Requirement of Homocitrate for the Transfer of a
49V-Labeled Precursor of the Iron-Vanadium Cofactor from
VnfX to nif-apodinitrogenase*
Carmen
Rüttimann-Johnson,
Priya
Rangaraj,
Vinod K.
Shah, and
Paul W.
Ludden
From the Department of Biochemistry, College of Agricultural and
Life Sciences, University of Wisconsin, Madison,
Wisconsin 53706
Received for publication, August 10, 2000, and in revised form, October 23, 2000
 |
ABSTRACT |
A vanadium- and iron-containing cluster has been
shown previously to accumulate on VnfX in the Azotobacter
vinelandii mutant strain CA11.1
(
nifHDKvnfDGK::spc). In the present study, we show the
homocitrate-dependent transfer of 49V label
from VnfX to nif-apodinitrogenase in vitro.
This transfer of radiolabel correlates with acquisition of acetylene
reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of
alternative nitrogenases. Structural analogues of homocitrate prevent
the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB cofactor (-co) or a source of vanadium
(Na3VO4 or VCl3) does not increase
nitrogenase activity. Our results suggest that there is in
vitro incorporation of homocitrate into the V-Fe-S cluster
associated with VnfX and that the completed cluster can be inserted
into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into
nif-apodinitrogenase (
2
2
2) do not require
MgATP. Attempts to achieve FeV-co synthesis using extracts of other
FeV-co-negative mutants were unsuccessful, showing that earlier steps
in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not
occur in vitro.
 |
INTRODUCTION |
Azotobacter vinelandii and other members of the family
Azotobacteriaceae harbor three genetically distinct nitrogenase systems encoded by the nif, vnf, and anf genes
(1). Expression of the three nitrogenases is regulated by the metal
content of the culture medium (2). Dinitrogenase contains a unique
iron-sulfur-heterometal cofactor that is the active site of the enzyme.
The iron-molybdenum cofactor
(FeMo-co)1 constitutes the
active site of the nif-encoded, molybdenum-containing dinitrogenase. It is composed of molybdenum, iron, sulfur, and the
organic acid homocitrate in a 1:7:9:1 ratio (3, 4). The active site
cofactor of the vnf-encoded, vanadium-containing nitrogenase
(FeV-co) is believed to be structurally analogous to FeMo-co, with an
atom of vanadium in FeV-co in the position of molybdenum in FeMo-co.
The anf-encoded dinitrogenase contains iron as the
only metal in its active site cofactor (FeFe-co).
The products of several nif genes, including
nifB, nifE, nifN, nifH,
nifV, and nifX have been shown to be involved in
the synthesis of FeMo-co. The genes coding for the structural
components of dinitrogenase (nifDK) are not required (5).
FeMo-co is assembled elsewhere and then inserted into dinitrogenase.
The metabolic product of the protein encoded by nifB is
NifB-co, a FeMo-co precursor that has been shown to be the iron and
sulfur donor to the cofactor (6, 7). NifB-co is most probably also a
precursor of FeV-co and FeFe-co, because nifB is required
for vanadium-dependent and iron only-dependent
diazotrophic growth of A. vinelandii (8). The gene products
of nifE and nifN form a heterotetrameric protein (NifNE) that has been shown to bind NifB-co (9) and is proposed to
serve as a scaffold during cofactor synthesis (10). The counterparts of
NifE and NifN in the vnf system (VnfE and VnfN) probably
serve a similar function during the synthesis of FeV-co. The product of
nifH (and vnfH), dinitrogenase reductase, has
multiple functions in the nitrogenase system. Besides being the
obligate electron donor to dinitrogenase during catalysis, it is also
required for FeMo-co (FeV-co) biosynthesis (11-13). Its exact role in
this process is not understood. The nifV gene codes for
homocitrate synthase (14). Homocitrate is a structural component of
FeMo-co and presumably FeV-co and FeFe-co, because nifV is
required for full functionality of all three dinitrogenases (15). The
products of nifX and its homologue vnfX have also
been shown to be involved in FeMo-co and FeV-co synthesis,
respectively, although their exact role remains to be established. A
vanadium- and iron-containing cluster having similar EPR
characteristics to FeV-co accumulates on VnfX during FeV-co synthesis
(16). NifX stimulates the synthesis of FeMo-co in an in
vitro system (17). Both proteins are able to bind NifB-co
(16).2 The complete
biosynthetic pathways of FeMo-co and FeV-co have not yet been
elucidated, and other not yet identified components have been shown to
stimulate the synthesis of FeMo-co in vitro (17).
