(Received for publication, August 30, 1996, and in revised form, November 13, 1996)
From the Department of Biochemistry and Center for the Study of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
The vnf-encoded apodinitrogenase
(apodinitrogenase 2) from Azotobacter vinelandii is an
2
2
2 hexamer. The
subunit (the VNFG protein) has been characterized in order to further
delineate its function in the nitrogenase 2 enzyme system. Two species
of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG associated with the VNFDK polypeptides, and the other is a homodimer of the VNFG protein. Both species of VNFG
are observed in extracts of A. vinelandii strains that accumulate dinitrogenase 2, whereas extracts of strains impaired in the
biosynthetic pathway of the iron-vanadium cofactor (FeV-co) that
accumulate apodinitrogenase 2 (a catalytically inactive form of
dinitrogenase 2 that lacks FeV-co) exhibit only the VNFG dimer on
native gels. FeV-co and nucleotide are required for the stable association of VNFG with the VNFDK polypeptides; this stable
association can be correlated with the formation of active
dinitrogenase 2. The iron-molybdenum cofactor was unable to replace
FeV-co in promoting the stable association of VNFG with VNFDK. FeV-co
specifically associates with the VNFG dimer in vitro to
form a complex of unknown stoichiometry; combination of this
VNFG-FeV-co species with apodinitrogenase 2 results in its
reconstitution to dinitrogenase 2. The results presented here suggest
that VNFG is required for processing apodinitrogenase 2 to functional
dinitrogenase 2.
The biological reduction of atmospheric N2 to NH4+ occurs via any one of three genetically distinct nitrogenase enzymes in the aerobe Azotobacter vinelandii (for reviews, see Refs. 1 and 2). Expression of the three nitrogenase enzymes, designated nitrogenase 1, 2, and 3, is regulated by the metal content of the growth medium (3). Nitrogenase 1, a molybdenum (Mo)-containing enzyme, is synthesized in medium containing Mo, while nitrogenases 2 and 3 are repressed by molybdenum. Nitrogenase 2, a vanadium (V)-containing enzyme, is expressed in the absence of molybdenum in medium containing vanadium, expression of nitrogenase 3 (containing only iron) requires medium depleted in both molybdenum and vanadium. Nitrogenases 1, 2, and 3 are encoded by the nif, vnf, and anf operons, respectively; certain nif gene products are necessary for the function of the molybdenum-independent nitrogenases (4-6).
All three nitrogenase enzymes are oxygen-labile iron-sulfur proteins comprising two components, dinitrogenase and dinitrogenase reductase. Dinitrogenase contains the active site cofactor of the enzyme, and dinitrogenase reductase serves as the obligate electron donor to dinitrogenase during catalysis in a MgATP- and reductant-dependent process (7, 8). In addition to reducing N2, all three nitrogenase enzymes reduce C2H2 and H+ (9).
The component proteins of nitrogenase 2 have been purified from both A. vinelandii and Azotobacter chroococcum (10-14). Dinitrogenase 2 contains an iron-vanadium cofactor (FeV-co)1 similar to the iron-molybdenum cofactor (FeMo-co) of nitrogenase 1 at the active site (15), and P clusters that are spectroscopically identical to those of dinitrogenase 1 (16). Although the biosynthetic pathway of FeMo-co and the processing of the component proteins of nitrogenase 1 to catalytically active forms are well documented (for review, see Ref. 17), steps in the biosynthesis of the cofactors of nitrogenases 2 and 3 and in the maturation of their component proteins remain uncharacterized.
Certain features of the Mo-independent nitrogenases distinguish them
from nitrogenase 1. The formation of C2H6 (in
addition to C2H4) as a minor product of
C2H2 reduction is a characteristic of
nitrogenase 2 and 3; in fact, C2H6 formation is
often taken as indicative of the presence of a Mo-independent
nitrogenase (18). Most relevant to this investigation, dinitrogenases 2 and 3 contain an additional subunit, , encoded by vnfG
and anfG, respectively (19-21), compared to the
2
2 structure of the
nif-encoded dinitrogenase 1. Kim and Rees (22) suggested
that the
subunits might be involved in stabilization of the
quaternary structures of dinitrogenases 2 and 3; subsequent studies,
however, illustrated more intriguing functions for the
subunits of
dinitrogenase 2 and 3. Waugh et al. (23) demonstrated that
VNFG and ANFG were required for diazotrophic growth by A. vinelandii but not for C2H2 reduction.
Recently, VNFG was shown to be required to convert apodinitrogenase 2 (in the presence of FeV-co) to a form capable of substrate reduction
(24); the requirement for VNFG could occur in the processing of
apodinitrogenase 2 to dinitrogenase 2, or in catalysis by the newly
formed holodinitrogenase 2 (24). The role of VNFG and ANFG might be
analogous to the function of the
protein in processing
nif-encoded apodinitrogenase 1 (dinitrogenase 1 that does
not contain FeMo-co) to dinitrogenase 1. The form of apodinitrogenase 1 that is activable by FeMo-co has an
2
2
2 composition (25); the
protein, a non-nif-encoded gene product, has been shown
to specifically associate with FeMo-co and perhaps function as a
chaperone-insertase in the maturation of apodinitrogenase 1 to
dinitrogenase 1 (26).
