©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii(*)

(Received for publication, November 13, 1995; and in revised form, January 12, 1996)

Ranjini Chatterjee Ronda M. Allen Paul W. Ludden (§) Vinod K. Shah (¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The vnf-encoded apodinitrogenase (apodinitrogenase 2) has been purified from Azotobacter vinelandii strain CA117.30 (DeltanifKDB), and is an alpha(2)beta(2)(2) hexamer. Apodinitrogenase 2 can be activated in vitro by the addition of the iron-vanadium cofactor (FeV-co) to form holodinitrogenase 2, which functions in C(2)H(2), H, and N(2) reduction. Under certain conditions, the alpha(2)beta(2)(2) hexamer dissociates to yield the free subunit (the VNFG protein) and a form of apodinitrogenase 2 that exhibits no C(2)H(2), H, or N(2) reduction activities in the in vitro FeV-co activation assay; however, these activities can be restored upon addition of VNFG to the FeV-co activation assay system. No other vnf-, nif-, or non-nif-encoded proteins were able to replace the function of VNFG in the in vitro processing of alpha(2)beta(2) apodinitrogenase 2 (in the presence of FeV-co) to a form capable of substrate reduction. Apodinitrogenase 2 is also activable in vitro by the iron-molybdenum cofactor to form a hybrid enzyme with unique properties, most notably the inability to reduce N(2) and insensitivity to CO inhibition of C(2)H(2) reduction.


INTRODUCTION

The biological conversion of atmospheric nitrogen to ammonia can occur via three distinct nitrogenase enzymes in Azotobacter vinelandii: the conventional molybdenum-containing enzyme (nitrogenase 1), a vanadium-containing enzyme (nitrogenase 2), and a third enzyme (nitrogenase 3) that is believed to contain only iron (for reviews see (1) and (2) ). Nitrogenases 1, 2, and 3 are genetically distinct, being encoded by the nif, vnf, and anf operons, respectively. Although specific structural genes encode each nitrogenase enzyme, the products of certain nif genes are also required for the full functionality of the vnf- and anf-encoded nitrogenases(3, 4) . A. vinelandii harbors all three nitrogenases; the expression of the individual nitrogenase enzymes is dependent on the metal content of the growth medium. Nitrogenase 1 is synthesized in medium containing molybdenum. Expression of nitrogenase 2 requires medium depleted of molybdenum and containing vanadium; nitrogenase 3 is expressed in medium deficient in both molybdenum and vanadium(2) .

All three nitrogenase enzymes are two-component systems consisting of dinitrogenase and dinitrogenase reductase. Dinitrogenase 1, an alpha(2)beta(2) tetramer of the nifD and nifK gene products, respectively, has been extensively characterized(5, 6) . Dinitrogenase 2 (encoded by vnfDGK) has been purified from both Azotobacter chroococcum(7) and A. vinelandii(8) . Two species of dinitrogenase 3 (encoded by anfDGK) and a hybrid form of dinitrogenase 3 have also been characterized in A. vinelandii(9, 10) . The presence of vnfG and anfG (encoding the subunits of dinitrogenases 2 and 3, respectively) is a genetic feature of the molybdenum-independent nitrogenases only; however, the presence of the subunit in purified dinitrogenases 2 and 3 appears to vary(7, 8, 9, 10) . The role of the subunit in the functioning of dinitrogenase 2 was recently addressed by Waugh et al., who found that an A. vinelandii strain lacking vnfG was unable to grow diazotrophically, suggesting that the subunits might be required for N(2) reduction(11) .

Apodinitrogenase 1 (lacking the iron-molybdenum cofactor) is an alpha(2)beta(2)(2) hexamer in certain mutant strains of A. vinelandii; the protein (a non-nif gene product(12) ) was shown to be involved in the processing of apodinitrogenase 1 to the holoenzyme(13) . By analogy to the nitrogenase 1 enzyme system, a possible role of the subunits of nitrogenases 2 and 3 might be in the processing of these apodinitrogenase proteins to their respective holoenzyme forms. The subunit composition of apodinitrogenase 2 is therefore of interest, as it might provide insight into additional roles of the subunit (the VNFG protein) of dinitrogenase 2. A second property that distinguishes the molybdenum-independent nitrogenases from nitrogenase 1 is their substrate reduction characteristics. All three enzymes reduce N(2) to NH(3), C(2)H(2) to C(2)H(4), and H to H(2); however, in addition to reducing C(2)H(2) to C(2)H(4), nitrogenases 2 and 3 also catalyze the formation of C(2)H(6) (as a minor product) from C(2)H(2)(14, 15) .

The substrate reduction characteristics of the nitrogenases appear to be dictated both by the cofactor at the active site and their polypeptide environments. Certain altered forms of the iron-molybdenum cofactor (FeMo-co) resulted in a loss of N(2) and/or C(2)H(2) reduction activities of nitrogenase 1, indicating the involvement of the cofactor in substrate specificity (16, 17) . The role of the polypeptide in the substrate specificity of nitrogenase 1 was demonstrated by the conversion of glutamine 191 (of the nifD-encoded subunit) to lysine, which caused a loss of N(2) reduction and the atypical reduction of C(2)H(2) to both C(2)H(4) and C(2)H(6)(18) . The requirement for specific polypeptide-cofactor interactions was also observed with dinitrogenase 1 containing the iron-vanadium cofactor (FeV-co); (^1)the hybrid enzyme was unable to reduce N(2), and C(2)H(2) was reduced to both C(2)H(4) and C(2)H(6), the latter being a property of nitrogenase 2(19) .