An in vitro FeMo-co synthesis system has been described
(18). It typically involves mixing of extracts from two different mutants defective in the synthesis of the cofactor or a mutant extract
complemented with the purified missing component(s). In vitro synthesis of FeV-co has not been accomplished yet. Here we report the homocitrate-dependent in vitro
incorporation of a VnfX-associated V-Fe-S cluster into
nifapodinitrogenase.
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EXPERIMENTAL PROCEDURES |
Materials--
All materials used for growth media
preparation were of analytical grade. 49V (in 6 N HCl, 0.5-1.0 mCi/ml) was purchased from Los Alamos
National Laboratories. Sodium orthovanadate, sodium metavanadate,
homocitric acid, isocitric acid, and malic acid were from Sigma. Tris
base and glycine were purchased from Fisher. Acrylamide/bisacrylamide solution was obtained from Bio-Rad. Ammonium tetrathiomolybdate was a
gift from Dr. D. Coucouvanis.
Bacterial Strains and Culture Conditions--
A.
vinelandii strains CA12 (
nifHDK (19)), CA11.1
(
nifHDKvnfDGK::spc (20)), CA117.3
(
nifDK
nifB (21)), DJ42.48
(
nifENvnfE705::kan (22)), CA11.8
(
nifHDK
vnf 72 (23)), UW45 (nifB
(8)), and CA11.6.82 (tungsten-tolerant,
vnfD82::Tn5-B21
nifHDK
Rifr (24)) used in this work have been described. The
strains were grown in 20-liter carboys containing 15 liters of Burk's
modified medium with 40 µg/ml nitrogen in the form of ammonium
acetate and 1 µM NaVO3, or 1 mM Na2WO4, or 1 µM
Na2MoO4. The cultures were incubated at
30 °C and aerated vigorously. They were monitored for the depletion
of ammonium, following which derepression of nif or
vnf genes was allowed for 4-5 h. Cells were collected using a Pellicon cassette system equipped with a filtration membrane (0.45 µm, Millipore Corporation) and frozen at
80 °C. For growth in
the presence of vanadium or tungsten, all glassware used to prepare the
culture medium and for cell growth was washed with 4 N HCl
and rinsed thoroughly with deionized water. Cultures in the presence of
49V were grown in 1-liter flasks with 250 ml of growth
medium containing NaVO3 (0.1 µM) spiked with
radioactive 49VCl5 (0.05 µCi/ml) and 40 µg/ml nitrogen in the form of ammonium acetate. Cells were grown
overnight at 30 °C with shaking, collected by centrifugation, and
resuspended in nitrogen-free medium containing NaVO3 and
49VCl5 (same concentrations as above). They
were then incubated for 5 h at 30 °C for derepression of the
vnf-nitrogenase system. Cells were harvested by
centrifugation and frozen at
80 °C.
Buffer Preparation--
All buffers were sparged with purified
N2 for 20-30 min, and sodium dithionite was added to a
final concentration of 1 mM. Buffers used for protein
purification contained 0.5 µg/ml leupeptin and 0.2 mM
phenylmethylsulfonyl fluoride.
