 7.5, containing 0.1 mM EDTA and 150 mM NaCl) were added. The gel slices were crushed with a clean pestle, and the tubes were shaken in a rotary shaker at 30 °C overnight. The solutions were centrifuged at 10,000 rpm for 10 min, and the supernatants were removed into Eppendorf tubes, to which an equal volume of SDS-sample buffer was added. The entire procedure was performed under aerobic conditions.

Electrophoresis-- SDS-PAGE was performed as described (12) with a 2.8% stacking gel and a 12% resolving gel. The proteins were electrophoresed at 100 V until the bromphenol blue entered the resolving gel and then electrophoresed at 200 V until the dye front reached the bottom of the gel. Low molecular weight markers (Bio-Rad) were used. Proteins were separated on anaerobic native gels with a 7-20% acrylamide and 0-20% sucrose gradient as previously described (26).

Iron Stain-- Native gels were stained for iron as described previously (27).

Antibodies and Immunoblot Analysis-- Antibodies to the various proteins were prepared at the University of Wisconsin antibody facility. The protocols for immunoblotting and developing with modifications by Brandner et al. (28) have been described.

Visualization of Radioactivity-- Gels were exposed to a phosphor screen for 1-2 days and were scanned using a Molecular Dynamics Model 425e PhosphorImager.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Incorporation of 55Fe-Label from 55Fe-NifB-co into Extracts of Various A. vinelandii Mutants-- FeMo-co is unstable in aqueous solutions; thus it is likely that precursors of FeMo-co accumulate on proteins during the course of cofactor biosynthesis. Though use of the in vitro FeMo-co biosynthesis system has yielded considerable information regarding the biosynthesis of FeMo-co, not much is known about the proteins that are associated with precursors of the cofactor. Previous studies with 55Fe- and 35S-labeled NifB-co have shown that NifB-co serves as a specific iron and sulfur donor to FeMo-co (18). The in vitro FeMo-co synthesis system, together with native anoxic gel electrophoresis, was employed to monitor the incorporation of radiolabel from purified 55Fe-labeled NifB-co into FeMo-co and its precursors.

Our strategy for the identification of proteins that accumulate FeMo-co precursors was as follows: (i) perform in vitro FeMo-co biosynthesis in the presence of purified 55Fe-NifB-co, (ii) separate the proteins on anoxic native gels, and (iii) visualize proteins accumulating radiolabel by phosphorimaging analyses of these gels. One limitation in this procedure is that only precursor·protein complexes that are sufficiently stable to survive non-denaturing gel electrophoresis can be visualized. The in vitro FeMo-co synthesis reaction was performed as described under "Experimental Procedures" by the addition of purified 55Fe-labeled NifB-co to extracts of A. vinelandii strains carrying deletion of one or more genes that are required for FeMo-co synthesis. The results of this study are illustrated in Fig. 1. Upon the addition of 55Fe-labeled NifB-co to an in vitro FeMo-co synthesis reaction mixture containing the extract of an A. vinelandii strain carrying a lesion of nifB (strain UW45), FeMo-co is synthesized and inserted into apodinitrogenase forming dinitrogenase (Fig. 1, lane 1), the migration of which was identified by immunoblot analysis (data not shown). A C2H2 reduction activity of 5.0 nmol/min/mg of protein was obtained for this reaction, consistent with the formation of holodinitrogenase. We estimate that this activity is equivalent to greater than 10% of the specific activity of a crude extract of wild-type A. vinelandii. Furthermore, this represents an activity equivalent to our best in vitro FeMo-co synthesis.


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Fig. 1.   PhosphorImager analysis of anoxic, native gel illustrating the incorporation of 55Fe label from 55Fe-NifB-co into extracts of various A. vinelandii strains. In vitro FeMo-co synthesis reactions, including 55Fe-labeled NifB-co and extracts of various A. vinelandii strains were performed as described under "Experimental Procedures." Lane 1, reaction including UW45 (nifB-); lane 2, including CA117.3 (Delta nifDKDelta nifB); lane 3, including CA117.3 plus purified apodinitrogenase (50 µg of protein); lane 4, including CA11.3 (Delta nifHDKDelta nifB::kan); lane 5, including CA142 (Delta nifDKYDelta nifENX::kan); and lane 6, including UW (NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-grown). The table below the figure indicates the components known to be present in the reaction mixture. The C2H2 reduction activities (nmol/min/mg of protein) of the assays are reported where appropriate. -, refers to less than 0.1 nmol/min/mg of protein.

