From the Departments of Biochemistry and
Bacteriology and the § Center for the Study of
Nitrogen Fixation, University of Wisconsin,
Madison, Wisconsin 53706
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ABSTRACT |
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The biosynthesis of the iron-molybdenum cofactor
(FeMo-co) of dinitrogenase was investigated using
99Mo to follow the incorporation of Mo into
precursors. 99Mo label accumulates on dinitrogenase only
when all known components of the FeMo-co synthesis system, NifH, NifNE,
NifB-cofactor, homocitrate, MgATP, and reductant, are present.
Furthermore, 99Mo label accumulates only on the gamma
protein, which has been shown to serve as a chaperone/insertase for the
maturation of apodinitrogenase when all known components are present.
It appears that only completed FeMo-co can accumulate on the gamma
protein. Very little FeMo-co synthesis was observed when all known
components are used in purified forms, indicating that additional
factors are required for optimal FeMo-co synthesis. 99Mo
did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is
not observed. 99Mo accumulates on several unidentified
species, which may be the additional components required for FeMo-co
synthesis. The molybdenum storage protein was observed and the
accumulation of 99Mo on this protein required nucleotide.
The iron-molybdenum cofactor
(FeMo-co)1 of dinitrogenase
(Fig. 1) constitutes the active site of
the nif-encoded, molybdenum-containing dinitrogenase protein
in Azotobacter vinelandii and other nitrogen-fixing organisms (1-3). FeMo-co can be isolated by extraction from the purified dinitrogenase protein (2), and the isolated cofactor can be
used to activate FeMo-co-deficient forms of dinitrogenase (referred to
hereafter as "apodinitrogenase") that accumulate in strains unable
to synthesize the cofactor (2, 4, 5). FeMo-co consists of Mo, Fe, and S
atoms in a 1:7:9 ratio; in addition, the organic acid homocitrate is an
integral component of the compound (6), serving as a nonprotein ligand
to the molybdenum atom. The structure of FeMo-co in the protein was
determined by Kim and Rees (7) and Chan et al. (8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The structure of FeMo-co, after Kim and Rees
(7).
Genetic studies have revealed that functional copies of the nifB, nifN, nifE, nifH, and nifV genes are required for synthesis of FeMo-co in vivo (9-11); the nifQ gene is also required under conditions of molybdenum limitation (12). The nifKD genes, which encode the subunits of dinitrogenase (NifKD), are not required for FeMo-co synthesis, and thus FeMo-co is not synthesized "in place" but rather is preformed and then transferred to its site in dinitrogenase (13). In the absence of NifKD, completed FeMo-co accumulates on a protein called gamma, which serves as a chaperone/insertase for the maturation of NifKD and for insertion of FeMo-co (14).
An in vitro FeMo-co synthesis system was devised to address the biochemical roles of these genes and to identify other factors required for FeMo-co synthesis (15). In this system, at least homocitrate, molybdenum (supplied as molybdate), MgATP, NifB-co, NifH (dinitrogenase reductase), reductant, and NifNE are required to achieve FeMo-co synthesis. Each of these components is available in a purified form, but when they are added together, very little FeMo-co synthesis is observed, suggesting that additional factor(s), yet to be discovered, are required.
Previous studies have shown that NifB-co contains iron and sulfur (16, 17). Purified, 55Fe- and 35S-labeled NifB-co has been shown to bind to NifNE in the absence of other factors, and the label can be shown to accumulate on the gamma protein and in dinitrogenase (17, 18). Thus NifB-co is believed to be the primary, if not sole, iron donor to FeMo-co.
The entry point of molybdenum into the pathway of FeMo-co synthesis has
not been established. Pienkos and Brill (19) used 99MoO42 to study the
incorporation of molybdenum in A. vinelandii
cells in vivo. They observed the accumulation of molybdenum
in a non-nif protein that they termed the molybdenum storage
protein (Mo-Sto) and in the dinitrogenase protein in vivo.
