A Vanadium and Iron Cluster Accumulates on VnfX during
Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase in
Azotobacter vinelandii*
Carmen
Rüttimann-Johnson,
Christopher R.
Staples,
Priya
Rangaraj,
Vinod K.
Shah, and
Paul W.
Ludden
From the Department of Biochemistry, College of Agriculture and
Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
The vnf-encoded nitrogenase from
Azotobacter vinelandii contains an iron-vanadium cofactor
(FeV-co) in its active site. Little is known about the synthesis
pathway of FeV-co, other than that some of the gene products required
are also involved in the synthesis of the iron-molybdenum cofactor
(FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found
that VnfX, the gene product of one of the genes contained in the
vnf-regulon, accumulates iron and vanadium in a novel V-Fe
cluster during synthesis of FeV-co. The electron paramagnetic resonance
(EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are
consistent with a VFe7-8Sx precursor of
FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly
resembles that of isolated FeV-co and a model
VFe3S4 compound. The V-Fe cluster accumulating
on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be
involved in the early steps of FeV-co synthesis, suggesting that it
corresponds to a precursor of FeV-co. VnfX purified from a
nifB strain incapable of FeV-co synthesis has a different
electrophoretic mobility in native anoxic gels than does VnfX, which
has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co
(and presumably FeV-co), binds to VnfX purified from the
nifB strain, producing a shift in its electrophoretic
mobility on anoxic native gels. The data suggest that a precursor of
FeV-co that contains vanadium and iron accumulates on VnfX, and thus,
VnfX is involved in the synthesis of FeV-co.
 |
INTRODUCTION |
Interest in vanadium as a biologically relevant element has
increased in the last two decades. Although it had been known for some
time that vanadium occurs in a highly enriched form in marine organisms
and mushrooms (for a review, see Ref. 1), the discovery of its presence
and functional role in haloperoxidases (reviewed by Butler (2)) and
nitrogenases (reviewed by Eady (3)) is recent. Nitrogenase, the
bacterial enzyme that catalyzes the conversion of N2 into
NH4+, contains a unique
iron-sulfur-heterometal cofactor, which is the site of substrate
reduction. The most widely studied nitrogenases contain molybdenum as
the heterometal in their active site cofactor (the iron-molybdenum
cofactor (FeMo-co)1). The
discovery of molybdenum-independent nitrogenases by Bishop et
al. (4, 5) has led to the isolation and characterization of
nitrogenases containing an iron-vanadium cofactor (FeV-co) (see Ref. 3
for a review), as well as nitrogenases in which no metal other than
iron has been detected in the active site cofactor (6). These three
nitrogenase systems are genetically distinct, the genes encoding them
being contained in different regulons (designated nif,
vnf, and anf genes for the molybdenum, vanadium,
and iron-only nitrogenases, respectively). Azotobacter vinelandii harbors all three nitrogenase systems, and their
expression is regulated by the metal content of the culture medium (7). Each nitrogenase consists of two easily separable protein components: dinitrogenase and dinitrogenase reductase. The reduction of substrate is catalyzed by dinitrogenase, whereas dinitrogenase reductase serves
as the obligate electron donor to dintrogenase.
The nif, vnf, and anf regulons encode
not only the nitrogenases but also products involved in cofactor
biosynthesis and insertion in the cofactorless dinitrogenases. Among
the genes known to be required for FeMo-co synthesis are
nifB, nifE, nifN, nifV, and nifH. The protein encoded by nifB synthesizes
NifB-co, a precursor that is the iron and sulfur donor to FeMo-co (8,
9) and most probably to FeV-co, because nifB is required for
vanadium-dependent diazotrophic growth (10). The gene
products of nifE and nifN form a tetrameric
protein (NifEN), that is able to bind NifB-co (11). Analogs of the
nifE and nifN genes exist in the vnf
regulon (vnfEN (12)), probably serving a similar role during
FeV-co synthesis. The gene product of nifV is homocitrate
synthase (13). Homocitrate is a structural component of FeMo-co (14)
and most probably FeV-co, because nifV is also required for
full functionality of the vnf-dinitrogenase (15). The
product of nifH, dinitrogenase reductase, plays multiple
roles in the nitrogenase system. In addition to reducing dinitrogenase
during nitrogenase turnover, NifH (VnfH) is required for FeMo-co (and
presumably FeV-co) biosynthesis (16, 17), but its exact role in this
process remains to be established. It is important to note that the
structural gene products for dinitrogenase (nifDK) are not
required for accumulation of FeMo-co and not involved in its synthesis
(18).