In this report, we describe the characterization of VNFG (the subunit of apodinitrogenase 2) and present results that indicate a role
for VNFG in the processing of apodinitrogenase 2 to dinitrogenase 2.
All reagents used for A. vinelandii growth media were of analytical grade or higher purity. Sodium metavanadate (NaVO3, 99.995% in purity), Tris base, and glycine were purchased from Fisher. Sodium dithionite (DTH) was from Fluka Chemicals. DEAE-cellulose was a Whatman DE-52 product. Octyl-Sepharose CL-4B, Sephadex G-25, and the Superose 12 gel filtration column were from Pharmacia Biotech, Inc. Nitrocellulose membrane and acrylamide/bis solution were from Bio-Rad. Citric acid was obtained from Mallinckrodt. All other chemicals were from Sigma. The fast protein liquid chromatography system was a LKB instrument.
A. vinelandii Strains and Growth ConditionsA.
vinelandii strains UW45 (nifB; Ref. 27), CA12
(
nifHDK; Ref. 28), CA11.1
(
nifHDKvnfDGK1::spc; Ref. 23), CA119
(vnfG, Cys-17
stop; Ref. 23), CA117.30
(
nifDKB; Ref. 24), DJ42.48 (
nifENXvnfE;
Ref. 29), CA11.80 (
nifHDKvnfH; Ref. 19), and CA11.6.82
(W-tolerant,
vnfD82::Tn5-B21
nifHDKrifr;
Ref. 30) have been described. 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 CA12, CA11.1, CA119, CA117.30, DJ42.48,
CA11.80, and CA11.6.82 were grown in Burk's medium that lacked sodium
molybdate and contained 10 µM NaVO3 for
derepression of the vnf system. Cultures of CA12, CA119, and
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
depletion of ammonium, following which they were derepressed for 4 h. The cells were concentrated using a Pellicon cassette system
equipped with a filtration membrane (0.45 µM, Millipore
Corp., Bedford, MA) and were centrifuged. The cell pellets were frozen
in liquid N2 and stored at
80 °C. Strains CA11.1,
DJ42.48, CA11.80, and CA11.6.82 were grown in 250-ml cultures; cells
were concentrated by centrifugation at 10,000 rpm for 10 min, and
derepression was initiated for 4 h by suspending the cell pellets
in nitrogen-free Burk's medium. Cells were harvested by centrifugation
and were frozen as described above.
Strain UW45 was grown and derepressed on tungsten-containing medium (molybdenum-free) as described previously (31). For derepression of the nif system, strains were grown on Mo-containing medium as described previously (32). Cell-free extracts of all A. vinelandii strains were prepared by the osmotic shock method (32). When necessary, small molecules were removed from the extracts by Sephadex G-25 gel-filtration chromatography.
Buffer PreparationAll buffers were sparged with purified N2 (and degassed on a gassing manifold where appropriate) for 10-30 min, and 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 fast protein liquid chromatography were filtered through a 0.45 µM filter. Tris-HCl used was at pH 7.4 unless indicated otherwise.
ElectrophoresisThe procedure for SDS-PAGE has been described (33); all SDS gels were 15% acrylamide (0.39% bisacrylamide). Proteins being analyzed by anoxic native gel electrophoresis were resolved on 7-14% acrylamide (37.5:1, acrylamide:bisacrylamide) and 0-20% sucrose gradient gels at 4 °C; 65 mM Tris-glycine (pH 8.5) sparged with N2 and containing 1.7 mM DTH was used as the electrophoresis buffer. Gels were prerun for at least 60 min at 120 V for initial reduction, and proteins were electrophoresed for approximately 1900 V × h (at 120 V). Unless indicated otherwise, all samples applied onto the gel contained 8% glycerol.
Antibodies and Immunoblot AnalysisVNFG (the protein)
was separated from the
2
2
2
apodinitrogenase 2 hexamer by SDS-PAGE. The pure VNFG protein was
extracted from the gel and injected into rabbits to produce polyclonal
antibodies at the Animal Care Unit of the University of
Wisconsin-Madison Medical School. Antibodies to VNFDK (the
and
polypeptides of dinitrogenase 2) were produced similarly. Immunoblot
analysis was performed as described by Brandner et al.
(34).
Dinitrogenase 2 was partially purified from A. vinelandii strain CA119 (vnfG, Cys-17 stop) by
DEAE-cellulose chromatography, followed by heat treatment as described
by Hales et al. (11). Partially purified dinitrogenase 2 was
concentrated by ultrafiltration using a XM100-A membrane. Partial
purification of apodinitrogenase 2 from A. vinelandii strain
CA117.30 (
nifDKB) was performed through the
octyl-Sepharose chromatography step as described previously (24).