Ugalde et al. (13) noted the accumulation of
99Mo on a 65-kDa protein in K. pneumoniae in
nifKD mutants; this protein may be the nifY gene
product or an analog of the gamma protein. The accumulation of
99Mo on dinitrogenase and on possible precursors during
in vitro FeMo-co synthesis was investigated by Hoover
et al. (3) using various homologs of homocitrate in the
synthesis system.
In this study, the incorporation of 99Mo into protein-bound
precursors of FeMo-co during in vitro FeMo-co synthesis was
investigated. In vitro FeMo-co synthesis mixtures containing
extracts and cofactors were mixed with
99MoO42 under anoxic
conditions and following incubation to allow FeMo-co synthesis; the
mixtures were electrophoresed on nondenaturing polyacrylamide
gels. Protein bands containing 99Mo were detected by
phosphorimage analysis. Where possible, 99Mo-labeled
protein bands were identified by co-migration with purified proteins
known to be involved in FeMo-co synthesis and by reaction with
antibodies against those known proteins on immunoblots.
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EXPERIMENTAL PROCEDURES |
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Strains and Growth Conditions-- A list of the Azotobacter vinelandii mutant strains used in this study is presented in Table I. In order to avoid dilution of the 99Mo with nonradiolabeled molybdenum, all A. vinelandii strains in this work (except as specified) were grown and derepressed in medium containing tungsten in place of molybdenum as described previously (15). Cells grown in medium containing tungsten in place of molybdenum accumulate apodinitrogenase and all other identified components required for FeMo-co synthesis (19).
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Preparation of Extracts-- Cell-free extracts were prepared as described (20). Small molecules (i.e. homocitrate and nucleotides) were removed from the cell extracts by Sephadex G-25 column chromatography. Where necessary, crude extracts were chemically oxidized as described previously (21).
Anoxic Native Gel Electrophoresis, Western Immunoblots, and Visualization of Radioactivity-- Gel electrophoresis, Western immunoblot procedures, and 99Mo visualization by phosphorimaging were performed as described previously (17).
In Vitro FeMo-co Synthesis Assay-- Nine-ml serum vials were flushed with purified argon and rinsed with anoxic, 25 mM Tris-HCl (pH 7.4) containing 1.7 mM sodium dithionite (DTH). Complete FeMo-co synthesis reaction mixtures (defined below) were prepared by combining the following: 100 µl of anoxic 25 mM Tris-HCl (pH 7.4) (containing 1.7 mM DTH, 10 µl of 10 µM Na2MoO4, 20 µl of 5 mM homocitrate (which had been treated with base to cleave the lactone, pH 8.0)) and 200 µl of an ATP-regenerating mixture (containing 3.6 mM ATP, 6.3 mM MgCl2, 51 mM phosphocreatine, 20 units/ml creatine phosphokinase, and 6.3 mM DTH in 25 mM Tris-HCl; pH of the mixture was 7.4). These mixtures were incubated anoxically at room temperature for 10-15 min. Two hundred µl of the appropriate A. vinelandii cell-free extract, 10 µl of a solution containing an excess of NifB-co as Fe and S donor for FeMo-co (16), and 0.1 mg of purified dinitrogenase reductase (10 µl) were added to the reaction mixtures. One-half µCi (carrier free) of Na299MoO4 (10 µl) (Nordion, Ontario, Canada) prepared in 25 mM Tris (pH 7.4) containing 1.7 mM DTH was then added to the reaction. The total volume of each assay mixture was 560 µl. The vials were incubated in a rotary water-bath shaker at 30 °C for 30 min to allow time for FeMo-co synthesis and its subsequent insertion into apodinitrogenase. After this incubation (designated the "incubation phase" of the assay), samples to be applied onto native polyacrylamide gels were placed on ice until loading. To demonstrate that all components of the assay were functional, the synthesis of FeMo-co was monitored in duplicate vials using the acetylene reduction assay (17, 22) to detect newly formed active dinitrogenase. Demonstration that a significant portion of the apodinitrogenase molecules present in the assay are activated by binding of newly synthesized FeMo-co was accomplished by demonstrating that acetylene reduction activity of the mixture was comparable to mixtures to which an excess of purified FeMo-co was added.