Any other gene products involved in FeMo-co/FeV-co biosynthesis remain
unknown, and the complete biosynthetic pathway is yet to be understood.
An important unanswered question regards the gene product(s) providing
the specificity for the incorporation of the heterometal (vanadium,
molybdenum, or iron) into the cofactor. We report here that a
V-Fe-containing cluster accumulates on the gene product of
vnfX during synthesis of FeV-co. Our data suggest that this
cluster is a precursor of FeV-co.
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EXPERIMENTAL PROCEDURES |
Materials--
All materials used for growth medium preparation
were analytical grade. Tris base and glycine were purchased from
Fisher. DEAE-cellulose (DE-52) was from Whatman. Sephadex G-75 and
phenyl-Sepharose were obtained from Amersham Pharmacia Biotech.
49V (V(VI) in 6 N HCl, 0.5-1.0 mCi/ml) was
purchased from Los Alamos National Laboratories. Sodium metavanadate
(99.995% purity) and all other chemicals were from Sigma.
Nitrocellulose membranes and acrylamide/bisacrylamide solution were
obtained from Mallinckrodt.
Bacterial Strains and Growth Conditions--
A.
vinelandii strains CA12 (
nifHDK (19)), CA11.1
(
nifHDKvnfDGK::spc (20)), CA117.3
(
nifDKB (21)), CA48
(vnfE705::kan (12)), DJ42.48
(
nifENvnfE705::kan (12)), and CA11.8
(
nifHDK
vnfH (22)) have been described. All glassware
used to prepare the culture medium and for cell growth was washed with
4 N HCl and rinsed thoroughly with deionized water. The
strains were grown in Burk's modified medium lacking molybdenum and
containing Na51VO3 (0.1 µM)
spiked with radioactive 49VCl6 (0.05 µCi/ml)
and 400 µg/ml nitrogen in the form of ammonium acetate at 30 °C
with shaking. In order to ensure maximum incorporation of
49V into the cells, starter cultures that had been depleted
of vanadium (by subculturing three times in molybdenum-free,
vanadium-free medium) were used to inoculate the
49V-containing medium. Cells were grown overnight,
collected by centrifugation, and resuspended in nitrogen-free medium
containing Na51VO3 and
49VCl6 (same concentrations as above). The
cultures were incubated then for 5 h at 30 °C for derepression
of the vanadium nitrogenase system. Cells were harvested by
centrifugation and frozen at
80 °C. For large scale purification
of VnfX, A. vinelandii strain CA11.1 was grown in 20-liter
carboys containing 15 liters of medium containing 0.1 µM
NaVO3 and 40 µg/ml nitrogen in the form of ammonium acetate. The cultures were incubated at 30 °C and aerated
vigorously. Cultures were monitored for the depletion of ammonium,
following which, derepression of nitrogenase was allowed for 5 h.
Cells were collected using a Pellicon cassette system equipped with a
filtration membrane (0.45 µM, Millipore Corporation) and
frozen at
80 °C. Cell-free extracts were prepared by osmotic
shock, as described earlier (23).
Buffer Preparation--
All buffers were sparged with purified
N2 for 20-30 min, and sodium dithionite was added to a
final concentration of 1-1.7 mM. Buffers used during
protein purification contained 0.5 µg/ml leupeptin and 0.2 mM phenylmethylsulfonyl fluoride.
Anoxic Native Gel Electrophoresis and Phosphorimaging--
The
procedure for anoxic native gel electrophoresis and phosphorimaging has
been described (8). Proteins were resolved on 7-20% acrylamide
(37.5:1 acrylamide:bisacrylamide) and 0-20% sucrose gradient gels.
After electrophoresis for 15 h at 100 V, the gels were dried and
exposed to a phosphor screen for 1-3 days. Screens were scanned using
a Molecular Dynamics model 425e PhosphorImager.
EPR Analysis--
EPR was performed at a microwave frequency of
9.23 GHz and a modulation amplitude of 12.5 Gauss using a Varian E-15
EPR spectrometer equipped with a Hewlett-Packard 5255A frequency
converter, a Varian E102 microwave bridge, and an Oxford 3120 temperature controller.
Purification of VnfX--
VnfX was purified from A. vinelandii CA11.1 grown and derepressed in the absence of
molybdenum and presence of vanadium. All procedures were performed
anaerobically. Cell-free extract from 150 g of cells grown in the
presence of NaVO3 and 6 g of cells grown in the
presence of 49VCl6 plus NaVO3 were
mixed and loaded on a DE-52 column (4 × 24 cm) equilibrated with
25 mM Tris-HCl, pH 7.4, containing 0.1 M NaCl.