Cell-free Extracts and Sephadex G-25
Chromatography--
Cell-free extracts were prepared by osmotic shock,
as described earlier (25). For some experiments the extract of A. vinelandii strain CA11.1 grown in vanadium-containing medium was
chromatographed on Sephadex G-25 to remove small molecular weight
molecules. Forty ml of extract were loaded on a Sephadex G-25 column
(2.5 × 51 cm) equilibrated with 25 mM Tris-HCl (pH
7.4), 10% glycerol. All procedures were performed anaerobically.
Purification of Other Components--
Apodinitrogenase was
purified in its hexameric form
(
2
2
2) (26) from A. vinelandii UW45, as described by Shah et al. (17). It
was ~70% pure, as judged by a densitometric scan of an SDS gel.
Unlabeled NifB-co and 55Fe-NifB-co were purified according
to Shah et al. (7) and Allen et al. (6), respectively.
In Vitro Cofactor Synthesis and Nitrogenase Assay--
Stoppered
9-ml serum vials that had been washed with 4 N HCl and
thoroughly rinsed with deionized water were used for the reactions. The
assay consists of two phases. During the first phase (35 min) cofactor
synthesis and insertion into apodinitrogenase are allowed to occur. The
second phase is the reduction of acetylene by the nitrogenase
holoenzyme formed during the first phase. The reactions were carried
out under argon. The standard reaction mixture contained the
following ingredients (first phase) in a total volume of 0.75 ml: 0.1 ml of 25 mM Tris-HCl (pH 7.4) containing 0.5 mM
sodium dithionite, 20 µl of 5 mM homocitrate (pH 8), 10 µl of 1 mM Na3VO4, 0.2 ml of an
ATP-regenerating mixture (27) containing 0.1 mM dithionite,
NifB-co (0.75 nmol of iron), dinitrogenase reductase (52 µg of
protein), 0.2 ml each of the extracts to be tested (between 1.7 and 2.5 mg of protein), and, when stated, purified
nif-apodinitrogenase (34 µg of protein). When
reconstituted with an excess of purified FeMo-co, 34 µg of purified
nif-apodinitrogenase produced 36 nmol of ethylene/min, and
the nif-apodinitrogenase present in 200 µl of UW45 (W)
extract produced 35 nmol of ethylene/min. To ensure that the quantity
of nif-apodinitrogenase used in the assays was saturating,
titration experiments were performed using a fixed amount of CA11.1 (V)
extract (0.2 ml, 1.7 mg of protein) and increasing amounts of
nif-apodinitrogenase (purified or as extract of UW45 (W))
(data not shown). The amount of nif-apodinitrogenase (purified or as extract of UW45 (W)) used in the assays was saturating. For some control reactions 10 µl of 1 mM
Na2MoO4 were added instead of
Na3VO4. This mixture was incubated for 35 min
at 30 °C with shaking. After this incubation the second phase of the
assay was initiated by injection of an additional 0.8 ml of
ATP-regenerating solution (containing 4 mM dithionite) and
52 µg of dinitrogenase reductase to each vial. The nitrogenase assay
was started by injecting 0.5 ml of acetylene into the vial. The vials
were incubated at 30 °C for 30 min with shaking. The reaction was
stopped by adding 0.1 ml of 4 N NaOH, and the ethylene and
ethane formed were measured with a Shimadzu model GC8A gas
chromatograph equipped with a Porapak N (Waters Associates) column.
Activities are expressed as nanomoles of ethylene (or ethane) formed
per min per assay. When studying the effect of ATP during the cofactor
synthesis and insertion phase, 10 µl of ammonium tetrathiomolybdate
(1 mM in N-methylformamide, pH 8)
(28) were added right after the first phase of the assay, and the vials
were incubated for 10 min at room temperature before starting the
second phase of the assay, to prevent further insertion of the cofactor
into apodinitrogenase. Reactions to be analyzed by anoxic native gel
electrophoresis only underwent the first phase of the reaction
(cofactor synthesis and insertion), after which they were placed on
ice. Aliquots (100 µl) of the reaction mixture were subjected to
anoxic native gel electrophoresis, as described below.