A reaction mixture containing the extract of strain CA117.3 (Delta nifDKDelta nifB) showed the incorporation of the radiolabel from 55Fe-NifB-co into a faster-migrating species (Fig. 1, lane 2), identified as NifNE by immunoblot analysis (data not shown). Two other faster-migrating radiolabeled bands were observed apart from the radiolabel associated with NifNE, which were named as 55Fe-labeled upper and lower bands, as shown in Fig. 1 (lane 2). It is essential to note that strain CA117.3 is able to support FeMo-co synthesis upon the addition of NifB-co alone, because NifKD proteins are not required for cofactor biosynthesis (13, 14). Results from previous studies indicate that completed FeMo-co is bound by the gamma protein (15). Thus, the presence of FeMo-co bound to gamma (gamma  ·FeMo-co) is expected when the in vitro FeMo-co synthesis reaction is carried out with the extract of strain CA117.3. However, under the electrophoretic conditions used in this study, completed FeMo-co bound to gamma  comigrated with the upper band and was separated during the course of purification of the upper band (data not shown). Upon the addition of purified apodinitrogenase (alpha 2beta 2gamma 2) to the reaction mixture containing the extract of strain CA117.3, the incorporation of the radiolabel into dinitrogenase was observed (Fig. 1, lane 3), suggesting that FeMo-co had been synthesized and inserted into the added apodinitrogenase. This is supported by a C2H2 reduction activity of 4.7 nmol/min/mg of protein for this reaction.

Strain CA11.3 (Delta nifHDKDelta nifB::kan) showed the incorporation of the radiolabel from 55Fe-NifB-co into NifNE and into one of the faster-migrating bands termed lower band (Fig. 1, lane 4) but not into the upper band. This suggested that the presence of NifH was required for the accumulation of the radiolabel on the band termed upper band. Note that the only difference between the CA117.3 and CA11.3 genotypes is the presence or absence of nifH; the nifH gene is present in strain CA117.3 and is deleted in strain CA11.3.

When the strain CA142 (Delta nifDKYDelta nifENX::kan) was used in the in vitro FeMo-co synthesis reaction, no incorporation of the radiolabel from 55Fe-NifB-co into either the upper or the lower bands could be observed, suggesting that NifNE or NifX or both may be required for the incorporation of radiolabel into these bands (Fig. 1, lane 5).

A reaction mixture containing the extract of the wild-type UW strain grown in presence of excess fixed nitrogen (nif-repressed) did not show either the upper or lower bands, suggesting that nif proteins were required for the incorporation of radiolabel from 55Fe-NifB-co into the above-mentioned bands (Fig. 1, lane 6). A slow-migrating protein that incorporated 55Fe-label from radiolabeled NifB-co was observed in extracts of several A. vinelandii strains. This band, labeled "? " in Fig. 1, was observed even under conditions when FeMo-co synthesis was not allowed to occur and thus represents a protein that is most likely not involved in FeMo-co synthesis.

At least four lines of evidence suggest that the 55Fe-labeled upper and lower bands contain proteins that are bound to precursors of FeMo-co: (i) The accumulation of radiolabel from 55Fe-NifB-co on these bands was seen only under conditions when FeMo-co synthesis occurred. For example, these bands were not observed when air-oxidized cell-free extracts of A. vinelandii mutants were used in the in vitro FeMo-co synthesis reaction (data not shown). As shown in Fig. 2, the incorporation of radiolabel from 55Fe-NifB-co into these bands did not occur; (ii) when O2-denatured 55Fe-NifB-co was used in the in vitro FeMo-co synthesis reaction, suggesting that a native form of NifB-co was necessary for the formation of the 55Fe-labeled upper and lower bands (Fig. 2, lane 4); nor did it occur (iii) when 55Fe-labeled FeCl3 was used in place of 55Fe-labeled NifB-co (Fig. 2, lane 5). This suggests that the radiolabel on these bands was not adventitiously bound 55Fe. (iv) The radiolabel could be transferred to apodinitrogenase from these bands upon the addition of purified apodinitrogenase to an in vitro FeMo-co synthesis reaction mixture lacking NifKD (compare lanes 2 and 3 of Fig. 1). This not only suggests the association of FeMo-co precursors with the 55Fe-labeled upper and lower bands, but also indicates that when purified apodinitrogenase was made available to the components of the in vitro FeMo-co synthesis reaction mixture, the newly synthesized FeMo-co was inserted into the added apodinitrogenase.