The column was washed with 300 ml of the same buffer, and protein was
eluted stepwise with 0.15, 0.25, and 0.35 M NaCl in 25 mM Tris-HCl, pH 7.4, containing 20% glycerol. The presence of VnfX in the fractions was assessed by phosphorimaging of anoxic native gels. The fractions containing VnfX (0.15 M NaCl)
were pooled and concentrated to 15 ml by ultrafiltration using a PM10 membrane (Amicon). The concentrated fraction was loaded on a Sephadex G-75 column (2.5 × 95 cm) equilibrated with 25 mM
Tris-HCl, pH 7.4, 10% glycerol, 50 mM NaCl. The fractions
from Sephadex G-75 containing VnfX were pooled, brought to 1 M (NH4)2SO4, and loaded on a phenyl-Sepharose column (1 × 15 cm) equilibrated with
Tris-HCl, pH 7.4, containing 1 M ammonium sulfate. The
column was washed with 1 column volume of the same buffer, and protein
was eluted stepwise with two column volumes each of 0.5, 0.25, and 0.0 M ammonium sulfate in 0.025 M Tris, pH 7.4. Fractions containing VnfX (0.5 M
NH4SO4) were pooled, concentrated by
ultrafiltration using the same membrane as above and used for metal and
EPR analyses.
Preparation of VnfX for N-terminal Sequencing--
One ml of
partially purified VnfX was subjected to preparative anoxic native gel
electrophoresis, the region of the gel containing the radioactivity was
sliced, and the protein was eluted from the acrylamide. SDS gel
electrophoresis of this sample showed a 20-kDa protein. The protein was
transferred to a polyvinylidene difluoride membrane (Problott, Applied
Biosystems), and N-terminal sequencing was done at the Department of
Biochemistry, Medical College of Wisconsin (Milwaukee, WI).
Antibodies and Immunoblot Analysis--
Pure VnfX was cut out of
preparative SDS-polyacrylamide gel electrophoresis gels. The protein
was eluted from the acrylamide and injected into a rabbit to produce
polyclonal antibodies at the Animal Care Unit of the University of
Wisconsin-Madison Medical School. Immunoblot analysis was performed as
described by Brandner et al. (24).
Purification of VnfX from a nifB
Strain--
VnfX
was partially purified from A. vinelandii CA117.3
(
nifDKB) grown and derepressed in the absence of
molybdenum and presence of vanadium. Extract of 40 g of cells was
chromatographed on a DE-52 column (2.5 × 17 cm) equilibrated with
25 mM Tris-HCl, pH 7.4, containing 20% glycerol. The
column was washed with 80 ml of the same buffer, and protein was eluted
stepwise with 0.075, 0.15, and 0.30 M NaCl in 25 mM Tris, pH 7.4, containing 20% glycerol. The presence of
VnfX in the fractions was assessed using immunoblot analysis of anoxic
native gels. The fractions containing VnfX were pooled and concentrated
to 15 ml by ultrafiltration using a PM10 membrane (Amicon). The
concentrated fraction was loaded on a Sephadex G-75 column (2.5 × 95 cm) equilibrated with 25 mM Tris-HCl, pH 7.4, 10%
glycerol, 50 mM NaCl. The fractions from Sephadex G-75
containing VnfX were pooled and frozen in liquid N2.
Incubations with NifB-co--
NifB-co was obtained from
Klebsiella pneumoniae UN1217 (nifN4536 (25)) as
has been described (9). NifB-co and 55Fe-NifB-co were in 25 mM Tris-HCl, pH 7.4, containing 2% SB12 (3-(dodecyldimethylammonio)propane-1-sulfonate, Fluka). Incubations of
VnfX with NifB-co were done for 30 min at 30 °C.