Anoxic Native Gel Electrophoresis and Phosphorimaging--
The
procedures for anoxic native gel electrophoresis and phosphorimaging
have been described (6). Proteins were resolved on 7-16% 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 Cyclone storage phosphor system (Packard Instruments).
 |
RESULTS AND DISCUSSION |
To investigate in vitro FeV-co synthesis, extracts of
different mutant strains defective in FeV-co (or FeMo-co) synthesis were mixed and incubated in conditions similar to those that have been
described for in vitro FeMo-co synthesis (18). When an extract of vanadium-grown A. vinelandii CA11.1, which lacks
the structural components of vnf-nitrogenase, was
complemented with an extract of tungsten-grown A. vinelandii
UW45 (nifB
) (in the presence of other
components; see "Experimental Procedures"), acetylene reduction
activity was observed (Table I,
line 1). High activities were also observed when the extract
of A. vinelandii UW45 was replaced by purified
nif-apodinitrogenase (Table I, line 2). Both the
extract of UW45 (W) and the purified
nif-apodinitrogenase were added in amounts that were
saturating in the assay, as determined by titration (see
"Experimental Procedures"). The enzyme responsible for the
acetylene reduction activity observed is probably a hybrid of
nif-dinitrogenase and FeV-co. The activity detected is
characteristic of alternative nitrogenases, because ethane as well as
ethylene were produced from acetylene. The ratio of ethylene to ethane produced (C2H6/C2H4 × 100) ranged from 2.2 to 2.9. These values are in the same range that
has been described for vnf-nitrogenases (29, 30) and for a
hybrid of nif-dinitrogenase containing FeV-co (31). A
control assay containing Mo and all factors necessary for in
vitro FeMo-co synthesis showed high ethylene production activity
but no detectable ethane, as expected (Table I, line 3).
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Table I
Acetylene reduction activities of mixtures of extracts of different
A. vinelandii mutant strains
Assays contained homocitrate (0.1 mM), ATP-regenerating
mix, Na3VO4 (20 µM), NifB-co (0.75 nmol
of iron), NifH (52 µg of protein), 0.2 ml of each extract (1.7-2.5
mg of protein), and nif-apodinitrogenase (34 µg) when
stated. Thirty-four µg of purified nif-apodinitrogenase
produced 36 nmol of ethylene/min when reconstituted with an excess of
purified FeMo-co. The nif-apodinitrogenase present in 200 µl of UW45 (W) extract produced 35 nmol of ethylene/min when
reconstituted with an excess of purified FeMo-co. The amounts of
nif-apodinitrogenase (purified or as extract of UW45 (W))
are saturating in the assays. Total volume equals 0.75 ml. (V) or (W)
indicate that the cells used were grown in the presence of vanadium (V)
or tungsten (W).
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Because the reaction mixture used contains all factors necessary for
FeMo-co synthesis, except for Mo, it is important to show that the
activity seen (or part of it) is not due to FeMo-co being synthesized
using contaminating Mo present in the system. The level of activity
arising from contaminating Mo present in the assay solutions and in the
extract of UW45 grown in tungsten is very low, as can be seen in Table
I, line 9. This control assay contains all components
necessary for in vitro FeMo-co synthesis, except for Mo. A
control experiment using an air-oxidized extract of vanadium-grown
A. vinelandii CA11.1 and all other factors necessary for the
reaction exhibited significantly reduced activity (Table I, line
5) compared with the experimental values, showing that the
activities seen in Table I, lines 1 and 2 do not
arise from contaminating Mo supplied by the extract of A. vinelandii CA11.1. Furthermore, when the assay was carried out
with an extract of a tungsten-tolerant A. vinelandii strain
that is incapable of Mo accumulation (CA11.6.82, tungsten-tolerant,
vnfD82::Tn5-B21
nifHDK Rifr, (24)), similar levels of activity were observed
(Table I, line 4).