In this report, we describe the purification and characterization of apodinitrogenase 2 from A. vinelandii strain CA117.30 (DeltanifKDB). Purified apodinitrogenase 2 has an alpha(2)beta(2)(2) subunit composition and is activable in vitro by FeV-co. Under certain conditions, the subunit (VNFG) dissociates from the alpha(2)beta(2)(2) hexamer to yield free VNFG and a form of apodinitrogenase 2 that is inactive in the in vitro FeV-co-activation assay. Apodinitrogenase 2 activity can be restored upon addition of VNFG to the FeV-co-activation assay system. In addition to being activable by FeV-co, apodinitrogenase 2 is also activable in vitro by FeMo-co. The activation of apodinitrogenase 2 by FeFe-co (the putative iron-only cofactor of dinitrogenase 3) was also attempted; the substrate reduction characteristics of the resulting hybrid enzymes were investigated.


EXPERIMENTAL PROCEDURES

Materials

All reagents used for A. vinelandii growth media were of analytical grade or higher purity. Sodium metavanadate (NaVO(3), 99.995% in purity) was obtained from Fisher Scientific Company. Ammonium sulfate and citric acid were from Mallinckrodt. Ethylene glycol was from Sigma. DEAE-cellulose was a Whatman DE-52 product. Octyl-Sepharose CL-4B, Sephacryl S-100, and the Superose 12 gel filtration column were purchased from Pharmacia Biotech Inc. The HPLC system used was a Beckman product, as was the TSK-phenyl hydrophobic column.

A. vinelandii Strains and Growth Conditions

A. vinelandii strain CA117.30 (DeltanifKDB) was constructed in the laboratory of Dr. P. E. Bishop by transforming A. vinelandii strain CA (wild type) with crude DNA from strain DJ33 (20) ; transformants were selected for rifampicin resistance. The rifampicin-resistant transformants were screened for inability to grow on N-free Burk's medium containing molybdenum. One of the nif, rifampicin-resistant transformants (designated CA117) was transformed with a 9.5-kilobase EcoRI fragment containing the nifB::Tn5 insertion mutation from CA30(21) . Kanamycin-resistant transformants were selected, and one of these transformants was designated CA117.30.

A. vinelandii strains CA12 (DeltanifHDK), CA11.6 (W-tolerant, DeltanifHDK), CA11.1 (DeltanifHDKvnfDGK1::spc), and UW45 (nifB) have been described(9, 11, 21, 22) . All strains were grown in Burk's medium prepared in deionized water; all vessels used in preparing media and for cell culture were rinsed thoroughly in 4 N HCl and then in deionized water. Strains CA117.30, CA12, and CA11.1 were grown on Burk's medium that lacked sodium molybdate and contained 10 µM sodium metavanadate. Cultures of strain CA117.30 (15 liters) were grown in 20-liter polycarbonate carboys with vigorous aeration at 30 °C in Burk's medium containing 40 µg of nitrogen/ml as ammonium acetate; the cultures were monitored for the depletion of ammonium, following which they were derepressed for 4 h. The cells were concentrated using a Pellicon cassette system containing a filtration membrane (0.45 µM, Millipore Corp., Bedford, MA) and were centrifuged; the cell pellets were frozen in liquid N(2) and stored at -80 °C. Strain CA12 was grown in 15-liter cultures (described above) on nitrogen-free Burk's medium; the cultures were harvested (as described above) when cell density reached 1.6-1.8 absorbance units at 600 nM. Strain CA11.6 was grown on Burk's medium containing only iron (with no added sodium molybdate or sodium metavanadate) as previously reported(9) . Strain CA11.1 was grown and derepressed on Burk's medium containing only iron by the method described for CA11.6. Cell-free extracts of all A. vinelandii strains were prepared by the osmotic shock method (23) .

Buffer Preparation

All buffers were sparged for 10-30 min with purified N(2) (and degased on a gassing manifold where appropriate) and sodium dithionite (DTH) was added to a final concentration of 1.7 mM. All buffers used in column chromatography contained 0.2 mM phenylmethylsulfonyl fluoride and 0.5 µg/ml leupeptin. Buffers used in HPLC and fast protein liquid chromatography were filtered through a 0.45-µm filter. Tris-HCl used was at pH 7.4 unless indicated otherwise.

Partial Purification of Dinitrogenases 2 and 3

Dinitrogenase 2 was partially purified from A. vinelandii strain CA12 (DeltanifHDK) by DEAE-cellulose chromatography followed by heat treatment as described by Hales et al.(8) . Partially pure dinitrogenase 2 was concentrated by ultrafiltration using a XM100-A membrane. Partial purification of dinitrogenase 3 from strains CA11.6 (W-tolerant, DeltanifHDK) and CA11.1 (DeltanifHDKDeltavnfDGK1::spc) was performed (up to the gel filtration chromatography step) by the method reported by Chisnell et al.(9) with the following modifications: 0.025 M Tris-HCl (pH 7.4) was used in place of 0.1 M HEPES (pH 7.1), and a XM100-A ultrafiltration membrane was used (instead of a PM-30 membrane) for the concentration of dinitrogenase 3 following heat treatment.

Preparation of FeV-co, FeMo-co, and FeFe-co

A source of FeV-co was prepared by citric acid treatment of partially pure dinitrogenase 2 (that exhibited a specific activity of 25.5 nmol C(2)H(4) formed/min/mg of protein) with variations to the method used by Allen et al. (24) for the preparation of FeMo-co from citric acid-treated dinitrogenase 1. In a typical reaction, 0.3 ml of a solution of 0.4 M citric acid (made anoxic by diluting a 2.0 M stock solution into water containing 1.7 mM DTH) was added to 2 ml (44.6 mg of protein) of partially pure dinitrogenase 2 in a screw-capped centrifuge tube (Beckman) fitted with rubber-stoppered inserts (to allow evacuation and flushing with argon on a gassing manifold). The solution was thoroughly mixed using a Vortex mixer and incubated on ice for 5 min, following which it was centrifuged at 5,000 times g for 5 min. The supernatant was discarded, and the pellet was suspended in 2 ml of 0.1 M Tris-HCl. This suspension of acid-denatured dinitrogenase 2 (hereafter referred to as FeV-co) was used for the in vitro activation of apodinitrogenase 2.