Analysis of VnfX for Homocitrate--
Approximately 80 ml of
49V-VnfX partially purified from A. vinelandii
CA11.1 (48 nmol of vanadium, as estimated by specific activity of
49V; 15 mg of total protein) was added dropwise to 900 ml
of acetone containing 6.75 ml of 4 N HCl at 4 °C with
continuous stirring over a 10-min period. The mixture was stirred for
an additional 15 min, and precipitated protein was removed by
filtration. The filtrate was evaporated to approximately 8 ml using a
rotary evaporator, and the pH was adjusted to 8.0-9.0 before
chromatography on an AG-1X8 (formate form, Bio-Rad) column (1 × 41 cm) equilibrated with water. The column was eluted with a gradient
of 0-6 N formic acid (400 ml total). In order to determine
in which fractions of the AG-1X8 column the homocitrate elutes, a
standard of [3H]homocitrate (0.1 mmol, 36,000 dpm) was
loaded previously on a comparable column and eluted under the exact
same conditions. The fractions containing the 3H label were
pooled, evaporated to dryness, redissolved in 5.0 ml of
H2O, and brought to pH 8. The presence of homocitrate in this fraction was confirmed by in vitro FeMo-co synthesis
assays (26), using extracts of A. vinelandii UW45
(
nifKDB) that had been desalted using Sephadex G-25 to
remove endogenous homocitrate. The corresponding fractions from the
AG-1X8 column on which the acidified acetone extract of VnfX had been
loaded were pooled, evaporated to dryness, redissolved in 0.2 ml of
double distilled H2O, and brought to pH 8.0. The presence
or absence of homocitrate in this fraction was assessed using in
vitro FeMo-co synthesis assays. The concentration of homocitrate
expected in this fraction if the V-Fe cluster associated to VnfX
contained homocitrate was calculated based on the concentration of
vanadium in the sample of VnfX used for homocitrate extraction
(considering 1 mol of homocitrate/mol of vanadium). The recovery of
standard homocitrate that underwent these same manipulations was
considered in this calculations (70%).
 |
RESULTS AND DISCUSSION |
In order to study which proteins play a role in FeV-co
biosynthesis, mutant strains of A. vinelandii were grown in
a medium depleted of molybdenum and containing 51V spiked
with 49V (radioactive). The labeled proteins present in
cell extracts were analyzed through phosphorimaging of anoxic native
gels (Fig. 1). In an extract of a strain
that is a wild-type for the vnf-nitrogenase system (CA12
(
nifHDK), the major protein band containing
49V was the vnf-dinitrogenase, as expected (Fig.
1, lane 1). In extracts of a mutant strain unable to
synthesize the structural proteins of either the molybdenum nitrogenase
or the vanadium nitrogenase (CA11.1
nifHDK
vnfDGK1::spc), one
prominent radiolabeled protein band was observed (Fig. 1, lane 2, VnfX). 49V did not accumulate on this protein in a
strain that lacks NifB-co, the iron and sulfur donor to FeMo-co (and
presumably FeV-co) (CA117.3
nifDK
nifB, Fig.
1, lane 3). Furthermore a strain lacking both NifH and VnfH
(CA11.8
nifHDK
vnfH) also failed to
accumulate 49V (Fig. 1, lane 4) on this protein.
NifH is known to be required for FeMo-co synthesis, and its
vnf-encoded homolog is proposed to play a similar role in
FeV-co synthesis. These observations suggest that a
49V-labeled precursor of FeV-co is associated with the
protein labeled VnfX. The same radiolabeled protein was also present,
although at a much lower intensity in the extract of the wild-type
strain (Fig. 1, lane 1). A 49V-labeled band was
seen in an extract of cells grown in the presence of
NH4+ (Fig. 1, lane 5, E). The
same band was also seen in all other extracts used. This band did not
stain for protein by Coomassie Blue or silver stain. Work is in
progress to determine the identity of this band. This band does not
correspond to H49VO42
(the
vanadium species expected to be in solution at this pH), because this
species migrated as a diffuse band at a different position in the gel
(Fig. 1, lane 6).

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Fig. 1.
Phosphorimage of a native anoxic gel of
extracts of A. vinelandii mutant strains grown and
derepressed in the presence of 49VCl6.
Lane 1, strain CA12 ( nifHDK, 81,000 cpm).
Lane 2, strain CA11.1
( nifHDKvnfDGK::spc, 32,000 cpm). Lane
3, strain CA117.3 ( nifDKB, 80,000 cpm). Lane
4, strain CA11.8 ( nifHDK vnfH, 80,000 cpm).
Lane 5, strain CA12 ( nifHDK, 81,000 cpm) grown
in the presence of ammonium. Lane 6, H249VO4 in 0.1 M Mops, pH 7.5. Lane 7, strain DJ42.48
( nifENX vnfE, 81,000 cpm). Lane
8, strain CA48 (vnfE705::kan, 80,000 cpm).
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Purification of VnfX--
The radiolabeled band present in
extracts of A. vinelandii CA11.1 (Fig. 1, VnfX)
was purified by following the radioactivity associated with it.