A preparation of FeV-co
was made by the citric acid treatment of partially purified
dinitrogenase 2 with modifications to the method described previously
(24). In a typical reaction, 0.4 ml of a solution of 0.4 M
citric acid (made anoxic by diluting a 2.0 M stock solution
into N2-sparged water containing 1.7 mM DTH)
was added to 2 ml (31.2 mg of protein) of dinitrogenase 2 (partially
purified from A. vinelandii strain CA119 (vnfG,
Cys-17 stop) in a screw-capped centrifuge tube (Beckman) fitted
with rubber-stoppered inserts to allow evacuation and flushing on a gassing manifold. The solution was mixed thoroughly using a Vortex mixer and incubated on ice for 10 min, following which it was centrifuged at 10,000 rpm for 5 min. The supernatant was discarded, and
the pellet was suspended in 4 ml of 0.025 M Tris-HCl. This suspension of acid-denatured dinitrogenase 2 was centrifuged at 10,000 rpm for 20 min, and the supernatant (hereafter referred to as FeV-co)
was used as a source of FeV-co in the in vitro activation of
apodinitrogenase 2 by the cofactor. Purified FeMo-co was prepared by
the method of Shah and Brill (32); when it was necessary to avoid any
denaturing effects of N-methylformamide (NMF), FeMo-co was
prepared by the citric acid treatment of dinitrogenase 1 as described
by Allen et al. (35).
The assay consists of two steps; in the first step, apodinitrogenase 2 is activated by FeV-co to form holodinitrogenase 2, and in the second step, the C2H2 reduction activity of the newly formed holodinitrogenase 2 is monitored. Reactions to be analyzed by native PAGE were only taken through the first phase of the assay (FeV-co insertion). Nine-ml rubber-stoppered serum vials were repeatedly evacuated and flushed with argon and rinsed with 0.3 ml of 0.025 M Tris-HCl (containing 1.7 mM DTH). The following components were added to the vials in the order indicated: 200 µl (4 mg of protein) of the appropriate cell-free extract or partially purified apodinitrogenase 2 (0.2 mg of protein), 200 µl of an anoxic solution of 50% glycerol (containing 1.7 mM DTH), 200 µl of a solution of FeV-co, and 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 in 0.025 M Tris-HCl, pH 8.0). The reaction-mixtures were incubated at room temperature for 20 min, and samples to be applied onto the native gels were placed on ice. The activity of the newly formed dinitrogenase 2 in the remaining vials was determined by the C2H2 reduction assay for nitrogenase (31) as follows: 800 µl of the ATP-regenerating mixture and 10 µl (0.1 mg of protein) of dinitrogenase reductase 1 were added to the vials, which were then brought to atmospheric pressure. C2H2 reduction was initiated by the addition of 0.5 ml of C2H2 to the vials; following a 30-min incubation at 30 °C in a rotary water-bath shaker, the reactions were terminated by the addition of 0.1 ml of 4 N NaOH.
When testing the requirement for a particular component in the FeV-co insertion assay, the given component was excluded and an equal volume of 0.025 M Tris-HCl was added. FeMo-co was added to the reactions (in place of FeV-co) as 40 µl of purified FeMo-co in NMF, or as 50 µl of acid-denatured dinitrogenase 1. Certain reactions contained FeV-co that had been air-oxidized. Reactions testing the requirement for dinitrogenase reductase contained either 40 µl (0.4 mg of protein) of purified dinitrogenase reductase 1, or 40 µl (0.1 mg of protein) of partially purified dinitrogenase reductase 2. Nucleotide (other than ATP) solutions contained the following: 15 mM XTP (or XDP), 30 mM MgCl2, and 6.3 mM DTH in 0.025 M Tris-HCl, pH 8.0; 200 µl of the appropriate nucleotide solution was added to certain reaction-mixtures in place of the ATP-regenerating solution. The final XTP (or XDP) concentration (other than ATP, which was added in the form of the ATP-regenerating mixture) in the FeV-co insertion reactions was 3.75 mM. When testing various cell-free extracts as a source of FeV-co, 200 µl of the appropriate extract (~3.6 mg of protein) was added to the reactions in place of acid-denatured dinitrogenase 2.
Molecular Mass DeterminationA Superose 12 column
(Vo = 8.5 ml) used in conjunction with a LKB fast
protein liquid chromatography system, was calibrated using bovine serum
albumin (67 kDa), ovalbumin (45 kDa), myoglobin (18 kDa), and
cytochrome c (12.5 kDa). Two hundred µl of partially
purified apodinitrogenase 2 (6.7 mg of protein) were applied to the
Superose 12 column that had been equilibrated in buffer containing 0.1 M NaCl in 0.025 M Tris-HCl. The column was
developed with the same buffer, and fractions (0.5 ml), collected anoxically, were monitored for VNFG (the protein) by SDS-PAGE and
anoxic native PAGE.
Protein concentrations of cell-free extracts and partially purified protein fractions were determined by the bicinchoninic acid method (36).