Purified FeMo-co was prepared by the method described by Shah and Brill (23) ; when it was necessary to avoid the denaturing effects of N-methylformamide, FeMo-co was prepared by the citric acid treatment of purified dinitrogenase 1 as described by Allen et al.(24) .

FeFe-co (the iron-only cofactor) was prepared from dinitrogenase 3 that was partially purified from A. vinelandii strains CA11.6 (W-tolerant, DeltanifHDK) and CA11.1 (DeltanifHDKDeltavnfDGK1::spc). The dinitrogenase 3 enzymes from CA11.6 and CA11.1 exhibited specific activities of 13.8 and 1.7 nmol of C(2)H(4) formed/min/mg of protein, respectively. Preparation of a source of FeFe-co was performed by the method described above for the preparation of FeV-co with one exception: 0.24 ml of 0.4 M citric acid was added to 2 ml (approximately 31 mg of protein) of partially pure dinitrogenase 3.

In Vitro Activation of Apodinitrogenase 2 by FeV-co (FeV-co Insertion Assay)

Apodinitrogenase 2 activity was monitored by C(2)H(2) reduction following the in vitro activation of apodinitrogenase 2 by FeV-co. The FeV-co insertion assays were performed in 9-ml, rubber-stoppered serum vials that were repeatedly evacuated and flushed with argon and rinsed with 0.3 ml of 0.025 M Tris-HCl. In a typical insertion assay, the following components were added to the vials in the order indicated: 100 µl of 0.025 M Tris-HCl, 200 µl of FeV-co (described above), 25-200 µl of cell-free extract or a partially pure fraction (as a source of apodinitrogenase 2). The reactions were incubated at room temperature for 15 min to allow the formation of holodinitrogenase 2. For the C(2)H(2) reduction phase of the assay, 800 µl of an ATP-regenerating mix (containing 3.6 mM ATP, 6.3 mM MgCl(2), 51 mM creatine phosphate, 20 units/ml creatine phosphokinase, and 6.3 mM DTH in 0.025 M Tris-HCl, pH 8.0) and 10 µl (20 µg of protein) of dinitrogenase reductase 1 were added to the vials, which were then brought to atmospheric pressure. C(2)H(2) reduction was initiated by the addition of 0.5 ml of C(2)H(2). The vials were incubated on a rotary water bath shaker at 30 °C for 30 min, following which the reactions were terminated by the addition of 100 µl of 4 N NaOH; C(2)H(4) and C(2)H(6) formed were quantitated by a gas chromatograph(25) .

The in vitro activation of apodinitrogenase 2 by FeMo-co or FeFe-co was carried out as outlined above with some exceptions. Purified FeMo-co (10 µl, in N-methylformamide) or acid-denatured dinitrogenase 1 (50 µl) was used as a source of FeMo-co (in place of FeV-co); acid-denatured dinitrogenase 3 (200 µl, described above) was used as a source of FeFe-co. Where indicated, 200 µl of CO gas was added to the vials prior to the addition of C(2)H(2).

N(2) and Proton Reduction Assays

N(2) reduction was performed by the N(2) fixation method(26) . Four ml of 99% N(2) were added to the 9-ml vials (which were evacuated and flushed with argon immediately before the addition of N(2)) following the activation of apodinitrogenase 2 (described above). The reactions were terminated by the addition of 0.5 ml of 4 N H(2)SO(4). Samples containing fixed N were digested and distilled(26) ; fixed N in the samples was then converted to N(2), which was quantitated as atom percent excess N in the samples by mass spectrometry using a MAT 250 isotope ratio mass spectrometer. Proton reduction assays were initiated by the addition of the ATP-regenerating mixture (described above) to the vials, which were then evacuated, flushed with argon, and brought to atmospheric pressure. H(2) formed was measured with a Shimadzu GC-8A thermal conductivity gas chromatograph equipped with a molecular sieve 5A 60/80 column. Where indicated, 200 µl of CO gas was added to the vials after they had been brought to atmospheric pressure. Both N(2) and proton reduction assays were performed for 30 min.