Definition of Assays--
In vitro synthesis
reactions in which all components known to be required for FeMo-co
synthesis are present, in addition to apodinitrogenase, will be
referred to as a "complete reaction." Components are supplied in
extracts or in purified form, as indicated. Although FeMo-co synthesis
does not require the presence of apodinitrogense, acetylene reduction
activity resulting from the activation of apodinitrogenase is used to
monitor FeMo-co synthesis. Reactions that utilized extracts of mutants
lacking specific gene products required for FeMo-co synthesis are
referred to by the known component that is excluded. For example, if
all known components required for FeMo-co synthesis are present except
NifNE, the reaction will be referred to as "NifNE." If this
reaction also lacks apodinitrogenase, it will then be referred to as
"
NifNE,
apodinitrogenase."
The purified in vitro FeMo-co synthesis reaction included NifNE, NifH, homocitrate, Na299MoO4, MgATP, DTH, and NifB-co. No apodinitrogenase was included in the experiments that employed the purified system; therefore, no label was detected at the position of dinitrogenase in those gels.
Purification of Other Components--
FeMo-co was purified as
described by Shah and Brill (2). Dinitrogenase and NifH (dinitrogenase
reductase) proteins were prepared as described previously (23). NifNE
was purified as described (18). Gamma protein, purified as described in
Ref. 14, was a gift from Mary Homer. Dinitrogenase reductase protein from Clostridium pasteurianum was a gift from Dr. Lance
Seefeldt. Antibodies against various proteins were prepared at the UW
antibody facility.
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RESULTS AND DISCUSSION |
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99Mo Is Detected on the Molybdenum Storage
Protein--
When 99Mo-labeled molybdate is supplied to
extracts capable of FeMo-co synthesis, two proteins become prominently
labeled (Fig. 2, lane 2).
These are the dinitrogenase protein and the molybdenum storage protein
(Fig. 2, Mo-Sto). Mo-Sto was previously shown to accumulate
99Mo in vivo and the protein was purified by
Pienkos and Brill (19). Mo-Sto is not co-regulated with nitrogenase,
and the protein purified as an 2
2
tetramer of 21 and 24 kDa subunits with approximately 15 Mo
atoms/tetramer, although the Mo content of the protein decreased during
purification. The protein labeled as Mo-Sto in Fig. 2 and elsewhere is
proposed to be the same protein based on its accumulation of Mo, its
position of migration on nondenaturing gels, and its expression in
NH4+-grown cells. Based on its size and
on results presented here, Mo-Sto would not appear to be the
periplasmic molybdate-binding protein, ModA, or the molybdate-binding
regulatory protein, ModE (24, 25). Mo-Sto is soluble, and thus it is
not likely to be the product of the modB, modC,
or modD genes, which encode the membrane-bound molybdate
transport system that has been described in A. vinelandii
and Escherichia coli.
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Nucleotide Is Required for the in Vitro Labeling of
Mo-Sto--
Previously, it had been shown that Mo-Sto was
99Mo-labeled during in vitro FeMo-co synthesis,
and studies here expand that observation to show that either ATP or ADP
is required for accumulation of 99Mo on Mo-Sto (Fig. 2)
(26). In the absence of nucleotide, label does not accumulate on any
protein observed by phosphorimage. At this point, it is unknown whether
the nucleotide is required to allow
MoO42 to bind to Mo-Sto or if it is
required to allow the removal of nonradioactive species
(MoO42
or
WO42
) already bound to the protein,
thereby making sites for binding of
99MoO42
available.