Association of FeV-co with VNFG (theThe interaction between FeV-co (in the form of acid-denatured dinitrogenase 2) and VNFG was monitored using the Superose 12 gel-filtration column (used in conjunction with a LKB fast protein liquid chromatography system). Two hundred µl of a solution of FeV-co were applied to the Superose 12 column (equilibrated in buffer containing 0.1 M NaCl in 0.025 M Tris-HCl); the column was eluted with the same buffer, and fractions (0.5 ml, collected anoxically) were tested for FeV-co using the FeV-co insertion assay. Two hundred µl (0.8 mg of protein) of a fraction containing VNFG that contained no VNFDK or FeV-co (and therefore exhibited no activity in the FeV-co insertion assay) were chromatographed on the Superose 12 column (equilibrated and eluted as described above), and 0.5-ml fractions (collected anoxically) were monitored for VNFG by SDS-PAGE. When examining the interaction between FeV-co and VNFG, 200 µl of a solution of FeV-co was incubated with 200 µl of the VNFG-containing fraction for 20 min at room temperature, and 200 µl of the reaction mixture were applied to the Superose 12 column (equilibrated as described above). The column was developed, and fractions were collected as described above; the fractions were monitored for both FeV-co and VNFG by the FeV-co insertion assay and by SDS-PAGE, respectively.
In a control experiment, 200 µl of a solution of FeV-co were
incubated with 200 µl (2.8 mg of protein) of cell-free extract of
NH4+-grown UW45
(nifB, conditions under which all three
nitrogen fixing systems are repressed), and 200 µl of the reaction
mixture were chromatographed as described above. Fractions were
monitored for FeV-co using the FeV-co insertion assay. In a second
control experiment, 200 µl of a solution of FeMo-co in NMF were
chromatographed on the Superose 12 column both in the presence and
absence of VNFG as described above; fractions were monitored for
FeMo-co using the FeMo-co insertion assay (32) and for VNFG by
SDS-PAGE.
The
presence of VNFG was monitored in several A. vinelandii
mutant strains by immunoblot analysis of cell-free extracts resolved by
SDS-PAGE; Fig. 1 is an immunoblot of a SDS gel developed
with antibody to VNFG. A. vinelandii strains CA12
(nifHDK), CA117.30 (
nifDKB), and DJ42.48
(
nifENXvnfE) accumulated VNFG when the strains were grown
and derepressed on V-containing medium (Fig. 1, lanes
1, 2, and 4). As reported previously, VNFG
is a component of the apodinitrogenase 2 complex (33), and was detected
in a purified sample of apodinitrogenase 2 (Fig. 1, lane 8).
No VNFG was detected in extract of strain CA117.30 grown and
derepressed on molybdenum-containing medium (Fig. 1, lane
5). As expected, strains CA11.1
(
nifHDKvnfDGK1::spc) and CA119
(vnfG, Cys-17
stop) did not accumulate VNFG (Fig. 1,
lanes 3 and 6); these data also show that the
antibodies to VNFG did not cross-react with VNFDK (present in extract
of strain CA119; Ref. 32).
Interestingly, a low molecular weight protein in a partially purified
fraction of the anf-encoded dinitrogenase 3 (purified from
CA11.1 grown and derepressed on Mo- and V-deficient medium) was also
detected with antibody to VNFG (Fig. 1, lane 7). The cross-reactive band seen in lane 7 was not VNFG since
dinitrogenase 3 was partially purified from a strain containing a
deletion in the vnfG gene (Fig. 1, lane 3); the
low molecular weight protein detected was most likely ANFG, the subunit of dinitrogenase 3. Given the 39.4% sequence identity at the
amino acid level between ANFG and VNFG (1), it is not completely
surprising that the antibody to VNFG is cross-reactive toward ANFG.
The forms of VNFG found in cell-free extracts were examined
by anoxic native PAGE. Fig. 2A is an
immunoblot of an anoxic native gel developed with antibody to VNFG, and
shows the VNFG forms present in various extracts. For simplicity, the
forms of VNFG, described below, are defined as the slow-migrating
species A, and the fast-migrating species B (Fig. 2A).
Species A consists of VNFG, which will be shown to be tightly
associated with the VNFDK polypeptides; species B was determined to be
a homodimer of VNFG. Accumulation of dinitrogenase 2 and
apodinitrogenase 2 in the various extracts was monitored by the
C2H2 reduction assay and the FeV-co insertion
assay, respectively (Table I).
|
Strain CA12 (nifHDK) accumulated both VNFG species A and
B (Fig. 2A, lane 1) and exhibited dinitrogenase 2 activity (Table I); no apodinitrogenase 2 was detected in the extracts
by the FeV-co insertion assay; however, it is possible that a low level of apodinitrogenase 2 is present but cannot be estimated due to the
high background activity of dinitrogenase 2 in the strain. The VNFDK
polypeptides were shown to migrate to the position of VNFG species A by
immunoblot analysis (using antibody to VNFDK) of extracts resolved by
native PAGE (Fig. 2B). The antibodies to VNFDK did not
cross-react with NIFDK (Fig. 2B, lane 6). Thus, VNFG species A in extract of CA12 constitutes VNFG associated with the
VNFDK polypeptides via interactions that are not disrupted under
conditions of the native PAGE procedure. The three forms of VNFG
observed in species A (Fig. 2A, lane 1) most
likely differ from each other in the stoichiometry of the VNFD, -G, and
-K polypeptides in each form. Recently, Blanchard et al.
reported the purification of two forms of dinitrogenase 2 from A. vinelandii: an
2
2 form and an
2 form; the
subunit was observed to be associated
with both forms but was not quantitated (12).