Purification of Apodinitrogenase 2

Cells (130 g of frozen cell paste) of strain CA117.30 were broken by osmotic shock using 0.025 M Tris-HCl containing 0.2 mM phenylmethylsulfonyl fluoride and 0.5 µg/ml leupeptin as described previously(23) . The cell-free extract was applied to a 4 times 23-cm DEAE-cellulose column that had been equilibrated using 1.5 liters of 0.1 M NaCl in 0.025 M Tris-HCl. All column chromatography procedures described except for the HPLC steps were performed at 4 °C. Following application of the extract, the column was washed with 2 bed volumes of the equilibration buffer; apodinitrogenase 2 was then eluted with 0.2 M NaCl in 0.025 M Tris-HCl. After the apodinitrogenase 2-containing fraction had been collected, the column was washed with 1 column volume of the same buffer. A second apodinitrogenase 2-containing fraction was then eluted using 0.3 M NaCl in 0.025 M Tris-HCl. We purified the apodinitrogenase 2 species that eluted with 0.2 M NaCl to near homogeneity; attempts to further purify the 0.3 M NaCl apodinitrogenase 2 fraction are currently underway. The 0.2 M NaCl apodinitrogenase 2-containing fraction from the DEAE-cellulose column was immediately applied to a 2.5 times 16-cm octyl-Sepharose column (equilibrated in 500 ml of 0.025 M Tris-HCl). The octyl-Sepharose column was washed with 2 bed volumes of 0.025 M Tris-HCl, followed by a wash with 1 bed volume of 40% ethylene glycol in 0.025 M Tris-HCl. Apodinitrogenase 2 was eluted with 70% ethylene glycol in 0.025 M Tris-HCl. The active fractions (approximately 50 ml) were concentrated on a 0.75 times 6-cm DEAE-cellulose column that was equilibrated with 0.1 M NaCl in 0.025 M Tris-HCl. Following application of the sample, the column was washed with 3 bed volumes of the equilibration buffer; apodinitrogenase 2 was eluted (as a light brown band) with buffer consisting of 0.3 M NaCl, 10% glycerol and 0.025 M Tris-HCl. A 25-30-fold concentration of the octyl-Sepharose fractions was typically achieved by this step. The final two steps in the purification of apodinitrogenase 2 employed a 0.75 times 7.5-cm TSK-phenyl column on an HPLC. The DEAE-cellulose fraction (1.8-2.0 ml) was applied to the TSK-phenyl column (equilibrated in buffer containing 0.3 M NaCl, 10% glycerol, and 0.025 M Tris-HCl), which was then washed with 2 bed volumes of the equilibration buffer. Two-ml fractions were collected, and apodinitrogenase 2 eluted with the wash. Although apodinitrogenase 2 did not bind to the TSK-phenyl column in NaCl (concentrations up to 2.0 M were tested), this step serves to remove certain contaminating proteins that could not be removed by a number of other columns tested on the HPLC. Ammonium sulfate was added to the apodinitrogenase 2-containing fraction to a final concentration of 1.0 M; this fraction was then reapplied to the TSK-phenyl column that was equilibrated in buffer containing 1.0 M (NH(4))(2)SO(4) in 0.025 M Tris-HCl. The column was washed with 1 bed volume of the equilibration buffer following which apodinitrogenase 2 was eluted with a 30-ml decreasing linear gradient from 1.0 M to 0 M (NH(4))(2)SO(4) (in 0.025 M Tris-HCl); 1-ml fractions were collected. Highly purified apodinitrogenase 2 eluted with 0.025 M Tris-HCl. Glycerol (containing 1.7 mM DTH) was added to the active fractions (to a final concentration of 20%), which were stored in 9-ml, serum-stoppered vials at -80 °C.

Protein Determination

Protein concentrations of cell-free extracts and all fractions in the purification of apodinitrogenase 2 were determined by the bicinchoninic acid method(27) . Protein concentrations of the highly pure apodinitrogenase 2-containing fractions were confirmed by the filter paper dye-binding assay for protein(28) .

Gel Electrophoresis

SDS gels were 10 or 15% acrylamide (0.89 and 0.39% bisacrylamide, respectively) with a 4.5% acrylamide (0.4% bisacrylamide) stacker; gels were stained with Coomassie Blue R-250 and/or silver. Two-dimensional native/SDS gel analyses of samples were performed as described previously(29) .

Sephacryl S-100 Chromatography

Two ml (54 mg of protein) of partially pure apodinitrogenase 2 (that had been concentrated on the 0.75 times 6-cm DEAE-cellulose column) were applied to a 1.5 times 80.0-cm Sephacryl S-100 column that had been equilibrated with 0.05 M NaCl in 0.025 M Tris-HCl. The column was developed with the same buffer at a flow rate of 18 ml/h. After 50 ml of buffer passed through the column, 4-ml fractions were collected. Glycerol (containing 1.7 mM DTH) was added to a final concentration of 20% to each fraction.

Molecular Mass Determination

A Superose 12 column (used in conjunction with an LKB fast protein liquid chromatography system) was calibrated with the following standards: catalase (240 kDa), aldolase (158 kDa), albumin (45 kDa), and chymotrypsinogen (25 kDa). The void volume of the column (V(0)) was determined to be 8.5 ml. Two-hundred µl (6.7 mg of protein) of partially pure apodinitrogenase 2 were applied to the Superose 12 column that had been equilibrated with 0.1 M NaCl in 0.025 M Tris-HCl. The column was developed with the same buffer, and apodinitrogenase 2 eluted between 8.2 and 8.4 ml (V(e)/V(0) was 0.96-0.99)


RESULTS AND DISCUSSION

The in Vivo Accumulation of Apodinitrogenase 2

An A. vinelandii strain containing deletions in the nifK, nifD, and nifB genes (CA117.30) was used for the purification of apodinitrogenase 2 because 1) lack of the nifB gene results in the accumulation of a cofactorless dinitrogenase protein because the product of the nifB gene is required for the synthesis of the cofactors of all three nitrogenases (3) and 2) the deletion in nifKD ensures that no apodinitrogenase 1 can accumulate under the specified growth conditions. The presence of apodinitrogenase 2 was monitored by C(2)H(2) reduction following its activation by FeV-co to form holodinitrogenase 2 (see ``Experimental Procedures''); an excess of dinitrogenase reductase 1 was included in the assays to quantitate the holodinitrogenase 2 formed. It has been shown previously that dinitrogenase reductase 1 serves as an electron donor to dinitrogenase 2 and that this hybrid complex functions effectively in substrate reduction(8) .

Conditions under which apodinitrogenase 2 was detected (by the FeV-co insertion assay) are summarized in Table 1. Apodinitrogenase 2 was detected in cell-free extracts of CA117.30 (DeltanifKDB) only under derepressing (nitrogen-fixing) conditions in media lacking molybdenum and containing 10 µM NaVO(3) (compare lines a-c, Table 1). Varying the length of derepression of strain CA117.30 showed that apodinitrogenase 2 levels were highest at approximately 4 h from the start of derepression (data not shown). Extracts of CA117.30 derepressed on NaVO(3) exhibited no cross-reactive material to antibodies prepared against dinitrogenase 1, indicating that no contaminating apodinitrogenase 1 was present (data not shown), and confirming a previous report that dinitrogenases 1 and 2 are immunologically distinct(8) . As predicted, extracts of an A. vinelandii strain deleted in the structural genes for dinitrogenase 2 (CA11.1(DeltanifHDKvnfDGK1::spc)) exhibited no activity in the FeV-co insertion assay (Table 1, line d). In cell-free extracts, apodinitrogenase 2 was inactivated by oxygen (upon exposure to air) and was also denatured by heat treatment at 60 °C (Table 1, lines e and f), the latter appears to be characteristic of the apoprotein, since holodinitrogenase 2 is stable under similar conditions(8) . (^2)



Purification of Apodinitrogenase 2

The purification scheme for apodinitrogenase 2 is outlined in Table 2. Highly purified apodinitrogenase 2 (approximately 94% in purity) exhibited a specific activity of 614 nmol C(2)H(4) formed/min/mg of protein following activation with FeV-co. A 384-fold purification and a final yield of 19% was obtained. The specific activities reported in Table 2represent one of three independent data sets collected for three reproducible purifications. The purity of apodinitrogenase 2 (determined by scanning densitometry of a Coomassie Blue R-250-stained SDS gel) was estimated at approximately 94% in the highly pure fraction (Fig. 1, lane 3).