Previous studies showed that 185W accumulated in
185WO42
-grown cells on a
protein that copurified with Mo-Sto (19). The extracts used in
experiments reported here were prepared from cells grown in the absence
of Na2MoO4 and the presence of
Na2WO4 to prevent the accumulation of
molybdenum-containing intermediates in the FeMo-co synthetic pathway.
Thus, it is possible that nucleotide is required to allow unlabeled W
species to vacate sites for
99MoO42
. This question
will be addressed in future studies.
ATP Is Required for 99Mo to Enter the FeMo-co Biosynthetic Pathway-- Previous studies have shown that ATP is required for the biosynthesis of FeMo-co and for the insertion of FeMo-co. Results in Fig. 2 show that ATP (Fig. 2, lane 2), but not ADP (Fig. 2, lane 3), will support synthesis of FeMo-co when all other required factors are present. In the absence of nucleotide, 99Mo does not accumulate on any protein band and when ADP is supplied, Mo-Sto becomes labeled, but Mo does not enter the FeMo-co biosynthetic pathway. Thus, the requirement for nucleotide does not represent merely a requirement for label to pass through Mo-Sto, as that protein can be labeled in the presence of ADP.
99Mo Accumulates on the Dinitrogenase Protein--
As
shown in Figs. 2 and 3, 99Mo
accumulates on the protein identified as the dinitrogenase protein when
a complete in vitro FeMo-co synthesis mixture was used. This
band has been identified as dinitrogenase by comigration with purified
dinitrogenase detected by cross reaction with anti-dinitrogenase
antibodies. The amount of Mo incorporated into FeMo-co and inserted
into dinitrogenase in a typical complete reaction mixture (for example,
Fig. 3, lane 1) was estimated to be approximately 1.2 atoms
of Mo per molecule of dinitrogenase. The amount of dinitrogenase
present was estimated by quantitation of the protein band on
immunoblots compared with the standards of known quantity of purified
dinitrogenase. The amount of Mo incorporated was estimated from the
known specific activity of 99Mo in the reaction mixture and
the quantitation of the amount of radioactivity in the band by
comparison with known amounts of 99Mo blotted on a piece of
filter paper. ImageQuant (Applied Biosystems) software was used in the
measurement. Furthermore, an assay equivalent to that shown in Fig. 3,
lane 1, exhibits > 80% of the acetylene reduction
activity of a mixture to which an excess of purified FeMo-co is added
(e.g. Fig. 3, lane 6). When one or more
components of the FeMo-co synthesis reaction mixture are left out of
the assay, 99Mo does not accumulate on dinitrogenase, and
acetylene reduction activity is not observed. As noted above, ATP is
also essential for label to appear in the dinitrogenase band (Fig. 2).
When any of the following are eliminated from the reaction mixture,
accumulation of 99Mo on dinitrogenase is not observed:
NifNE (Fig. 3, lane 4), NifB-co (Fig. 3, lane 5),
and NifH (Fig. 3, lane 7). Furthermore, when an extract of
nif-repressed (NH4+-grown),
wild type A. vinelandii is used in the assay, only Mo-Sto is
observed to accumulate 99Mo, and neither dinitrogenase nor
any other protein is labeled. Finally, if the assay is saturated with
unlabeled FeMo-co, 99Mo does not accumulate on
dinitrogenase (Fig. 3, lane 6); this indicates that all the
sites for FeMo-co on dinitrogenase are occupied and any
99Mo-labeled FeMo-co that is synthesized accumulates
elsewhere. In the reaction mix used for Fig. 3, lane 6, the
label accumulates on gamma protein and on an unidentified band
(band B). The migration of gamma protein at this position
has been established by comigration with purified gamma protein and by
cross reaction with anti-gamma protein antibodies. When homocitrate is
left out of the reaction mixture, label fails to accumulate as
completed FeMo-co on gamma protein (Fig. 3, lane 8; in this
reaction, both homocitrate and apodinitrogenase were left out of the
mix). This result suggests that only completed FeMo-co is capable of
being stably bound to gamma protein.