Extracts of strains CA117.30 (nifDKB), CA11.80
(
nifHDKvnfH), and DJ42.48 (
nifENXvnfE)
exhibited VNFG species B only (Fig. 2A, lanes
3, 5, and 6) despite the presence of the
VNFDK polypeptides in all three strains (Fig. 2B,
lanes 3-5). All three strains exhibited apodinitrogenase 2 activity (as determined by the FeV-co insertion assay) but no
dinitrogenase 2 activity (Table I). Therefore, a strain that
accumulates dinitrogenase 2 contains both VNFG species A and B, whereas
strains impaired in FeV-co biosynthesis that accumulate
apodinitrogenase 2 exhibit only VNFG species B. Species B might result
from the dissociation of VNFG from the
2
2
2 apodinitrogenase 2 hexamer under the native PAGE and gel-filtration chromatography conditions; however, the possibility that a population of species B is
not part of the apodinitrogenase 2 hexamer cannot be excluded. Species
B observed in extract of CA12 might be due to the presence of low
levels of apodinitrogenase 2. VNFG did not accumulate in strains CA11.1
(
nifHDKvnfDGK1::spc) and CA119
(vnfG, Cys-17
stop), which contain lesions in
vnfG (Fig. 2A, lanes 2 and
7).
Previously, we had determined that VNFG did not comigrate with VNFDK as
a complex when purified apodinitrogenase 2 (2
2
2) was analyzed by
anoxic native PAGE (24); it was also demonstrated that VNFG could be
dissociated from VNFDK when apodinitrogenase 2 was chromatographed on a
gel-filtration column (24). Thus, although the VNFDK and VNFG subunits
copurified in a 1:1:1 ratio, suggesting (together with molecular mass
analysis) that apodinitrogenase 2 is an
2
2
2 hexamer (24), the weak
interactions between VNFG (the
subunits) and VNFDK results in the
dissociation of VNFG from the apodinitrogenase 2 hexamer under
conditions of native PAGE and gel-filtration chromatography. In order
to characterize VNFG species B, and to determine whether this species
resulted from dissociation of VNFG from VNFDK, partially purified
apodinitrogenase 2 (
2
2
2)
was analyzed by gel-filtration chromatography using a Superose 12 column (see "Experimental Procedures"). VNFG reproducibly exhibited
an elution profile consistent with a molecular mass of ~26.3 kDa,
which is approximately twice the predicted mass (13.8 kDa) for the
vnfG gene product (19). Calibration of the Superose 12 column (see "Experimental Procedures") and the elution profile of
VNFG from the Superose 12 column are indicated in Fig. 3A. VNFG in the Superose 12 fractions
comigrated with VNFG species B in extract of strain CA12
(
nifHDK, vnf-derepressed) on an anoxic native gel (Fig.
3B, lanes 1-3). Thus, VNFG exists as a homodimer when it is not associated with the VNFDK polypeptides. VNFG in a sample
of purified apodinitrogenase 2 (
2
2
2) coelectrophoresed to
the position of VNFG species B in cell-free extract of CA12 (Fig. 3,
lanes 1 and 4), indicating that VNFG species B in
cell-free extracts of the strains tested is a dimer that is not tightly associated with any other protein(s).
Although apodinitrogenase 2 was purified as an
2
2
2 hexamer (24), the
interactions between VNFG (
) and VNFDK (
) appear to be readily
disrupted under conditions of gel-filtration chromatography and native
PAGE (24). In this respect, the physical properties of the
2
2
2 apodinitrogenase 2 complex are quite distinct from those of the
2
2
2 form
ofnif-encoded apodinitrogenase 1, since the
protein
comigrates with the
and
subunits on native PAGE, and is
dissociable only upon treatment of the
2
2
2 complex with reagents
such as urea (37). Dinitrogenase 1 and 2 also differ from each other
with respect to the affinities of the
protein and VNFG for
dinitrogenase 1 and 2, respectively. In cell-free extracts, the
protein dissociates from the
2
2
2 apodinitrogenase 1 complex upon activation with FeMo-co (26), whereas the
subunits (VNFG) became tightly associated with the
and
subunits of dinitrogenase 2 in the presence of FeV-co (Fig. 2A,
lane 1; results discussed below).
The in vitro FeV-co insertion assay system
together with anoxic native PAGE was employed to investigate conditions
under which VNFG species B would associate stably with VNFDK to form
species A (VNFG tightly associated with VNFDK). In vitro
FeV-co insertion reactions from which components were excluded, one at
a time or in combination, were applied onto the native gel, and the
VNFG species accumulated in the various reactions were compared to those accumulated in a complete FeV-co insertion reaction (see "Experimental Procedures"). Fig. 4 is an immunoblot
of an anoxic native gel (developed with antibody to VNFG) that
illustrates the requirements for the stable association of VNFG with
VNFDK. Cell-free extract (desalted by Sephadex G-25 chromatography) of strain CA117.30 (nifDKB) was used as a source of
apodinitrogenase 2 in the FeV-co insertion assays. Extract of strain
CA12 (
nifHDK), which exhibited dinitrogenase 2 activity
(Fig. 4, lane 1), shows the presence of both VNFG species A
(VNFG tightly associated with VNFDK) and B (dimer of VNFG). A FeV-co
insertion reaction that excluded both FeV-co and the ATP-regenerating
mixture (defined in "Experimental Procedures") exhibited VNFG
species B, the form exhibited by all strains containing
apodinitrogenase 2 (Fig. 4, lane 2). Upon addition of FeV-co
to a reaction that excluded only the ATP-regenerating mixture, the
formation of VNFG species A was observed (Fig. 4, lane 3).