Purification steps included DEAE-cellulose, Sephadex G-75, and
phenyl-Sepharose chromatography, as shown in Table
I. Purified VnfX showed a specific
activity of 133,948 cpm/mg. A purification fold of 165.5 and a final
yield of 26% were obtained. After this purification procedure, the
protein was not homogenously pure. In order to obtain pure protein, 1 ml of the fraction obtained after phenyl-Sepharose chromatography was
electrophoresed in an anoxic native gel, the region of the gel
containing the radioactivity was sliced, and the protein was eluted
from the acrylamide. This step is not shown in the purification table,
because VnfX lost the 49V label upon exposure to air, when
eluting from the gel. The purified protein was identified as
VnfX, based on its N-terminal sequence identity to the amino acid
sequence predicted from the vnfX gene (12) (MIKVAFASN). In
A. vinelandii, the vnfX gene is located downstream of the vnfEN genes, which are also proposed to be
involved in FeV-co synthesis (12).
Characterization of VnfX--
The temperature dependence at 10.0 mW of the X-band EPR spectrum of dithionite-treated (as isolated) VnfX
is shown in Fig. 2A. The
spectral properties can be readily interpreted as arising from a
majority S = 3/2 species that is of maximum observed
intensity at 4.8 K (Fig. 2B) and a minority
S = 1/2 species that increases in intensity with
increasing temperature to 50 K. The g values of the majority S = 3/2 species can be analyzed using a spin Hamiltonian, He, with D and E as the axial
and rhombic splitting parameters, and an isotropic g-tensor,
g0, as given by Equation 1.
|
(Eq. 1)
|
On the basis of their temperature dependence behavior, the
features at g = 4.82, g = 3.36, and g ~1.90 are attributed
to resonances from the lower doublet of the S = 3/2
system with a rhombicity parameter (E/D) of 0.15 and D > 0, using g0 = 2.00. An
accurate assessment of the high field g value is hindered by the
diffuse nature of the resonance and the presence of the minority species. As Fig. 2B demonstrates, power-saturation of the
majority species (determined by following the intensity of the g = 4.82 resonance) only becomes evident at temperatures below 10.0 K when using a 10.0 mW microwave power. A plot of the normalized intensity of
the g = 4.82 resonance divided by the square root of the microwave power versus the log of microwave power at a temperature of
4.8 K, according to Equation 2, is shown in Fig.
3A (27).
|
(Eq. 2)
|
P1/2 is the power for half-saturation, and
b is the inhomogeneity parameter. Analysis of the data
reveals a P1/2 of 4.3 mW using b = 1, which provided the best fit. Fig. 3A shows that at 4.8 K
and 1.0 mW, the S = 3/2 species is largely not
power-saturated. Because the minority S = 1/2 species
was not observed at 4.8 K and 1.0 mW (Fig. 3B), it can be
assumed that the g = 2 region of the spectrum is arising from only
the majority species. Magnetic hyperfine structure due to
51V (I = 7/2; the majority isotopic species
of vanadium present) is apparent in the g = 2 region under these
conditions (Fig. 3B, inset). Because the observation of this
hyperfine structure is maximized at the same conditions as is the
intensity of the S = 3/2 species, it is probable that
the hyperfine structure is due to 51V incorporated into the
species that produces the S = 3/2 resonances, rather
than due to a protein-bound 51V4+
(S = 1/2) species. The ground state spin and spectral
line shapes of the majority species resemble those of isolated FeV-co
(28), the reduced vnf-dinitrogenase of A. chroococcum (29), and a synthetic
[VFe3S4] cubane cluster (30). The minority
S = 1/2 component (g = 2.05, 1.96, 1.89) was most
clearly observable at higher temperatures (Fig. 2A), and its
relative abundance varied from sample to sample. The minority species
may represent a damaged version of the majority species. Analogous
signals have been seen in the vnf-dinitrogenases of A. chroococcum (31) and A. vinelandii (32) and have been
suggested to represent damaged FeV-co in those proteins. Therefore, the
spectroscopic properties of the S = 3/2 species are
consistent with an [Fe-V-S] cluster with similar properties to both
FeV-co (proposed to be [VFe7S9]) and a
[VFe3S4] cluster. Metal analysis revealed
that there are 8 ± 1 mol of Fe and 0.5 mol of vanadium bound per
mol of VnfX. The levels of vanadium and Fe are more consistent with a
model in which a [VFe7Sx] cluster is bound to
VnfX, as opposed to a [VFe3S4] cluster.