Figure 1: SDS-PAGE of highly purified apodinitrogenase 2 (lanes 2 and 3). Lanes 1 and 2 were from a 10.5% gel, and lanes 3 and 4 were from a 15% gel, which allowed visualization of VNFG (the subunit). Approximately 14 µg of protein was loaded in lanes 2 and 3. Molecular weight standards (lanes 1 and 4) are indicated.



The 14-kDa protein (Fig. 1, lane 3) that copurified with the alpha and beta polypeptides (encoded by vnfD and vnfK, respectively) was identified as VNFG (the subunit encoded by vnfG) by a comparison of its N-terminal amino acid sequence (the 2nd to 12th amino acids were identified as SQSHLDDLFAY) with the predicted amino acid sequence for the protein product of vnfG(30) . Scanning densitometry of the highly pure apodinitrogenase 2 fraction (Fig. 1, lanes 2 and 3) showed that the VNFK, VNFD, and VNFG polypeptides are present in equimolar ratios. The molecular mass of apodinitrogenase 2, estimated by Superose 12 gel filtration chromatography, is consistent with a protein of approximately 200 kDa. These data indicate that apodinitrogenase 2 has an alpha(2)beta(2)(2) subunit composition.

In order to determine whether VNFG (the subunit) formed a complex with the alpha and/or beta subunits, a native/SDS two-dimensional gel analysis was performed. Highly pure apodinitrogenase 2 was first subjected to anaerobic native PAGE as described previously(29) ; a lane from the native gel that contained apodinitrogenase 2 was cut lengthwise, and the proteins were resolved (in the second dimension) by SDS-PAGE. The native/SDS two-dimensional gel (visualized both by Coomassie Blue R-250 and silver stain) revealed that VNFG (the subunit) was not in a complex with the alpha and beta polypeptides (data not shown). These observations, together with the fact that highly pure apodinitrogenase 2 is an alpha(2)beta(2)(2) species, suggests that some weak interactions (such as ionic and/or hydrogen bonding) exist between and the alpha and/or beta subunits and that these interactions are not stable under the native PAGE conditions. In this aspect, the physical properties of the apodinitrogenase 2 complex differ from those of the alpha(2)beta(2)(2) complex of apodinitrogenase 1 in which the subunits were dissociable from the alpha and beta subunits only upon treatment with 6 M urea (29) . Purified dinitrogenase 2 from A. vinelandii and A. chroococcum exhibit different subunit compositions. The A. chroococcum enzyme was purified as an alpha(2)beta(2)(2) hexamer(7) , while the A. vinelandii enzyme has an alpha(2)beta(2) subunit composition(8) . The purification of apodinitrogenase 2 from A. vinelandii provides a framework for understanding the function(s) of VNFG (the subunit) and the processing of the apoprotein to the holoenzyme.

Apodinitrogenase 2 in cell-free extracts of CA117.30 (DeltanifKDB) separated into two distinct fractions upon DEAE-cellulose chromatography, the first species eluted with 0.2 M NaCl (and was purified to near homogeneity) and the second with 0.3 M NaCl (Table 2). The difference between the two apodinitrogenase 2 species is most likely not a result of the presence or absence of VNFG (the subunit) because both DEAE-cellulose fractions retain their activity, and loss of VNFG was seen to result in a loss in activity in the FeV-co insertion assay (discussed below). We are currently examining chromatography methods that might be used to purify and characterize the 0.3 M NaCl apodinitrogenase 2 species.

Requirement of VNFG for Detection of Apodinitrogenase 2 (by the FeV-co Insertion Assay)

The results of the native/SDS two-dimensional PAGE analysis, which indicated that VNFG (the subunit) was not in a stable complex with the alpha and/or beta subunits, prompted us to examine the effect of separating VNFG from the alpha(2)beta(2) form (using gel filtration chromatography) on apodinitrogenase 2 activity (defined as the activity obtained upon addition of FeV-co to convert apodinitrogenase 2 to the holoenzyme, which is active in substrate reduction); the results of this experiment are summarized in Table 3. Chromatography of partially pure apodinitrogenase 2 (purified up to the first HPLC step) on a Sephacryl S-100 column resulted in an 8% recovery of activity, and the two fractions that exhibited apodinitrogenase 2 activity eluted at the void volume of the column (Table 3, lines a and b). These observations suggested that a low molecular weight protein necessary for apodinitrogenase 2 activity was being separated from proteins of 100 kDa or greater molecular weights (Fig. 2). We combined fractions that eluted at the void volume (Fig. 2, lanes 2 and 3, fractions 2 and 3) with seven succeeding fractions that had no apodinitrogenase 2 activity and found two fractions (Fig. 2, lanes 7 and 8, fractions 7 and 8) that stimulated the activity of fractions 2 and 3 by approximately 30-45-fold (Table 3, lines e-h).