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Fig. 2.   PhosphorImager analysis of an anoxic native gel illustrating the transfer of radiolabel from NifNE to NifH. In vitro FeMo-co synthesis reactions, including 55Fe-labeled NifB-co and extract of strain CA11.3 (Delta nifHDKDelta nifB::kan) were performed as described under "Experimental Procedures." Three anoxic vials containing the extract of strain CA11.3 were used. Purified NifH (45 µg of protein) was added to the second and the third vials. The vials were incubated for 30 min for in vitro FeMo-co synthesis. At the end of 30 min, purified apodinitrogenase (50 µg of protein) was added to the third vial. The vials were incubated for an additional 10 min before application onto the anoxic native gel. Reaction mixtures in lanes 4 and 5 contained the extract of strain CA11.3, purified NifH (45 µg of protein), and purified apodinitrogenase (50 µg of protein) but not 55Fe-labeled NifB-co. Lane 1, reaction including extract of strain CA11.3; lane 2, same as in lane 1 plus purified NifH; lane 3, same as in lane 2 plus purified apodinitrogenase; lane 4, same as in lane 3 minus 55Fe-labeled NifB-co but plus anoxic O2-denatured NifB-co; and lane 5, same as in lane 3 minus 55Fe-labeled NifB-co but plus anoxic 55FeCl3. The table below the figure indicates the components known to be present in the reaction mixture. The C2H2 reduction activities (nmol/min/mg of protein) of the assays are reported where appropriate. -, refers to less than 0.1 nmol/min/mg of protein. The position of dinitrogenase, NifNE, NifH, and 55Fe-lower band are indicated by arrows.

Purification and Identification of the 55Fe-Labeled Upper Band-- A 1200-fold scale up of the in vitro FeMo-co synthesis reaction containing the extract of strain CA117.3 (Delta nifDKDelta nifB) and 55Fe-NifB-co was carried out to identify the protein in the upper 55Fe-labeled band, as described under "Experimental Procedures." The 55Fe-labeled upper band was purified by following the radioactivity associated with it. Purification steps included DEAE-cellulose, Sephadex G-75, phenyl-Sepharose chromatography, and preparative native gel electrophoresis. The region of the gel containing radioactivity was sliced, and the protein was eluted from the acrylamide. The protein was identified as NifH based on its migration on a SDS-gel (Fig. 3, lanes 1 and 2) and its cross-reactivity with anti-NifH antibody (Fig. 3, lanes 3 and 4). These results suggest that a FeMo-co precursor actually accumulated on NifH during cofactor biosynthesis.


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Fig. 3.   SDS-PAGE of 55Fe-upper band. Coomassie Blue-stained SDS-gel. Lane 1, purified NifH (10 µg of protein); and lane 2, 55Fe-upper band purified by preparative native gel electrophoresis. Anti-NifH immunoblot. Lane 3, purified NifH (2 µg of protein); and lane 4, 55Fe-upper band purified by preparative native gel electrophoresis. Numbers at the left show molecular masses in kilodaltons.

The involvement of NifH in FeMo-co biosynthesis was examined using the extract of CA11.3 (Delta nifHDKDelta nifB::kan). As seen in Fig. 2 (lane 1), the extract of this strain showed the accumulation of radiolabel on NifNE and on the lower band when the in vitro FeMo-co biosynthesis reaction was performed with 55Fe-NifB-co, as described under "Experimental Procedures." Upon the addition of purified NifH to the above reaction (Fig. 2, lane 2), the accumulation of radiolabel on NifH was observed with the concomitant decrease in the amount of radiolabel accumulating on NifNE. These results suggest that FeMo-co biosynthesis was blocked before the addition of NifH and that the addition of NifH to the reaction mixture allowed cofactor biosynthesis to proceed beyond NifNE. The addition of purified NifH and purified apodinitrogenase to the extract of CA11.3 allowed the accumulation of the radiolabel at the dinitrogenase position with the concomitant decrease in the radiolabel on NifH and on the lower band (Fig. 2, lane 3). This demonstrates that the radiolabel associated with NifH and the lower band can be transferred to apodinitrogenase. The C2H2 reduction activity (4.7 nmol/min/mg of protein) of this reaction mix was also consistent with the formation of holodinitrogenase.