To show that the cofactor present in this nitrogenase contains
vanadium, the assay was carried out under standard conditions using
purified nif-apodinitrogenase and an extract of A. vinelandii CA11.1 that had been grown in the presence of
49V (a radioactive isotope of vanadium). The assay mixture
was electrophoresed under anoxic native conditions, and the gel was
analyzed by phosphorimaging. A transfer of label from VnfX to the
position of dinitrogenase was observed (Fig.
1, lane 2). An extract of
A. vinelandii CA12 (
nifHDK) grown in the
presence of 49V was loaded in Fig. 1, lane 4 as
a standard to show the migration of vnf-dinitrogenase in the
gel. 55Fe-labeled nif-dinitrogenase obtained by
in vitro FeMo-co synthesis using 55Fe-NifB-co is
shown in Fig. 1, lane 5. Both nitrogenases run as multiple
bands in anoxic native gel conditions. The reason for this is unknown.
It has been described previously that in A. vinelandii CA11.1 most of the 49V accumulates on VnfX, as seen in Fig.
1, lane 1, in the form of a vanadium-containing Fe-S cluster
(16).

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Fig. 1.
Phosphorimage of a native anoxic gel showing
transfer of 49V to dinitrogenase. Reaction mixtures
shown in lanes 1-3 contained an ATP-regenerating mixture
(see "Experimental Procedures"), homocitrate (0.1 mM),
NifB-co (0.75 nmol of iron), NifH (52 µg of protein) and the
following: lane 1, extract of A. vinelandii
CA11.1 ( nifHDK vnfDGK::spc) grown
in 49V-containing medium; lane 2, extract of
A. vinelandii CA11.1 grown in 49V-containing
medium and purified nif-apodinitrogenase; lane 3,
extract of A. vinelandii CA11.1 grown in
49V-containing medium and extract of A. vinelandii CA117.3 ( nifDK nifB) grown
in vanadium-containing medium. Lane 4 contains an extract of
A. vinelandii CA12 ( nifHDK) grown in
49V-containing medium. Lane 5, complete in
vitro FeMo-co synthesis reaction containing
55Fe-labeled NifB-co, 1 µM
MoO4 2, and extract of A. vinelandii UW45 (nifB ) grown in
tungsten-containing medium.
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Experiments in which a source of vnf-apodinitrogenase was
provided instead of nif-apodinitrogenase were also
conducted. Extracts of A. vinelandii CA117.3
(
nifDK
nifB) and CA11.8
(
nifHDKvnfH) were used as sources of
vnf-apodinitrogenase. Unexpectedly, very low nitrogenase
activity was observed in these experiments (Table I, lines 6 and 7). Accordingly, no 49V was seen associated
to dinitrogenase by phosphorimage analysis of anoxic native gels of
these incubations (Fig. 1, lane 3). The reason for the
failure to see nitrogenase activity in these conditions is currently
unknown. All the factors necessary for the synthesis of FeV-co should
be present in the extract of A. vinelandii CA11.1 grown in
vanadium, because the addition of purified
nif-apodinitrogenase alone is enough to see the activity.
Thus, the lack of activity observed when using
vnf-apodinitrogenase could be due to inefficient insertion
of the cofactor. It is possible that an unknown factor required for
insertion is labile and does not withstand the cell lysis or assay
conditions. It has been shown that the product of vnfG is
required for insertion of FeV-co into vnf-dinitrogenase (21). VnfG was present in the extracts used as a source of
vnf-apodinitrogenase, as estimated by Western blot analysis
(data not shown). What protein, if any, was facilitating the insertion
of the newly formed FeV-co into nif-apodinitrogenase in the
experiments described earlier is not known. VnfG was not present in the
incubation mixtures containing nif-apodinitrogenase, because
the mutant strain used (CA11.1) has a deletion in vnfDGK.