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99Mo Is Not Observed on NifNE-- It is surprising that no 99Mo is detectable on NifNE in these experiments. From previous studies, it is known that NifNE migrates a little faster than dinitrogenase in the gel system employed here (17, 18). It is known that purified NifNE and NifNE in an in vitro FeMo-co synthesis mixture will bind NifB-co and that no additional factors are required for NifNE to bind NifB-co (17, 18). 55Fe-labeled NifB-co is known to donate Fe atoms to FeMo-co (17). A significant degree of sequence similarity is observed between nifNE and nifKD, and the cysteine residue that serves as the ligand to FeMo-co in dinitrogenase is conserved in the deduced sequence of NifNE (27). Thus it has been proposed that NifNE is the scaffold upon which FeMo-co is completed. The results presented here do not support that hypothesis, as 99Mo is not observed at the position of the NifNE band under any conditions. If Mo did bind to NifNE, accumulation of 99Mo on NifNE might have been expected in a mixture in which a component that performed a step in FeMo-co synthesis subsequent to NifNE was omitted from the reaction mixture; no reaction mixture lacking a single component of the assay mixture resulted in accumulation of 99Mo on NifNE (Fig. 3). Furthermore, binding of NifB-co to NifNE is not inhibited by FeMo-co (17).
It is very important to note that only those complexes of proteins with 99Mo that are sufficiently stable to remain intact during electrophoresis will be observed with the approach employed here. Therefore, it is possible that Mo does enter the pathway at the NifNE step but that the resulting intermediate is not stable during electrophoresis. Our working hypothesis is that Mo enters the FeMo-co synthesis pathway at a step subsequent to NifNE.
99Mo Accumulates on the Gamma Protein--
The gamma
protein serves as a chaperone/insertase for the maturation of
apodinitrogenase and the insertion of FeMo-co (14, 28). It was first
observed as a protein that co-purified with apodinitrogenase from
nifB mutants (5). Subsequent work has shown that gamma
protein is associated with the nifKD gene products in
extracts of nifB mutants. Gamma protein becomes associated with apodinitrogenase in the presence of NifH and MgATP
(apodinitrogenase from nifH mutants accumulates as an
2
2 tetramer of nifK and nifD gene products) and is required for the insertion of
FeMo-co (28). Free gamma protein exists as a dimer and binds purified FeMo-co in the absence of any additional factors; upon binding FeMo-co,
gamma dimers dissociate into monomeric gamma:FeMo-co units (14). In the
in vitro FeMo-co synthesis system, 99Mo
accumulates at the position of the gamma protein in a system that
includes all components required for FeMo-co synthesis, but lacks the
apodinitrogenase (Fig. 3, lane 2). When the apodinitrogenase is available, most of the label accumulates on dinitrogenase as a
result of insertion of completed FeMo-co into the apodinitrogenase. In
these experiments, the position of gamma protein with FeMo-co bound was
confirmed by co-electrophoresis with purified material. 99Mo is not observed at the position of the gamma protein
if NifNE, NifB-co, NifH, ATP, or homocitrate is excluded from the
reaction mixture (Fig. 3, lanes 4, 5, 7, and 8;
Fig. 2, lane 1). Because purified gamma protein will bind
FeMo-co, these results are consistent with the hypothesis that the
label accumulating at the position of gamma protein represents
completed FeMo-co bound to gamma protein. It is possible that the label
represents an immature form of FeMo-co bound to gamma protein, but
there are no data to suggest that hypothesis. Accumulation of
99Mo on gamma protein does not occur when an extract of
nif-repressed, wild type A. vinelandii is used in
the assay (Fig. 3, lane 3) even though that extract contains
gamma protein; expression of gamma protein is not co-regulated with the
nif regulon (14). Thus, the accumulation of 99Mo
on the gamma protein requires products of the nif regulon
that perform known steps of FeMo-co synthesis. Note that the assay with
the NH4+-grown extract did contain added
ATP, homocitrate and NifB-co; these plus the gamma protein are not
sufficient to allow accumulation of 99Mo on any protein in
the extract other than Mo-Sto. When excess, unlabeled FeMo-co is added
to the complete FeMo-co synthesis mixture, a decreased amount of
99Mo is observed on gamma protein (Fig. 3, lane
6), indicating that most of the sites are occupied by the added
FeMo-co.