Figure 2: SDS-PAGE of Sephacryl S-100 fractions 2-8 (lanes 2-8) and VNFG ( subunit)-containing HPLC fraction 5 (lane 9). The alpha and beta subunits in fractions 2 and 3 (lanes 2 and 3) are indicated by a and, VNFG ( subunit) in fractions 7-9 (lanes 7-9) is indicated by b. The 15% SDS gel does not allow optimal separation of the alpha and beta subunits. Molecular weight standards (lane 1) are indicated.



In order to establish that the stimulating factor was VNFG, a side fraction (from the final HPLC step in the purification of apodinitrogenase 2) containing VNFG (Fig. 2, lane 9) as the only detectable low molecular weight protein, was added to fractions 2 and 3. Addition of the VNFG-containing fraction (designated fraction 5 (HPLC) in Table 3) resulted in a 14-20-fold stimulation of activity of fractions 2 and 3 (Table 3, lines i and j), indicating that the low molecular weight protein involved in reconstitution of apodinitrogenase 2 activity (in the presence of FeV-co) was indeed VNFG (the subunit). The low levels of activity in fractions 2 and 3 (Table 3, lines a and b) can be attributed to the presence of some VNFG that was visible upon silver staining a SDS gel of the Sephacryl S-100 fractions (data not shown). Apodinitrogenase 2 activity of fractions 2 and 3 was dependent on the level of the VNFG-containing fraction added; the addition of increasing levels of fractions 7 and 8 (Fig. 2, lanes 7 and 8) to fractions 2 and 3, respectively, resulted in a linear increase in apodinitrogenase 2 activity of the latter fractions, indicating that VNFG was most likely the limiting component in the assay (Fig. 3). The ability of VNFG to stimulate apodinitrogenase 2 activity (in the presence of FeV-co) was observed to be both heat- and O(2)-labile (Table 3, lines k and l). The difference in the level of stimulation obtained by the addition of the Sephacryl S-100 fractions (fractions 7 and 8) and the HPLC fraction (fraction 5) to fractions 2 and 3 might result from a lower VNFG concentration in the HPLC fraction (the protein concentration of the HPLC fraction was approximately half that of the Sephacryl S-100 fractions).


Figure 3: Titration of Sephacryl S-100 fractions 2 and 3 with VNFG ( subunit)-containing fractions 7 and 8, respectively. The dotted line indicates the titration of fraction 2 with fraction 7, and the solid line indicates the titration of fraction 3 with fraction 8.



The stimulation of apodinitrogenase 2 activity (in the presence of FeV-co) was further characterized by the addition of various extracts/fractions to Sephacryl S-100 fractions 2 and 3; data obtained with only fraction 2 are shown, as similar results were obtained with fraction 3 (Table 3, lines m-p). That the addition of CA11.1 (DeltanifHDKDeltavnfDGK1::spc, derepressed on NaVO(3)) extract did not stimulate apodinitrogenase 2 activity (Table 3, line n) demonstrates that alpha(2)beta(2) apodinitrogenase 2 specifically requires VNFG as the stimulatory component in the in vitro assay system (as CA11.1 contains a deletion in vnfG) and that other vnf proteins do not function to restore apodinitrogenase 2 activity in vitro. Extracts of CA11.1 derepressed on W-containing medium (growth conditions allowing the expression of nif proteins) and NH(4)-grown UW (wild-type A. vinelandii) also failed to stimulate activity (Table 3, lines n and o), indicating that nif proteins and non-nif proteins (specifically the protein (a non-nif protein) which is required for the activation of apodinitrogenase 1 with FeMo-co (13) ) do not replace VNFG in vitro. The addition of partially purified (73% in purity, 0.04-0.31 mg total protein added) to fraction 2 had no effect on the activity of the fraction (Table 3, line p), indicating that although the partially pure protein (and present in extracts of CA11.1 and UW) was competent in binding FeMo-co(12) , it was unable to function in place of VNFG in vitro.

Separation of VNFG (the subunit) from the alpha(2)beta(2)(2) form of apodinitrogenase 2 also resulted in a loss of both H and N(2) reduction activities of Sephacryl S-100 fractions 2 and 3 (as detected by the FeV-co insertion assay). Both H and N(2) reduction activities were restored upon addition of VNFG-containing fractions to the FeV-co insertion assays (Table 4). These data indicate a requirement for VNFG (in addition to FeV-co) in the in vitro assay system.



In summary, the results discussed above indicate that VNFG (the subunit) can be dissociated (to a large extent) from the alpha(2)beta(2)(2) form of apodinitrogenase 2 by gel filtration chromatography. The separation of VNFG results in an inability to convert the alpha(2)beta(2) form of apodinitrogenase 2 (in the presence of FeV-co) to a form capable of C(2)H(2), H, and N(2) reduction; substrate reduction activities are restored upon addition of VNFG alone to the in vitro FeV-co insertion assays. The ability of VNFG to stimulate apodinitrogenase 2 activity was observed to be heat- and O(2)-sensitive. Various vnf, nif, and non-nif proteins (in cell-free extracts) failed to replace the function of VNFG in vitro. These results indicate a specific requirement for VNFG for the in vitro activation of apodinitrogenase 2 by FeV-co, and/or for substrate reduction by holodinitrogenase 2.

The dissociation of VNFG under the chromatography conditions of the Sephacryl S-100 column could be a result of the prolonged length of time that the apoprotein is exposed to the matrix in the absence of glycerol. It is interesting that in the purification of dinitrogenase 2 from A. vinelandii, Hales et al. (8) observed an irreversible loss in dinitrogenase 2 activity when Sephacryl S-200 chromatography was performed using a long column; whether this was due to the slow dissociation of VNFG under the experimental conditions remains unknown. It should be noted that in the final HPLC step in the purification of apodinitrogenase 2, fractions immediately preceding the highly pure apodinitrogenase 2-containing fractions contained significant levels of VNFG but did not exhibit activity when monitored using the FeV-co insertion assay (data not shown). This suggests that VNFG might be dissociating from a population of the alpha(2)beta(2)(2) species in the presence of 1.0 M (NH(4))(2)SO(4) (see ``Experimental Procedures'') or that a population of free VNFG might be copurifying with the alpha(2)beta(2)(2) species up to the last HPLC step, during which they separate. Addition of a VNFG-containing side fraction (from the final HPLC step) to the highly pure alpha(2)beta(2)(2)-containing fraction did not result in a stimulation in activity of the latter fraction, implying that VNFG was not limiting the activity of the highly pure alpha(2)beta(2)(2) fraction. We have recently demonstrated that VNFG cannot be dissociated from holodinitrogenase 2 under the Sephacryl S-100 chromatography conditions used to separate VNFG from apodinitrogenase 2, indicating that apodinitrogenase 2 is distinct from holodinitrogenase 2 with respect to the ability of VNFG to dissociate from the former.