The radioactive species accumulating on NifH was O2-labile, because exposure of the identical sample to air eliminated the detection of this band (data not shown). The amount of radiolabel on NifH diminished during the course of purification, suggesting a transient association of a FeMo-co precursor with NifH. The continual loss of radiolabel has so far prevented the definitive identification and the characterization of the cluster on NifH. However, at least three lines of evidence support the hypothesis that the label associated with NifH is in the form of a FeMo-co precursor and is not adventitiously bound Fe: 1) No accumulation of radiolabel on NifH was observed upon the addition of radiolabeled NifB-co to strains containing NifH, such as UW45 (nifB) and CA142 (Delta nifDKYDelta nifENX) (Fig. 1, lanes 1 and 5). If the radiolabel on NifH were adventitiously bound iron, then the presence of radiolabel on NifH in all strains containing NifH would be expected. 2) The radiolabel on NifH was transferred to apodinitrogenase upon the addition of purified apodinitrogenase to an extract lacking nifDK (compare lanes 2 and 3 in both Figs. 1 and 2) strongly suggesting the association of NifH with a FeMo-co precursor. 3) The absence of a band that co-migrated with NifH in samples where O2-inactivated 55Fe-NifB-co or 55FeCl3 was utilized demonstrates that active NifB-co was required for incorporation of 55Fe into NifH (Fig. 2, lanes 4 and 5).

Purification and Identification of the 55Fe-Labeled Lower Band-- The radiolabeled lower band that formed upon the incubation of 55Fe-labeled NifB-co with the extract of CA117.3 (Delta nifDKDelta nifB::kan) in a FeMo-co synthesis reaction was purified by following the radioactivity associated with it. Purification steps of the 55Fe-labeled lower band from a 1200-fold scale-up of the in vitro FeMo-co synthesis reaction included DEAE-cellulose, Sephadex G-75, phenyl-Sepharose chromatography, and preparative native gel electrophoresis. The portion of the gel containing radioactivity was sliced, and protein was eluted from the acrylamide, as described under "Experimental Procedures." The protein was identified as NifX, based on the N-terminal sequence obtained and on its identity to the sequence predicted from nifX (MSSPTRQLQVLD). The nifX gene is located downstream of nifE and N, in the nifENX operon of A. vinelandii. We have shown, in a separate study, the requirement of NifX for cofactor biosynthesis by the in vitro FeMo-co synthesis reaction (12). The data presented here suggest the association of NifX with a FeMo-co precursor during the course of cofactor biosynthesis. This species will be termed 55Fe-NifX for the remainder of the manuscript.

Transfer of Radiolabel from 55Fe-NifX to Apodinitrogenase-- To determine if the radiolabeled species on NifX can be donated to apodinitrogenase, purified NifX bound to the radiolabeled FeMo-co precursor (55Fe-NifX) was incubated with purified apodinitrogenase (alpha 2beta 2gamma 2), as described under "Experimental Procedures." A solution substantially enriched in 55Fe-NifX obtained from a 1200-fold scale-up of the in vitro FeMo-co synthesis reaction was used for this study. The results, presented in Fig. 4, show that the radiolabeled species on NifX could be transferred to apodinitrogenase, although not completely (Fig. 4, lane 4). The transfer of the radiolabel from 55Fe-NifX to apodinitrogenase correlated with C2H2 reduction activity of 224 nmol/min/mg of 55Fe-NifX, strongly suggesting the presence of completed FeMo-co on NifX. In support of this hypothesis, the binding of purified FeMo-co to purified NifX has been observed in vitro.2 The partial transfer of radio label from 55Fe-NifX to apodinitrogenase suggested that 55Fe-NifX is comprised of a mixture of either NifX bound to finished FeMo-co or to NifX bound to a FeMo-co precursor that is yet to be completed. Increasing the concentration of purified apodinitrogenase in the reaction mixture did not increase the amount of transferred label from 55Fe-NifX, suggesting that apodinitrogenase is not limiting in the reaction mix (data not shown). A matter of concern here is that the C2H2 reduction activity observed might result from the presence of gamma  ·FeMo-co in the partially purified 55Fe-NifX sample; gamma  has been shown to bind FeMo-co (15). However, no gamma  was observed, as judged by anti-gamma immunoblot analysis of the 55Fe-NifX solution. That the radiolabel at the apodinitrogenase position is not adventitiously bound iron is evidenced by the lack of transfer of the radiolabel when O2-denatured 55Fe-NifX was used in the reaction (Fig. 4, lane 5).