What Is the Role of NifH in FeMo-co Synthesis?-- NifH (dinitrogenase reductase) plays several roles in nitrogen fixation. In addition to serving as the unique electron donor to dinitrogenase for N2 reduction, NifH is required for both FeMo-co synthesis and insertion (10, 28, 29). In the absence of NifH, no 99Mo accumulates on any protein other than Mo-Sto in the in vitro FeMo-co synthesis system (Fig. 3, lane 7); thus, Mo does not appear to enter the FeMo-co synthesis pathway in the absence of NifH. In the gel system employed here, NifH migrates in the region of the protein bands labeled C and D in Fig. 3. At this time, it is not possible to state definitively that one or the other of these 99Mo-labeled bands contains NifH. Identification of the proteins forming bands C and D will require at least partial purification of the labeled proteins, and this is being pursued at this time. It is interesting that a 55Fe-labeled band is observed in the same region of the gel as bands C and D when 55Fe-labeled NifB-co is used in the in vitro system (i.e. lanes 2 and 5 of Fig. 3 of Ref. 17). Like bands C and D, that 55Fe-labeled band appeared in reactions lacking homocitrate, consistent with the hypothesis that they represent FeMo-co precursors.
Collectively considered, these results suggest a model in which
NifB-co, synthesized by NifB, is donated to NifNE. In a MgATP- and
NifH-dependent step, molybdenum is incorporated and
accumulates on the protein bands marked as C and D. Homocitrate is
added last either as a prerequisite to transfer of completed FeMo-co to
gamma protein or as the final step of FeMo-co synthesis on gamma
protein. FeMo-co accumulates on gamma protein until NifH-mediated
association of gamma protein and apodinitrogenase occurs, whereupon
gamma protein transfers FeMo-co to apodinitrogenase. At some point, perhaps when MoO42 enters the pathway,
it must be reduced from Mo(VI) to Mo(IV), the formal oxidation state
proposed to be in FeMo-co. In previous experiments, a requirement for
reductant for in vitro FeMo-co synthesis was demonstrated
(21), although it has not been shown that the reductant is consumed
stoichiometrically with the amount of FeMo-co produced. Likewise, it is
shown here and elsewhere that ATP is required for FeMo-co synthesis,
although it is not known which step in the synthesis requires ATP. It
is not known whether ATP is hydrolyzed.
99Mo Incorporation Using a System of Purified
Components--
Experiments described above have utilized centrifuged,
desalted crude extracts of appropriate strains of A. vinelandii. Purified components known to be required for FeMo-co
synthesis include NifNE, NifB-co, Na2MoO4,
NifH, MgATP, homocitrate, and reductant (dithionite). When purified
samples of each of these are mixed with purified gamma protein and
purified apodinitrogenase, very little active dinitrogenase is formed.
The incorporation of 99Mo into components of the purified
system was investigated, and the results are shown in Fig.
4. No 99Mo accumulated on any
band in the absence of NifB-co (Fig. 4, lane 1), NifH
(lane 2), NifNE (lane 3), or ATP (lane
4). In the complete purified system, a small amount of label
accumulated in the region of bands C and D, similar to the bands C and
D of Fig. 3. In the absence of homocitrate, 99Mo
accumulated in a band that moves more slowly than band C and D; this
band was not observed in the crude system. Although complexes of
citrate with MoO42 and homocitrate
with vanadate have been reported (30-32),
MoO42
:homocitrate did not form a
species that migrates as a distinct band in our gel system; for
example, such a band would be expected in Fig. 4, lanes
1-4. It is difficult to avoid the conclusion that one or more of
the purified proteins added to the system is responsible for the
99Mo-labeled bands seen in lanes 5 and
6. These bands are in the approximate position of NifH, and
this protein is the best candidate for these bands, although we lack a
positive demonstration of NifH at exactly these positions. From other
work, it is known that binding of metal clusters to proteins can affect
the migration of the protein on gels (18).