There are at least two possible explanations for the requirement of VNFG for in vitro apodinitrogenase 2 activity. Analogous to the role of the protein in the activation of apodinitrogenase 1, VNFG might be required for the activation process whereby apodinitrogenase 2 is converted to holodinitrogenase 2 in the presence of FeV-co. Alternatively, VNFG might play a role in substrate reduction by the newly formed holodinitrogenase 2. Recently, Waugh et al. (11) noted that a vnfG strain was unable to grow diazotrophically but that cell-free extracts of the strain exhibited some C(2)H(2) reduction activity; our results indicate a loss in C(2)H(2), H, and N(2) reduction activity in fractions lacking VNFG, implying that VNFG might be involved in the activation of apodinitrogenase 2 by FeV-co in vitro. A direct comparison, however, cannot be made between our results and those of Waugh et al., as our studies were performed on an in vitro system. It is interesting that while DeltanifK and/or nifD strains synthesize and accumulate FeMo-co in vivo on a 65-kDa protein(31, 32) , strain CA11.1 (DeltanifHDKDeltavnfDGK1::spc) did not accumulate FeV-co in vivo when derepressed on NaVO(3) (data not shown). It is possible that the in vivo accumulation of FeV-co requires the presence of VNFG; however, the possibility that FeV-co synthesis does not occur in the absence of the structural polypeptides for dinitrogenase 2 cannot be excluded. Future studies are aimed at examining the interactions of VNFG (the protein) with FeV-co and the alpha(2)beta(2) form of apodinitrogenase 2 in order to investigate the role of VNFG in the processing of apodinitrogenase 2 to the holoenzyme.

Substrate Reduction Patterns of FeV-co- and FeMo-co-activated Apodinitrogenase 2

In addition to being activable in vitro by FeV-co, apodinitrogenase 2 was also activable by FeMo-co. The substrate reduction properties of the FeV-co- and FeMo-co-activated proteins are compared in Table 5. As predicted, purified apodinitrogenase 2 (activated with FeV-co) reduced C(2)H(2) (to C(2)H(4) and C(2)H(6)), H, and N(2). As observed with holodinitrogenase 2(33) , C(2)H(2) reduction by FeV-co-activated apodinitrogenase 2 was inhibited by CO (71% inhibition of activity was observed in the presence of 2.5% CO), whereas H reduction remained unaffected. The addition of FeV-co to apodinitrogenase 1 in cell-free extracts of UW45 (derepressed on molybdenum) resulted in the formation of a hybrid enzyme that reduced C(2)H(2) but not N(2), confirming the studies of Smith et al.(19) , who observed a lack of N(2) reduction activity when apodinitrogenase 1 from Klebsiella pneumoniae was activated with FeV-co.



The activation of apodinitrogenase 2 by FeMo-co resulted in a hybrid enzyme with properties distinct from both dinitrogenase 1 and dinitrogenase 2. FeMo-co-activated apodinitrogenase 2 was unable to reduce N(2), and C(2)H(2) reduction by the hybrid enzyme was resistant to inhibition by CO under conditions where C(2)H(2) reduction by FeMo-co-activated apodinitrogenase 1 was inhibited by 98%. The C(2)H(2) and H reduction activities of FeMo-co-activated apodinitrogenase 2 were approximately 6- and 1.5-fold lower, respectively, than that of the FeV-co-activated protein. The lower activities of FeMo-co-activated apodinitrogenase 2 might result from the inefficient activation of the apoprotein by FeMo-co. The low activities are probably not due to FeMo-co being a limiting component of the assay, as apodinitrogenase 1 (in cell-free extracts of UW45) was activated to 20.3 nmol C(2)H(4) formed per min per assay by the level of FeMo-co added. Our results are in disagreement with those of Moore et al. (33) who reported that FeMo-co-activated apodinitrogenase 2 reduced N(2) as effectively as dinitrogenase 1 and that the hybrid enzyme retained CO sensitivity of C(2)H(2) reduction. C(2)H(2) reduction by FeMo-co-activated apodinitrogenase 2 was characterized by the formation of C(2)H(6) as a minor product; the C(2)H(6):C(2)H(4) ratio of the hybrid enzyme was increased (0.21) compared with the FeV-co-activated protein (0.08). Pau et al. (10) noted C(2)H(2) reduction to both C(2)H(4) and C(2)H(6) in a hybrid form of dinitrogenase 3 that contained FeMo-co at its active site; Gollan et al. (34) independently observed an increase in the C(2)H(6):C(2)H(4) ratio of dinitrogenase 3 from Rhodobacter capsulatus upon the in vivo incorporation of FeMo-co into the anf1 polypeptides. In both cases it was unclear whether the formation of C(2)H(6) arose from FeMo-co-containing dinitrogenase 3 or from a mixed population of dinitrogenase 3 containing both FeMo-co and FeFe-co.