The level of VNFG species A formed was increased in a complete FeV-co
insertion reaction (Fig. 4, lane 4) in comparison to the
reaction that excluded the ATP-regenerating mixture (Fig. 4, lane
3).
The appearance of species A can be correlated with the formation of
holodinitrogenase 2 in the reactions that were tested in the
C2H2 reduction assay (see "Experimental
Procedures"); the dinitrogenase 2 activity indicated in lane
3 where the ATP-regenerating mixture was excluded from the FeV-co
insertion reaction applied onto the native gel is probably due to the
addition of excess ATP-regenerating mixture to all the reactions that
were monitored for C2H2 reduction activity (see
"Experimental Procedures"). When FeV-co was oxidized by exposure to
air prior to its addition to the FeV-co insertion reaction, formation
of species A was not observed (data not shown). Thus, the presence of
FeV-co that is able to reconstitute apodinitrogenase 2 to active
dinitrogenase 2 promotes the association of VNFG with VNFDK, and the
association is stimulated by the addition of the ATP-regenerating
mixture to the FeV-co insertion assay. The stable association of VNFG with VNFDK might arise from a conformational change (induced in the
apodinitrogenase 2 complex upon insertion of FeV-co into the active
site) that increases the affinity of VNFG for VNFDK; alternatively, VNFG might function in associating with and inserting FeV-co into the
active site of apodinitrogenase 1, upon which VNFG forms a stable
complex with VNFDK. The latter possibility is addressed in a later
section. It is worth noting that, although the desalted extracts of
CA117.30 (nifDKB) used in this particular study contained high levels of dinitrogenase reductase 2, use of partially purified apodinitrogenase 2 (and also extract of strain CA11.80
(
nifHDKvnfH)) free of dinitrogenase reductase 2 in later
studies indicated that dinitrogenase reductase 2 is not necessary for
the association of VNFG with VNFDK (discussed below).
The level of species A formed in the absence of added ATP-regenerating
mixture (to reactions analyzed by native PAGE) during the insertion
phase of the assay (see "Experimental Procedures") is most likely
due the incomplete removal of nucleotides from the cell-free extract by
Sephadex G-25 chromatography; the requirement of nucleotide in the
stable association of VNFG with VNFDK was investigated further, and the
results are discussed below. When FeV-co was excluded from the
insertion reaction, the formation of species A was not observed (Fig.
4, lane 5), indicating that the presence of the
ATP-regenerating mixture alone does not promote the association of VNFG
with VNFDK. As anticipated, when extract of CA117.30
(nifDKB), the source of
2
2
2 apodinitrogenase 2 was
excluded from the FeV-co insertion assay, no VNFG was observed (Fig. 4,
lane 6).
The observation that the addition of FeMo-co (in place of FeV-co) to
the insertion reaction did not promote the stable association of VNFG
with VNFDK was surprising (Fig. 4, lane 7). Activation of
apodinitrogenase 2 with FeMo-co results in the formation of a hybrid
dinitrogenase 2 that exhibits low levels of
C2H2 reduction activity (Ref. 33; see also Fig.
4, lane 7). It is possible that the activation of
apodinitrogenase 2 by FeMo-co is inefficient, and therefore the level
of species A formed is below the detection limit of the visualization
method used. The level of FeMo-co added to the insertion reaction was
sufficient to activate apodinitrogenase 1 in extract of A. vinelandii strain UW45 (nifB) to a high
level (27.8 nmol of C2H4 formed/min/assay);
thus, the absence of species A is not a result of a limiting amount of
FeMo-co. When FeMo-co was added to the insertion reaction in the form
of acid-denatured dinitrogenase 1 (instead of purified FeMo-co in NMF),
species A formation was still not observed (data not shown), indicating
that its absence is not a result of any denaturing effects of NMF. A
second possibility that could account for the absence of species A
formation in the presence of FeMo-co is that VNFG might be involved in
specifically associating with FeV-co but not FeMo-co, and inserting the
cofactor into the active site pocket of apodinitrogenase 2. This
possibility is supported in part by studies described below.
In order to determine whether nucleotide was absolutely
required for the association of VNFG with VNFDK, partially purified apodinitrogenase 2 was used in place of desalted, cell-free extract of
CA117.30 in the FeV-co insertion assay. Fig. 5 is an
immunoblot of an anoxic native gel (developed with antibody to VNFG)
that illustrates the results of this study. In the absence of both FeV-co and nucleotide, species A was not observed (Fig. 5, lane 1). The addition of FeV-co alone to a reaction that excluded
nucleotide did not promote association of VNFG with VNFDK (Fig. 5,
lane 2). ATP and its analogs ADP and
,
-CH2-ATP functioned in the formation of species A
(Fig. 5, lanes 4-6). These data demonstrate that both
FeV-co and nucleotide are necessary for the stable association of VNFG
with VNFDK. When the components of the ATP-regenerating system
excluding ATP were added to a FeV-co insertion reaction, species A
formation did not occur (Fig. 5, lane 3), indicating that
Mg2+, creatine phosphate, and creatine phosphokinase were
not responsible for the stable association of VNFG with VNFDK.