However, it does not appear that FeV-co itself accumulates on VnfX, as
vanadium- and iron-containing VnfX (as purified) is not able to donate
49V label to FeV-co-deficient dinitrogenase (VnfDGK) (see
below). The low level of vanadium (0.5 mol/mol of VnfX) may indicate
that some vanadium was lost during purification or that a fraction of
the cluster on the isolated VnfX had not yet acquired an atom of
vanadium. It is possible that the minority species arises from a
damaged [Fe-S] cluster in which vanadium has been lost. As discussed below, NifB-co is able to bind to VnfX in the absence of vanadium. Isolated NifB-co contains only Fe and S, and is diamagnetic in the
presence of dithionite. Whereas it is possible that VnfX-bound NifB-co
is paramagnetic, it would not exhibit the magnetic hyperfine that the
S = 3/2 species described above does. Experiments are in progress
to determine the spectral properties of VnfX-bound NifB-co.

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Fig. 2.
Temperature dependence of the X-band EPR
spectrum of VnfX. A, temperature dependence of the EPR
spectrum at 10 mW of microwave power. Spectra are the sums of six
scans, with a six-scan cavity spectrum performed at the same
experimental conditions subtracted. VnfX purified from A. vinelandii CA11.1 ( nifHDKvnfDGK::spc) was
at a final concentration of 23 µM. B, plot of
g = 4.82 resonance intensity versus reciprocal
temperature.
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Fig. 3.
Microwave power dependence of the X-band EPR
spectrum of VnfX. A, power study of the
S = 3/2 signal. The intensity of the g = 4.82 resonance was divided by the square root of the microwave power in mW
(PmW), normalized, and plotted versus
the log of PmW. The data was fit using Equation 2, affording a P1/2 = 4.3 mW. Best fits were
obtained with b = 1. Microwave power levels used were
0.10, 0.20, 0.50, 1.0, 2.0, 3.5, 5.0, 10, 15, and 20 mW. B,
EPR spectrum of VnfX obtained at 4.8 K and 1.0 mW. The inset expands
the region of the spectrum containing the nuclear hyperfine resonances
(2600-3800 Gauss). The spectrum shown is the sum of six scans, with a
six-scan cavity spectrum performed at the same experimental conditions
subtracted. VnfX purified from A. vinelandii CA11.1
( nifHDKvnfDGK::spc) was at a final
concentration of 23 µM.
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As indicated above, vanadium- and iron-containing VnfX is not
able to donate the V-Fe cluster to FeV-co-deficient dinitrogenase (VnfDGK) under the conditions tested to date. Assay mixtures used for
this purpose contained 25 mM Tris-HCl, pH 7.4, extract of A. vinelandii CA117.3 (
nifKDB) grown in the
presence of vanadium (as a source of FeV-co-deficient
vnf-dinitrogenase, VnfH, and other vnf proteins),
an ATP generating system, MgCl2 (1.8 mM), and
partially purified VnfX with the 49V-Fe cluster bound
(122,000 cpm/assay). Assays were performed in the absence or presence
of homocitrate (104 µM). Purified NifH and partially
purified NifEN were added to some assays, in case the vnf
counterparts of these proteins were limiting in the extract of A. vinelandii CA117.3. Under none of the conditions used was the
49V-Fe cluster donated to apo-dinitrogenase, as estimated
by phosphorimaging of anoxic native gels of the assay mixtures, nor
could nitrogenase activity be detected.
The V-Fe Cluster Accumulating on VnfX Does Not Contain
Homocitrate--
Experiments were conducted to determine whether the
V-Fe cluster associated to VnfX contains homocitrate. Partially
purified VnfX was extracted with acidified acetone, and the extract was purified by AG-1X8 chromatography. As shown in Table
II, no homocitrate could be detected in
the extract of VnfX, as assessed by in vitro FeMo-co
synthesis assays. Table II also shows the activities obtained in
vitro FeMo-co synthesis assays when adding amounts of
homocitrate equivalent to the amounts expected to be in the VnfX
extract if this organic acid was a part of the cluster associated to
VnfX. Homocitrate is probably added to FeV-co at a later step than the one occurring on VnfX.