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Time Course of 99Mo-Labeling of Mo-Sto and
Dinitrogenase--
Fig. 5 shows a time
course of labeling of Mo-Sto and dinitrogenase using a crude extract of
A. vinelandii strain UW45 (nifB) plus added
NifB-co, homocitrate, and MgATP. Mo-Sto becomes labeled first, and
dinitrogenase fails to show marked labeling until the 10 min time
point. More importantly, it appears that the entry of Mo into the
FeMo-co biosynthetic pathway is a slow step in the process, as there is
no significant accumulation of label on gamma protein or bands C and D. Any Mo that makes it into the system is rapidly converted to completed
FeMo-co that is inserted into dinitrogenase.
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Use of NifH from C. pasteurianum--
NifH from C. pasteurianum was used in place of NifH from A. vinelandii to supplement a crude extract of A. vinelandii strain UW97 (nifH) that lacks NifH. NifH
from C. pasteurianum is known to form an inhibitory complex
with A. vinelandii dinitrogenase that is unable to reduce
substrates (33). It was thought that C. pasteurianum NifH
might fail to function in FeMo-co synthesis or might form an inhibitory
complex with a component of FeMo-co synthesis, leading to accumulation
of an intermediate in the pathway. As seen in Fig.
6, C. pasteurianum NifH
functions effectively in FeMo-co synthesis in the crude A. vinelandii system, indicating that it possesses the structural
features required for this role of NifH. Some 99Mo
accumulation on gamma protein is observed in this experiment, indicating that the C. pasteurianum NifH might not be fully
effective in mediating the association of gamma protein with A. vinelandii dinitrogenase. Previously, we have shown that VnfH from
A. vinelandii will substitute for A. vinelandii
NifH in FeMo-co synthesis and gamma protein-dependent
insertion (34).
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The results presented here show that 99Mo label accumulates
on dinitrogenase only when all known components of the FeMo-co
synthesis system are present. Furthermore, 99Mo label only
accumulates on the gamma protein, which has been shown to be a
chaperone/insertase for the maturation of apodinitrogenase when all
known components are present. It appears that only completed FeMo-co
can accumulate on the gamma protein. Using all known components in
purified form, very little FeMo-co synthesis was observed, and
therefore, additional factors are required for optimal FeMo-co synthesis. 99Mo was not observed on NifNE under any
conditions tested, suggesting that Mo enters the pathway at some other
step, although it remains possible that a Mo-containing precursor of
FeMo-co that is not sufficiently stable to persist during gel
electrophoresis occurs but was not observed. 99Mo
accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. A nucleotide dependence for accumulation of Mo on Mo-Sto was demonstrated.
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ACKNOWLEDGEMENTS |
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We thank M. Homer and R. Chatterjee for helpful discussions. Purified NifH from C. pasteurianum was kindly provided by Lance Seefeldt. Purified gamma protein was kindly supplied by Mary Homer.
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FOOTNOTES |
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* This work was supported by NIGMS, National Institutes of Health, Grant 35332 (to P. W. L.) and by National Science Foundation Grant MCB-9604446 (to G. P. R.).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.
¶ Current address: Stratagene, 11011 N. Torrey Pines Rd., La Jolla, CA 92037.
** 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.
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ABBREVIATIONS |
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The abbreviations used are: FeMo-co, iron-molybdenum cofactor of nitrogenase; Mo-Sto, molybdenum storage protein; NifB-co, iron- and sulfur-containing precursor to FeMo-co; DTH, sodium dithionite.
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REFERENCES |
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