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Fig. 4.   PhosphorImager analysis of an anoxic native gel illustrating the transfer of radiolabel from 55Fe-NifX to apodinitrogenase. The position of apodinitrogenase and 55Fe-NifX are indicated. Incubations were carried out as described under "Experimental Procedures." Lane 1, partially purified 55Fe-NifX (5,000 cpm); lane 2, anoxic O2-denatured 55Fe-NifX (5000 cpm); lane 3, purified apodinitrogenase (25 µg of total protein); lane 4, 55Fe-NifX (5000 cpm) incubated with apodinitrogenase; lane 5, anoxic O2-denatured 55Fe-NifX (5000 cpm) incubated with apodinitrogenase. The table below the figure indicates the components known to be present in the reaction mixture.

NifB-co Binding Perturbs the Migration of NifX-- Immunoblot analysis of anoxic native gels containing the extract of the A. vinelandii strain CA11.1 (Delta nifHDKDelta vnfDGK::spc) using anti-NifX antibody showed the presence of a slow-migrating and a fast-migrating NifX species (Fig. 5, lane 3). The fast-migrating NifX species comigrated with 55Fe-NifX (Fig. 5, lane 1) and was not observed upon exposure of the sample to air (Fig. 5, lanes 2 and 4). This is presumably due to the loss of the FeMo-co precursor bound to NifX upon O2 exposure. The extract of NifB-co-deficient strain CA117.3 (Delta nifDKDelta nifB::kan), on the other hand, showed the presence of only the slow-migrating species of NifX (Fig. 5, lane 5). Upon the addition of purified NifB-co to the extract of strain CA117.3, a shift in the electrophoretic mobility of NifX was observed (Fig. 5, lane 6). These data suggest that the electrophoretic mobility of NifX on anoxic, native gels is dependent upon the presence or absence of NifB-co. Roll et al. (25) have observed similar shifts on anoxic native gels for NifNE upon addition of purified NifB-co to extracts lacking nifB.


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Fig. 5.   Analysis of NifX by anoxic native gel electrophoresis. PhosphorImager analysis of an anoxic native gel. Lane 1, purified 55Fe-NifX (5000 cpm); lane 2, anoxic O2-denatured 55Fe-NifX (5000 cpm). Anti-NifX immunoblot of an anoxic, native gel. Lane 3, extract of A. vinelandii strain CA11.1 (Delta nifHDKDelta vnfDGK::spc; 260 µg of total protein); lane 4, anoxic O2-denatured extract of strain CA11.1 (260 µg of total protein); lane 5, extract of strain CA117.3 (Delta nifDKDelta nifB; 260 µg of total protein); and lane 6, extract of strain CA117.3 (260 µg of total protein) incubated with NifB-co (1.5 nmol of Fe).

To characterize the two electrophoretically distinct NifX species, NifX that is not bound to the FeMo-co precursor was purified partially from the extract of an A. vinelandii strain carrying a deletion of nifB, strain CA117.3 (Delta nifDKDelta nifB::kan). The purification of NifX was performed as described under "Experimental Procedures." NifX, purified in this manner, was ~60% pure as judged by scanning densitometry and showed a 6-fold stimulation of in vitro FeMo-co synthesis assays involving the extract of strain DJ42.48 (Delta nifENXDelta vnfE), as reported previously by Shah et al. (12). The results presented in Fig. 6 show that the purified NifX comigrated with the slow-migrating species of NifX (compare lanes 1 and 2). The addition of purified NifB-co caused a shift in the migration of NifX on anoxic, native gels; NifX now comigrated with the fast-migrating species (Fig. 6, compare lanes 2 and 3). These results suggest the binding of NifX with NifB-co. Fig. 6 also shows the incorporation of radiolabel from 55Fe-labeled NifB-co into NifX as judged by phosphorimaging (lane 6) and the incorporation of iron from unlabeled NifB-co as judged by iron staining (lane 7). These results confirm the binding of NifB-co to NifX. O2-inactivated NifB-co did not cause a shift in the migration of NifX; nor did a solution of SB-12, the detergent used in the extraction of NifB-co (data not shown). On the other hand, O2-exposed NifX (made anoxic after O2 exposure by degassing and flushing with argon gas and by the addition of DTH) still retained its ability to bind NifB-co (Fig. 6, lane 4). That NifNE is not required for the binding of NifB-co to NifX was determined by examination of the electrophoretic mobility of NifX in the extract of a nifE strain (DJ35), where in a portion of NifX migrated as the fast-migrating species (Fig. 6, lane 5).