VnfX Is Not Absolutely Required for FeV-co
Synthesis--
Surprisingly, A. vinelandii CA48
(vnfE705::kan), a strain that does not
express VnfX (as shown by immunoblot analysis, apparently due to
polarity of the mutation in vnfE on vnfX) is able
to grow diazotrophically in the presence of vanadium, although after a prolonged lag period (12). This strain does produce
vnf-encoded dinitrogenase, as shown in Fig. 1, lane
8. As expected, no label was seen in this extract at the position
of VnfX. It is possible that NifX or some other protein replaces VnfX
in the synthesis of FeV-co. Wolfinger and Bishop (12) have suggested
that NifEN replaces VnfEN in this strain during
vanadium-dependent diazotrophic growth. Immunoblot analysis
shows that both NifX and NifEN are expressed in vanadium-grown CA48
(data not shown). Nevertheless, no 49V was associated to
NifX in extract of this strain (Fig. 1, lane 8). Association
of 49V to NifX may be weaker than to VnfX and not withstand
the electrophoresis conditions. No 49V-labeled proteins
were seen in extracts of strain DJ42.48
(
nifENvnfE705::kan) (Fig. 1, lane
7). The mutations in this strain are polar on nifX and
vnfX.
Two Electrophoretically Distinct Species of VnfX--
Polyclonal
antibodies to VnfX were generated using pure VnfX, which was extracted
from an SDS gel. Immunoblot analysis of anoxic native gels of the
partially purified VnfX (from A. vinelandii CA11.1) showed a
major band cross-reacting with the antibody, which comigrated with the
49V-labeled VnfX (Fig. 4,
lanes 2 and 5). A minor band of slower electrophoretic mobility also cross-reacted with the VnfX antibody (labeled V-deficient VnfX in Fig. 4, lane 5).
After treatment of VnfX with air, the band corresponding to
49V-VnfX could no longer be seen, but the band migrating in the higher position remained (Fig. 4, lane 6). Treatment of VnfX
with air resulted in loss of vanadium and iron (data not shown). The slower migrating band in lane 5 probably arose from
dissociation of the V-Fe cluster from VnfX during purification (see
below). In extracts of a mutant strain incapable of NifB-co production (A. vinelandii CA117.3
(
nifDK
nifB)), there was no vanadium or iron
associated with VnfX (as estimated by phosphorimaging (Fig. 1,
lane 3) and iron staining of anoxic native gels, not shown). The vanadium-deficient species cross-reacting with the anti-VnfX antibody in extracts of this strain had a slower mobility in anoxic native gel electrophoresis (Fig. 4, lane 7) than the species
that has the V-Fe cluster bound. Vanadium-deficient VnfX was partially purified from an extract of A. vinelandii CA117.3. When
subjected to gel filtration electrophoresis using Sephadex G-75, this
form of VnfX eluted at a volume that corresponds to an apparent
molecular mass of 33 kDa, compared with an elution volume corresponding to an apparent molecular mass of 20 kDa for the species that has the
V-Fe cluster bound (the molecular mass predicted from the DNA sequence
of vnfX is 19 kDa (12)). The subunit molecular mass of both
vanadium-deficient VnfX and V-Fe-containing VnfX was determined to be
20 kDa by SDS-polyacrylamide gel electrophoresis. We hypothesize that a
dimeric form of VnfX monomerizes upon binding a V-Fe precursor of
FeV-co. This would be analogous to the monomerization of the
FeMo-co-binding protein,
, which is involved in insertion of FeMo-co
into the cofactorless nif-apodinitrogenase (33). Other
interpretations are also consistent with our observations, such as a
dramatic conformational change in a monomeric VnfX upon metal cluster
binding.

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|
Fig. 4.
Analysis of VnfX by anoxic, native gel
electrophoresis. Phosphorimage of an anoxic native gel. Lane
1, extract of A. vinelandii CA12 ( nifHDK)
grown in the presence of 49V (1.5 mg of total protein;
81,000 cpm). Lane 2, VnfX partially purified from A. vinelandii CA 11.1 ( nifHDK vnfDGK)
grown in the presence of 49V (263 µg of total protein;
28,500 cpm). Lane 3, VnfX partially purified from A. vinelandii CA117.3 ( nifDK nifB) grown
in the presence of vanadium incubated with 55Fe-NifB-co
(425 µg of total protein; 5,000 cpm). Lane 4, 55Fe-NifB-co (5,000 cpm). Immunoblot of an anoxic native
gel developed with antibody to VnfX. Lane 5, VnfX partially
purified from A. vinelandii CA 11.1 ( nifHDK vnfDGK) grown in the presence of
49V (1.0 µg of total protein). Lane 6, VnfX
partially purified from A. vinelandii strain CA 11.1 exposed
to air for 5 min before electrophoresis (1.0 µg of total protein).