The non-nif protein gamma has been shown to function as a
chaperone that aids insertion of FeMo-co into
nif-apodinitrogenase (26). Gamma is expressed under
vnf conditions and was present in the extracts of A. vinelandii CA11.1, as well as part of the
nif-apodinitrogenase used
(
2
2
2). A stable association of FeV-co with gamma has not been observed before and gamma
is not able to replace VnfG effectively in the insertion of FeV-co into
vnf-apodinitrogenase (21). It is possible that gamma can
transiently associate with FeV-co and allow its insertion into
nif-apodinitrogenase. Alternatively, some other protein or VnfX itself may be acting as the insertase in this system. Experiments with purified components where no gamma is present will provide insight
into this problem.
Effect of Homocitrate Analogues--
Because A. vinelandii CA11.1 is defective in the subunits for
vnf-dinitrogenase and not in any of the genes known
to encode proteins required for the biosynthesis of FeV-co, it seemed
possible that the activity seen in Table I (lines 1,
2, and 4) was due to FeV-co present in this
extract. It has been reported that the V-Fe-S cluster accumulating on
VnfX in extracts of A. vinelandii CA11.1 contains no or very
low levels of homocitrate (16). To test whether homocitrate
incorporation into the unfinished cluster occurs in vitro,
we studied the effect of homocitrate analogues on the reaction. These
organic acids are able to replace homocitrate in the FeMo-co synthesis
system (27, 32), resulting in the synthesis of aberrant forms of
FeMo-co that are known to exhibit altered substrate specificity (27,
32). The use of isocitrate and malate in FeMo-co synthesis results in
dinitrogenases with 8-16% of the wild type acetylene reduction
activities (27). When added in an excess in in vitro FeMo-co
synthesis assays containing homocitrate, these organic acids
cause a reduction in the level of the acetylene reduction activity of
the resulting dinitrogenase. Isocitrate and malate were added to the
assays containing an extract of vanadium-grown A. vinelandii CA11.1 and nif-apodinitrogenase (Table
II). When no organic acid was added to
the reaction, some residual activity was seen, presumably resulting
from the homocitrate present in the extract. High activity was observed
when 0.1 mM homocitrate (final concentration) was added.
This concentration has been shown to be saturating for the in
vitro FeMo-co synthesis reaction (32). In assay mixtures
containing 0.1 mM homocitrate and isocitrate (5 and 10 mM), acetylene reduction activities were decreased by 86 and 89%, respectively. The highest concentration of malate used
(20 mM) resulted in a 46% decrease in the acetylene reduction ability of nitrogenase. To ensure that the effect of the
organic acids observed was not an inhibition of the coupled acetylene
reduction assay, isocitrate and malate were added to control reactions
immediately before the acetylene reduction assays were started (Table
II, lines 10-13). The highest concentrations used of malate
and isocitrate only inhibited acetylene reduction by 10.4 and 5.9%,
respectively. Thus, the low activities observed when adding the organic
acids during the synthesis phase probably reflect the incorporation of
the homocitrate analogues into FeV-co during the reaction.
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Table II
Effect of homocitrate analogues on acetylene reduction activity by
resulting nitrogenase
In vitro FeV-co synthesis assays were carried out under
standard conditions using purified nif-apodinitrogenase (34 µg) and an extract of A. vinelandii CA11.1 grown in the
presence of vanadium (1.7 mg of protein), as described in
"Experimental Procedures." Thirty-four µg of purified
nif-apodinitrogenase produced 36 nmol of ethylene/min when
reconstituted with an excess of purified FeMo-co.
nif-apodinitrogenase is saturating in the assays. HC,
homocitrate.
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Requirements for the Homocitrate Incorporation Reaction--
To
further characterize the reaction taking place when incubating the
extract of vanadium-grown A. vinelandii CA11.1 with nif-apodinitrogenase, a crude extract was chromatographed on
Sephadex G-25 to remove low molecular
weight molecules. The desalted extract was used for the reactions described in Table III and Fig.