The role of the cofactor in specifying substrate reduction properties and inhibitor susceptibilities was observed in dinitrogenase 1 when homocitrate (an integral component of FeMo-co(16) ) was substituted with its analogs. Incorporation of threo-fluorohomocitrate resulted in a complete loss of N(2) reduction while C(2)H(2) and H reduction activities were retained(17) , substitution of homoisocitrate and 2-oxoglutarate resulted in a loss of both C(2)H(2) and N(2) reduction(16) . Altering the structure of FeMo-co by the incorporation of homocitrate analogs did not result in CO-resistance of C(2)H(2) reduction(17) , indicating that the CO-resistance of C(2)H(2) reduction exhibited by FeMo-co-activated apodinitrogenase 2 is a result of altered interactions between residues at the cofactor binding site of apodinitrogenase 2 and FeMo-co. That the polypeptide also dictates substrate specificity of nitrogenase 1 was demonstrated by the substitution of glutamine 191 in the alpha polypeptide of dinitrogenase 1 by lysine(18) ; the mutant strain was unable to grow diazotrophically, and cell-free extracts reduced C(2)H(2) to both C(2)H(4) and C(2)H(6), the latter being a property of the vnf- and anf-encoded nitrogenases ((14) ).^1 More recently, Kim et al. (35) have shown that substitution of alpha histidine 195 (of dinitrogenase 1) with glutamine results in an altered enzyme that binds but does not reduce N(2) and that exhibits hypersensitivity to CO inhibition of C(2)H(2) reduction.

The altered substrate reduction properties conferred by FeMo-co activation of apodinitrogenase 2 are possibly due to the inability of FeMo-co to properly contact the protein ligands necessary for optimal reduction of all three substrates. An altered orientation of the cofactor in the binding site could explain the lowered C(2)H(2) and H reduction and also the CO-resistance of C(2)H(2) reduction, which might arise from a decreased accessibility of CO to its binding site. Although the two amino acid residues implicated in covalently binding FeMo-co (cysteine 275 and histidine 442) are conserved in all three dinitrogenase proteins(36, 30) , specific amino acid-cofactor interactions unique to each dinitrogenase protein are probably required for optimal substrate reduction, the ligations necessary for N(2) reduction being the most stringent.

Substrate reduction by apodinitrogenase 2 activated with the putative iron-only cofactor (FeFe-co) was dependent on the source of dinitrogenase 3 used in the preparation of the cofactor. Partially pure dinitrogenase 3 (previously characterized(9) ) from A. vinelandii strain CA11.6 (W-tolerant, DeltanifHDK) was initially used in the preparation of FeFe-co. Acid-denatured dinitrogenase 3 (from CA11.6) preparations activated apodinitrogenase 2, forming an enzyme that exhibited C(2)H(2), H, and N(2) reduction activities, albeit 4-5-fold lower than FeV-co-activated apodinitrogenase 2 (data not shown). In order to confirm these results, we used partially purified dinitrogenase 3 from A. vinelandii strain CA11.1 (DeltanifHDKDeltavnfDGK1::spc) as a source of cofactor. It should be noted that although strains CA11.6 and CA11.1 were derepressed under identical conditions, the specific activity of dinitrogenase 3 from CA11.1 was repeatedly observed to be 8-10-fold lower than dinitrogenase 3 from CA11.6 (see ``Experimental Procedures''). No C(2)H(2), H, or N(2) reduction was detected upon addition of acid-denatured dinitrogenase 3 (from CA11.1) to apodinitrogenase 2. It is possible that the cofactor from CA11.1 dinitrogenase 3 activated apodinitrogenase 2, but the hybrid enzyme was unable to function in C(2)H(2), H, and N(2) reduction; however, the possibility that the cofactor failed to activate apodinitrogenase 2 also exists. The activities observed by activation of apodinitrogenase 2 by the cofactor from CA11.6 dinitrogenase 3 might be attributed to low levels of a molybdenum- or vanadium-containing cofactor. Establishing the substrate reduction pattern of FeFe-co-activated apodinitrogenase 2 awaits a more thorough characterization of the cofactors of dinitrogenase 3 from A. vinelandii strains CA11.6 and CA11.1.


CONCLUSIONS

In this study, we have shown that highly purified apodinitrogenase 2 from A. vinelandii is an alpha(2)beta(2)(2) protein and that the subunit (VNFG) is specifically required for the processing of apodinitrogenase 2 (in the presence of FeV-co) to a form capable of C(2)H(2), H, and N(2) reduction. The function(s) of VNFG might be elucidated by further characterization of apodinitrogenase 2 and its processing to the holoenzyme. We have also demonstrated that apodinitrogenase 2 is activable in vitro by FeMo-co to form a hybrid enzyme with unique substrate reduction properties, most notably a loss of N(2) reduction and insensitivity of C(2)H(2) reduction to inhibition by CO. The formation of hybrid dinitrogenase enzymes (both in vivo and in vitro) indicates a lack of specificity with respect to the activation of the apodinitrogenase proteins by a particular cofactor. Specificity does occur, however, at the level of cofactor biosynthesis(25) . A study of the ability of certain nif and vnf gene products involved in cofactor biosynthesis to function in the in vitro synthesis of FeMo-co and FeV-co might provide insight into the component(s) specifying the heterometal that is incorporated into the dinitrogenase proteins.


FOOTNOTES

*
This research was supported by U.S. Department of Agriculture Grant 93-373-9234. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 608-262-6859; Fax: 608-262-3453; ludden{at}biochem.wisc.edu

Supported by National Institutes of Health Grant GM35332 (to P. W. L.).

(^1)
The abbreviations used are: FeV-co, iron-vanadium cofactor; FeMo-co, iron-molybdenum cofactor; FeFe-co, iron-iron cofactor; HPLC, high pressure liquid chromatography; DTH, sodium dithionite; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. Chatterjee, R. M. Allen, P. W. Ludden, and V. K. Shah, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Paul Bishop and R. Premakumar for the construction of A. vinelandii strain CA117.30. We thank Robert Burris for performing the N(2) reduction assays and for reviewing this manuscript. Mary Homer is acknowledged for providing partially purified protein.


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