Lane 7, VnfX partially purified from NifB-co-deficient
A. vinelandii strain CA117.3 (43.0 µg of total protein).
Lane 8, VnfX partially purified from NifB-co-deficient
A. vinelandii strain CA117.3 (43.0 µg of total protein)
incubated with NifB-co (72.5 pmol of Fe). Lane 9, VnfX
partially purified from NifB-co-deficient A. vinelandii
strain CA117.3 (43.0 µg of total protein) incubated with
air-inactivated NifB-co (72.5 pmol of Fe). Lane 10, VnfX
partially purified from NifB-co-deficient A. vinelandii
strain CA117.3 (43.0 µg of total protein) incubated with SB12 (final
concentration, 0.03%) in 0.025 M Tris-HCl, pH 7.4. Lane 11, extract of A. vinelandii strain CA11.1
grown and derepressed in the presence of molybdenum (150 µg of total
protein). Lane 12, extract of A. vinelandii
CA11.1 grown in the presence of vanadium and ammonium acetate (150 µg
of total protein).
|
|
Vanadium-deficient VnfX (as Purified from the nifB
Strain) Is Able to Bind NifB-co--
Treatment of vanadium-deficient
VnfX with purified NifB-co caused a shift in the migration of VnfX in
anoxic native gels (Fig. 4, lane 8). The VnfX-NifB-co
complex migrated at the same position as does VnfX that has the V-Fe
cluster bound to it (e.g. the VnfX purified from A. vinelandii strain CA11.1) (Fig. 4, lane 2).
Oxygen-inactivated NifB-co did not produce the shift of VnfX (Fig. 4,
lane 9), nor did a solution of SB12 (Fig. 4, lane
10), a detergent that was present at that concentration in the
NifB-co solution. The binding of NifB-co to VnfX was confirmed by
incubating the partially purified protein with
55Fe-NifB-co. When this incubation mixture was subjected to
anoxic native gel electrophoresis and the gel was analyzed by
phosphorimaging, the 55Fe label was found associated with
VnfX (Fig. 4, lane 3).
Immunoblot analysis was also used to confirm that VnfX was not
expressed in A. vinelandii grown in the presence of
molybdenum (Fig. 4, lane 11) or in the presence of ammonium
acetate (Fig. 4, lane 12), which represses synthesis of all
three nitrogenase systems. Furthermore, anti-VnfX antibody did not
cross-react with NifX (data not shown). No association that is stable
to anoxic native gel electrophoresis was observed between VnfX and
dinitrogenase (VnfDGK) or VnfH (data not shown).
A homolog of vnfX exists in the nif regulon
(nifX). The product of nifX is required for
in vitro FeMo-co synthesis with purified components (34).
Nevertheless, nifX is not absolutely required in
vivo for FeMo-co synthesis, because strains of A. vinelandii and K. pneumoniae with mutations in
nifX are Nif+ (35, 36). The same seems to be
true for VnfX. A. vinelandii CA48
(vnfE705::kan) is able to grow diazotrophically in
the presence of vanadium, although after a prolonged lag period. It is
possible that some other gene product replaces VnfX (or NifX) in
vivo, although with lower efficiency.
As stated before, in extracts of strains with mutations in
nifB or a double mutation in vnfH and
nifH, no V-Fe cluster accumulates on VnfX. Thus, the
involvement of NifB and VnfH in FeV-co biosynthesis must occur earlier
in the biosynthetic pathway than the step occurring on VnfX. We
speculate that the gene products believed to play a role in FeV-co
synthesis would do so in the following order (Scheme
I).
Conclusions--
We have identified and characterized what appears
to be a precursor of FeV-co that contains the heterometal (vanadium).
This intermediate is associated with VnfX, which suggests that this protein is involved in FeV-co synthesis. The step occurring on VnfX
could be the addition of vanadium to the partially formed cluster,
because both a vanadium-containing cluster and NifB-co, its Fe-S
precursor, can accumulate on VnfX. Alternatively, a cluster already
containing vanadium could be transferred to VnfX, and a further
processing step could occur on this protein.
 |
ACKNOWLEDGEMENTS |
We thank Dr. George Reed for the use of his
EPR facility, Drs. Paul Bishop and Dennis Dean for generously providing
the mutant strains used in this study, Dr. Gary Roberts for helpful
suggestions, and Dr. L. Mende-Müller for N-terminal sequencing of VnfX.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM35332.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Wisconsin, 433 Babcock Dr., Madison, WI 53706. Tel.:
608-262-6859; Fax: 608-262-3453; E-mail:
ludden{at}biochem.wisc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
co, cofactor;
Mops, 4-morpholinepropanesulfonic acid;
EPR, electron paramagnetic
resonance.
 |
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