An interesting observation that arose from these studies was that dinitrogenase reductase appeared not to be necessary for the stable association of VNFG with VNFDK. The partially purified fraction of apodinitrogenase 2 used in the FeV-co insertion reactions contained no dinitrogenase reductase 1 (or dinitrogenase reductase 2) as detected by C2H2 reduction activity when complemented with dinitrogenase 1, and also by immunoblot analysis of the fraction using antibody to dinitrogenase reductase 1 (dinitrogenase reductase 2 can be detected by antibody to dinitrogenase reductase 1; data not shown). Dinitrogenase reductase 1 or dinitrogenase reductase 2 were included in certain FeV-co insertion reactions to determine whether an effect on species A formation would be observed in their presence. Reactions in which dinitrogenase reductase 1 or dinitrogenase reductase 2 were added in the absence of FeV-co did not result in species A formation (data not shown), indicating that the presence of dinitrogenase reductase and ATP (added in the form of an ATP-regenerating system) were insufficient for the stable association of VNFG with VNFDK. In a complete FeV-co insertion reaction (containing FeV-co), addition of dinitrogenase reductase 1 or dinitrogenase reductase 2 did not have an effect on the association of VNFG with VNFDK (data not shown).
The requirements for the stable association of VNFG with the VNFDK
polypeptides of apodinitrogenase 2 differ in two notable aspects from
the processing of 2
2 (NIFDK)
apodinitrogenase 1 to the
2
2
2 form of
apodinitrogenase 1: 1) FeV-co is required for the former process
whereas the presence of FeMo-co is not necessary for the association of
the
protein with the NIFDK polypeptides of apodinitrogenase 1 (25),
and 2) dinitrogenase reductase is not required for the stable
association of VNFG with VNFDK; Allen et al. (25)
demonstrated the requirement of dinitrogenase reductase 1 (NIFH) in the
formation of the
2
2
2
apodinitrogenase 1 complex. Both of the above processes are
nucleotide-requiring; however, association of
with
2
2 apodinitrogenase 1 requires ATP (25),
a non-hydrolyzable analog of ATP did not support the maturation
process.2 The precise roles of dinitrogenase
reductase 1 and nucleotide in the maturation of apodinitrogenase 1 have
not been established. The stable association of VNFG with VNFDK is
supported by ATP and its analogs ADP and
,
-CH2-ATP (a
non-hydrolyzable analog of ATP), suggesting that the hydrolysis of ATP
might not be necessary for the process. A number of functions might be
proposed for the nucleotide in the FeV-co-dependent
association of VNFG with VNFDK. Upon insertion of FeV-co into
apodinitrogenase 2, nucleotide might be necessary for maintaining a
certain conformational state that allows the stable association of VNFG
with VNFDK. Alternatively, nucleotide might be necessary for the
insertion of FeV-co into apodinitrogenase 2 to form the holoenzyme, or
for the formation of a VNFG-FeV-co complex. The latter possibility can
most likely be ruled out as the association of FeV-co with VNFG
in vitro does not require the addition of nucleotide
(discussed further below).
The interaction between FeV-co and VNFG was examined by
gel-filtration chromatography. A FeV-co-containing solution and a VNFG-containing fraction (that contained no VNFDK and thus exhibited no
activity in the FeV-co insertion assay) were fractionated separately on
a calibrated Superose 12 column (see "Experimental Procedures"). VNFG reproducibly eluted at Ve/Vo
1.65-1.82 (Fig. 3A), and FeV-co eluted at
Ve/Vo 1.06-1.24 (Fig.
6A, peak 1), most likely because
the cofactor remained associated with the acid-denatured dinitrogenase
2 protein (which is predicted to elute at approximately
Vo).
When a reaction mixture containing both the FeV-co-containing solution
and the VNFG-containing fraction was chromatographed on the Superose 12 column, the migration of FeV-co was retarded; FeV-co eluted at
Ve/Vo 1.59-1.76 (Fig.
6A, peak 2). The Superose 12 fractions that
contained FeV-co (as determined by the FeV-co insertion assay) were
analyzed by SDS-PAGE, and the presence of VNFG in these fractions was
demonstrated (Fig. 6B, lanes 2-5). When the
FeV-co-containing Superose 12 fractions were added to apodinitrogenase
2 (in extracts of CA117.30 (nifDKB)), the formation of
holodinitrogenase 2 was observed (data not shown). Upon fractionation
of a mixture containing both the FeV-co solution and extract of strain
UW45 ((nifB
), grown under
NH4+-sufficient conditions) on the
Superose 12 column, the migration of FeV-co remained unaltered (Fig.
6C, peaks 1 and 2), suggesting that
the change in the elution profile of FeV-co upon addition to VNFG is
quite likely due to a specific interaction of FeV-co with VNFG.