2. As expected, the complete system
showed high acetylene reduction activity (Table III, line 1)
and a shift of the 49V label to the position of nitrogenase
in the anoxic native gel (Fig. 2, lane 2). No activity was
observed when homocitrate was omitted from the reaction mixture (Table
III, line 4). Accordingly, the appearance of the label on
nitrogenase was dependent on the presence of homocitrate (Fig. 2,
lane 3). 55Fe-labeled nif
dinitrogenase obtained by in vitro FeMo-co synthesis using
55Fe-NifB-co is shown in Fig. 2, lane 6. The
hexameric form of nif-apodinitrogenase (
2
2
2), which is directly
activable by FeMo-co, was used in these reactions. Apodinitrogenase can
also exist in a tetrameric form (
2
2),
which is not activable by FeMo-co. The maturation of the
2
2 form into the
2
2
2 form requires ATP and
dinitrogenase reductase (26, 33) but not the insertion of FeMo-co into
the mature
2
2
2 form. In
our system, ATP was found not to be necessary for the homocitrate
incorporation reaction or for the insertion of FeV-co into
apodinitrogenase (Table III, line 5; Fig. 2, lane 5). The omission of NifB-co from the incubation mixture did not have a significant effect on the observed activity (Table III, line 6), nor did the exclusion of
Na3VO4 or its replacement by VCl3
(Table III, lines 7 and 8). Similarly, the
presence of unlabeled Na3VO4 in the reaction
mixture did not affect the intensity of the 49V label seen
on nitrogenase (Fig. 2, lane 4). These observations are
consistent with the idea that the reaction occurring in
vitro is the incorporation of homocitrate into the cluster
associated with VnfX that already contains vanadium, iron, and sulfur.
When the reaction was carried out using a desalted extract of A. vinelandii CA11.1 grown with a source of unlabeled vanadium, and
49VCl5 was included in the reaction mixture, no
49V was observed associated with dinitrogenase in
phosphorimages of anoxic native gels, despite high nitrogenase
activities (data not shown). Thus, the in vitro FeV-co
synthesis system seems to use only vanadium already associated
with the cluster. It is also conceivable that incorporation of
vanadium into the cluster occurs in vitro but that
the system is only able to use the chemical form of vanadium present in
the extract, which may be different from the form of vanadium supplied
externally. However, desalted 49V-labeled extracts show
homocitrate-dependent incorporation of 49V into
nitrogenase (Fig. 2, lane 2), and thus, we conclude that the
label is transferred from a macromolecule-bound form.

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Fig. 2.
Homocitrate-dependent
incorporation of 49V into dinitrogenase.
49V-labeled proteins were detected by phosphorimage
analysis after anoxic native gel electrophoresis. Reactions shown in
lanes 1-5 contained an extract of 49V-grown
A. vinelandii CA11.1 that had been desalted by Sephadex G-25
chromatography, nif-apodinitrogenase, and other components
of the complete reaction mixture described under "Experimental
Procedures," except as noted. Lane 1, minus
nif-apodinitrogenase; lane 2, complete system;
lane 3, minus homocitrate; lane 4, plus
Na3VO4; lane 5, minus MgATP;
lane 6, complete in vitro FeMo-co synthesis
reaction containing 55Fe-labeled NifB-co, 1 µM MoO4 2, and
extract of A. vinelandii UW45
(nifB ) grown in tungsten medium.
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Complementation of Crude Extracts of Other FeV-co-negative
Mutants--
To assess whether earlier steps in the synthesis of
FeV-co occur in vitro, we tried to complement extracts of
mutant strains defective in gene products known to be involved in
FeV-co synthesis with the missing components. VnfH (NifH) is known to
be involved in FeV-co (FeMo-co) synthesis (12, 13), although its exact role during the synthesis remains unknown. Thus, nifH vnfH
double mutants are unable to synthesize FeMo-co and FeV-co. No
acetylene reduction activities were seen when trying to complement an
extract of a mutant strain defective in VnfH and NifH (A. vinelandii CA11.8) grown in vanadium medium with an extract of
UW45 grown in tungsten medium, which contains NifH and
nif-apodinitrogenase (Table
IV, line 1). The same results
were obtained when purified apodinitrogenase and NifH were used instead
of the extract of UW45 grown in tungsten (Table IV, line 3)
or when a source of VnfH (an extract of CA117.3 grown in vanadium) was
added to these assays (Table IV, lines 2 and 4).