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Fig. 6.   NifX can bind NifB-co. Anti-NifX immunoblot of anoxic, native gel. Lane 1, extract of A. vinelandii strain CA11.1 (Delta nifHDKDelta nifDGK; 260 µg of total protein); lane 2, NifX partially purified from strain CA117.3 (Delta nifDKDelta nifB; 25 µg of total protein); lane 3, NifX partially purified from strain CA117.3 (25 µg of total protein) incubated with NifB-co (1.5 nmol of Fe); lane 4, anoxic O2-exposed NifX partially purified from strain CA117.3 (25 µg of total protein) incubated with NifB-co (1.5 nmol of Fe); and lane 5, extract of strain DJ35 (Delta nifE; 260 µg of total protein). PhosphorImager analysis of anoxic, native gel. Lane 6, NifX partially purified from strain CA117.3 (500 µg of total protein) incubated with 55Fe-NifB-co (10,000 cpm). Iron stain of anoxic, native gel. Lane 7, NifX partially purified from strain CA117.3 (500 µg of total protein) incubated with NifB-co (1.5 nmol of Fe).

By use of the in vitro FeMo-co synthesis system, NifX has been shown to be required for FeMo-co synthesis (12). Surprisingly, NifX is not absolutely required for the synthesis of the cofactor in vivo, because strains carrying a deletion of nifX are capable of diazotrophic growth (29) exhibiting total nitrogenase activity similar to that of wild-type. This suggests that some other gene product can replace NifX in the in vivo synthesis of FeMo-co.

Results similar to those presented here were obtained for VnfX, an NifX homolog in the vnf system (26). VnfX has been purified with a V-Fe cluster bound from extracts of strain CA11.1 (Delta nifHDKDelta vnfDGK::spc) grown in the presence of vanadium, thus suggesting a role for the protein in FeV-co synthesis (26). VnfX purified without the cluster bound has been shown to bind NifB-co specifically, and NifB-co binding caused a shift in its mobility on anoxic, native gels. Like NifX, VnfX is not absolutely required for FeV-co synthesis in vivo, suggesting that it can be replaced by some other gene product.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The major observations made in the present study are: 1) NifH and NifX accumulate FeMo-co precursors during the course of FeMo-co biosynthesis; 2) radiolabel from NifX can be transferred to apodinitrogenase, and the corresponding C2H2 reduction activity strongly suggests that NifX may be associated with mature FeMo-co; and 3) NifX can bind NifB-co and consequently migrates at a different position on anoxic native gels than does free NifX.

Based on these results and on other observations from previous studies, our working model for the biosynthesis of FeMo-co is as follows: the binding of NifB-co to NifNE occurs as the first step in the FeMo-co biosynthetic pathway. The FeMo-co precursor is then transferred from NifNE to NifH. We propose that the heterometal addition to the FeMo-co precursor occurs either on NifH or on NifX. The addition of homocitrate might occur as the last step during cofactor biosynthesis on NifX. The mature FeMo-co is bound by gamma, which delivers the cofactor to apodinitrogenase to form holodinitrogenase.

The binding of NifB-co to NifNE has been observed previously both in vivo as well as in vitro (25). That this binding is not dependent on the presence of MgATP, NifH, or NifX in vitro suggests that NifB-co binding to NifNE occurs as one of the early events in the FeMo-co biosynthetic pathway (25). In the present study, we have shown that, like NifNE, NifX also is capable of binding NifB-co and that this binding is not dependent on the presence of NifNE. However, whether the binding of NifB-co to NifX is physiologically relevant remains to be determined. Also, it is not yet known if either NifNE or NifX catalyze any modification of NifB-co upon binding to the cofactor. The structure of NifB-co has not yet been established, but recent results suggest that NifB-co may be a cluster of complex Fe-S species.3 The binding of NifB-co to NifNE and NifX might be better understood when the structure of NifB-co becomes available.