In a similar set of experiments performed using FeMo-co in place of FeV-co, the elution profile of FeMo-co remained unaffected in the presence of added VNFG (Fig. 6D, peaks 1 and 2), further suggesting that VNFG associates specifically with FeV-co. These data, together with the apparently reversible association of VNFG with the apodinitrogenase 2 complex, suggest that VNFG might function in binding and inserting FeV-co into apodinitrogenase 2 to form the holoenzyme.
Two A. vinelandii strains were examined for the in
vivo accumulation of FeV-co, strain CA11.6.82 (W-tolerant,
vnfD82:: Tn5B21nifHDKrifr)
and strain CA11.1 (
nifHDKvnfDGK1:: spc).
FeV-co was not detected in extracts of these strains
(vnf-derepressed) by the FeV-co insertion assay. Given the
in vitro data suggesting the specific association of FeV-co
with VNFG, it is reasonable to expect that detectable levels FeV-co
might accumulate in vivo only in the presence of VNFG;
extracts of both strains tested lacked VNFG (as determined by
immunoblot analysis using antibody to VNFG). In contrast to the
nif system, in which the dinitrogenase 1 polypeptides are not required for the synthesis and accumulation of FeMo-co (38, 39),
the presence of the dinitrogenase 2 polypeptides might be necessary for
the accumulation of detectable levels of FeV-co. The possibility that
FeV-co is assembled on the dinitrogenase 2 polypeptides cannot be
excluded. The fact that no FeV-co was observed to accumulate in
vivo in strains CA11.1
(
nifHDKvnfDGK1::spc) and CA11.6.82
(W-tolerant,
vnfD82::Tn5B21
nifHDKrifr),
despite the presence of significant levels of the
protein (as
detected by immunoblot analysis) in extracts of both strains, suggests
that the
protein is not able to function as a chaperone-insertase in the nitrogenase 2 enzyme system.
Based on our prior (24) and current observations, a model for the
formation of holodinitrogenase 2 can be proposed (Fig. 7). Two processes could lead to the association of VNFG
with FeV-co. 1) In the presence of FeV-co, the loosely associated VNFG
() subunits of the
2
2
2
apodinitrogenase 2 complex might dissociate (and dimerize) and bind
FeV-co; or 2) a VNFG dimer that is not a part of the apodinitrogenase 2 hexamer might associate with FeV-co. The in vitro
association of FeV-co with the VNFG dimer does not require the presence
of nucleotide; however, nucleotide is required for the stable
association of VNFG with VNFDK. Thus, in a subsequent
nucleotide-dependent process, the VNFG-FeV-co species could
donate FeV-co to apodinitrogenase 2, inducing a conformational change
in the overall quaternary structure of the complex that allows the
stable association of VNFG with the newly reconstituted dinitrogenase
2. The fact that VNFG remains stably associated with VNFDK polypeptides
in holodinitrogenase 2 might have implications for its role in
catalysis; studies of Waugh et al. (23) indicated that VNFG
was required for full catalytic activity of nitrogenase 2.
The association of FeV-co with VNFG might be further characterized by monitoring the ability of altered forms of VNFG to bind FeV-co. The predicted amino acid sequence of VNFG indicates the presence of a single cysteine residue that is conserved between both VNFG and ANFG (the third subunit of dinitrogenase 3). By analogy to the role of Cys-275 of dinitrogenase 1 (one of the ligands to FeMo-co; Ref. 22), the invariant cysteine residue of VNFG (Cys-17) might serve as a ligand to FeV-co. It will be interesting to site-specifically alter Cys-17 of VNFG, and study the effect of the mutation(s) on the ability of VNFG to associate with FeV-co both in vivo and in vitro.
In this study, we have characterized the forms of VNFG observed in
cell-free extracts of various A. vinelandii mutant strains, and correlated the VNFG forms with the presence of either
apodinitrogenase 2 or holodinitrogenase 2. The homodimeric form of VNFG
observed under native PAGE and gel-filtration chromatography conditions in extracts of strains that accumulate apodinitrogenase 2 most likely
results from VNFG dissociating from the
2
2
2 apodinitrogenase 2 complex due to weak interactions of VNFG with VNFDK. In the presence of
FeV-co and nucleotide, VNFG becomes associated stably with the VNFDK
polypeptides to yield the VNFG form observed in strains that accumulate
dinitrogenase 2. In addition, the stable association of VNFG with VNFDK
can be correlated with the formation of catalytically active
dinitrogenase 2. In vitro, FeV-co associates with the
dimeric form of VNFG (in the absence of nucleotide), and combination of
this VNFG-FeV-co species with apodinitrogenase 2 results in its
activation to the holoenzyme. These data suggest that VNFG has a role
in the formation of dinitrogenase 2. The differences that have emerged
between the modes of action of VNFG and the
protein illustrate that
the mechanisms utilized in the processing of apodinitrogenase 2 to a
catalytically active form are clearly distinct from those involved in
the maturation of apodinitrogenase 1.
We thank Ronda Allen and Gary Roberts for valuable discussions and for critically reading this manuscript. We also thank Paul Bishop and R. Premakumar for generously providing A. vinelandii mutant strains and for helpful discussions. We acknowledge Stefan Nordlund for insightful comments.