A mutant strain defective in VnfNE and NifNEX (A. vinelandii DJ42.48) was also used in complementation studies. Extracts of A. vinelandii DJ42.48 grown in vanadium medium were complemented either with purified NifNE and VnfX (in the presence of
nif-apodinitrogenase) (Table IV, lines 7 and
8) or with an extract of A. vinelandii UW45 grown
in tungsten medium and VnfX (Table IV, lines 5 and 6). The VnfX used in these assays was purified from a
nifB
strain and does not have a cluster bound
to it (16). The acetylene reduction activities observed in these
conditions were not significantly above background. These results
indicate that earlier steps in the synthesis of FeV-co do not occur
in vitro under the assay conditions used in this study. They
further strengthen the notion that the activities reported in Table I
correspond to the addition of homocitrate to an already formed cluster
that accumulates on VnfX in the mutant strain CA11.1. In contrast,
in vitro synthesis of FeMo-co starting from NifB-co is
routinely observed when using conditions similar to those tried here
for FeV-co synthesis. The failure to obtain earlier steps in FeV-co
synthesis in vitro is not currently understood.
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Table IV
Acetylene reduction activities of extracts of vnf
strains grown in vanadium medium
Assays are complemented with an extract of A. vinelandii
UW45 (nifB) grown in tungsten medium or with purified components.
Assays contained homocitrate (0.1 mM), ATP-regenerating
mix, Na3VO4 (20 µM), NifB-co (0.75 nmol
of iron), NifH (52 µg of protein), 0.2 ml of each extract (1.7-2.5
mg of protein), and purified components when stated. VnfX (4.5 µg/assay) was purified from a nifB strain and
does not have a cluster bound to it. NifNE (5 µg/assay) produced 16 nmol of ethylene/min when assayed with an extract of strain DJ35 (Mo)
( nifE). nif-apodinitrogenase (34 µg/assay)
produced 36 nmol of ethylene/min when reconstituted with an excess of
purified FeMo-co. The nif-apodinitrogenase present in 200 µl of UW45 (W) extract produced 35 nmol of ethylene/min when
reconstituted with an excess of purified FeMo-co. The amounts of
nif-apodinitrogenase (purified or as extract of UW45 (W))
are saturating in the assays. Total volume equals 0.75 ml. (V) or (W)
indicate that the cells used were grown in the presence of vanadium (V)
or tungsten (W).
|
|
Conclusions--
The results of this study suggest that there is
incorporation of homocitrate in vitro into the V-Fe-S
cluster associated with VnfX in A. vinelandii CA11.1. The
newly formed vanadium-containing cluster can be inserted into
nif-apodinitrogenase to yield an active enzyme that can
convert acetylene to ethylene and ethane. Thus, incorporation of
homocitrate into FeV-co would occur as a late step in the synthesis
pathway of the cluster. More research needs to be done to identify what
proteins are involved in the homocitrate incorporation reaction.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Paul Bishop and Dr. Dennis Dean
for providing the mutant strains used in this study. We are thankful to
Luis Rubio and Gary Roberts for critical reviewing of the manuscript and to Sara Lange for her assistance in growing the cells used for this study.
 |
FOOTNOTES |
*
This work was supported by NIGMS, National Institutes of
Health Grant GM35332 (to P. W. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 608-262-6859;
E-mail: ludden@biochem.wisc.edu.
Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M007288200
2
P. Rangaraj, personal communication.
 |
ABBREVIATIONS |
The abbreviation used is:
-co, cofactor.
 |
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