Our results with the addition of 55Fe-NifB-co and purified NifH to the extract of a strain lacking NifH showed that the radiolabel associated with NifNE diminished with the concomitant appearance of radiolabel on NifH, although there was no change in the radiolabel associated with NifX. This suggests that the step catalyzed by NifH occurs after the one catalyzed by NifNE. These results also suggest that the binding of NifB-co to NifX is most probably due to the presence of a FeMo-co precursor binding site on NifX. In support of the hypothesis that the action of NifH occurs after the reaction catalyzed by NifNE, we have shown the interaction of NifH with the NifNE·NifB-co complex (31). We propose that NifH may lose its affinity to the [Fe-Mo-S] species upon the addition of molybdenum to the cofactor precursor. This might explain the transient association of the FeMo-co precursor with NifH. We hypothesize that the addition of homocitrate occurs on NifX as the last step during cofactor biosynthesis. In this context, we note the presence of completed FeMo-co on NifX. In vivo, the finished FeMo-co is bound by gamma, which might possess greater affinity for FeMo-co than does NifX. FeMo-co is then delivered to apodinitrogenase to form holodinitrogenase.

One of the central claims in this study is that a FeMo-co precursor accumulates on NifH. Apart from its role in nitrogenase turnover, NifH has been known to function as a required participant in the biosynthesis of FeMo-co (9, 10, 32). NifH is thus a "moonlighting protein" (33) that serves as the electron donor to dinitrogenase and as a biosynthetic enzyme in FeMo-co biosynthesis. It has also been known that the properties of NifH required for its function in FeMo-co biosynthesis are different from those required for its function in electron transfer to dinitrogenase (34-36). For example, we have shown in a separate study that a redox-active 4Fe-4S cluster in NifH is not necessary for its function in FeMo-co biosynthesis (37). Nevertheless, the exact role played by NifH in cofactor biosynthesis remains unclear. In the present study, we have shown the accumulation of a radiolabeled FeMo-co precursor on NifH. However, this association seems weaker than that of NifX and NifB-co due to the loss of radiolabel during the course of purification of NifH bound to the FeMo-co precursor. Studies involving 99Mo might elucidate the role of NifH in FeMo-co biosynthesis and determine whether the cluster on NifH contains molybdenum. Besides its role in substrate reduction, FeMo-co synthesis and apodinitrogenase maturation, NifH has been implicated in the expression of the alternative nitrogenases in A. vinelandii (38). Our results help support the idea that the role played by NifH in the regulation of expression of alternative nitrogenases might be as an indicator of molybdenum sufficiency. Specifically, NifH bound to the Fe-Mo-S species might serve to signal the proper functioning of the nif system. Moreover, it has been shown that NifH is required for the expression of anf-nitrogenase in Mo- and V-deficient conditions (38). Under these conditions, it is expected that NifH would not be bound to the FeMo-co precursor and thus could serve to indicate the impairment of nif-nitrogenase, in turn promoting the expression of anf-nitrogenase.

Finally, there remain a number of gaps in our understanding of the biosynthesis of FeMo-co. It is not known whether the NifNE proteins perform any modification(s) to NifB-co. The identity of the molybdenum donor to the FeMo-co biosynthetic pathway and the chemical form of molybdenum as it enters the pathway are unknown. In addition, the exact roles played by NifH and NifX are not yet clear. We believe in vitro FeMo-co synthesis reactions, including 99Mo, the radioisotope of molybdenum, will clarify the roles played by NifH and NifX in FeMo-co biosynthesis and will reveal the step at which molybdenum is incorporated into the FeMo-co precursor.

    ACKNOWLEDGEMENTS

We thank Dr. Gary Roberts, Dr. Chris Staples, and Dr. Dennis Dean for helpful discussions, Dr. Mary Homer for providing anti-gamma antibody and Dr. Jon Roll for providing anti-NifNE antibody. Dr. Gary Hathaway (Caltech) is acknowledged for N-terminal sequencing of the lower band. We thank Dr. Paul Bishop and Dr. Dennis Dean for sharing strains.

    FOOTNOTES

* This work was supported by NIGMS, National Institutes of Health Grant 35332 (to P. W. L.).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: Department of Biochemistry, 433 Babcock Ave., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-6859; Fax: 608-262-3453; E-mail: ludden@biochem.wisc.edu.

Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M100907200

2 P. J. Goodwin and D. R. Dean, personal communication.

3 C. R. Staples, P. Rangaraj, C. Rüttimann-Johnson, S. J. Yoo, E. Munck, and P. W. Ludden, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: NifKD, dinitrogenase (also called MoFe protein); FeMo-co, iron-molybdenum cofactor; NifB-co, NifB-cofactor; DTH, sodium dithionite; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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