From the Department of Biochemistry and Center for the Study of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
Received for publication, January 31, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Iron-molybdenum cofactor (FeMo-co) biosynthesis
involves the participation of several proteins. We have used
55Fe-labeled NifB-co, the specific iron and
sulfur donor to FeMo-co, to investigate the accumulation of
protein-bound precursors of FeMo-co. The 55Fe label from
radiolabeled NifB-co became associated with two major protein bands
when the in vitro FeMo-co synthesis reaction was carried
out with the extract of an Azotobacter vinelandii mutant
lacking apodinitrogenase. One of the bands, termed
55Fe-labeled upper band, was purified and shown to be NifH
by immunoblot analysis. The 55Fe-labeled lower band was
identified as NifX by N-terminal sequencing. NifX purified from an
A. vinelandii nifB strain showed a different electrophoretic mobility on anoxic native gels than did NifX with the
FeMo-co precursor bound.
Nitrogenase, the enzyme responsible for the conversion of
dinitrogen to ammonium, is composed of two oxygen-labile
metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2).
During catalysis, dinitrogenase (also called MoFe protein or
NifKD)1 is specifically
reduced by dinitrogenase reductase (Fe-protein or NifH), one electron
at a time, until a sufficient number of electrons accumulates on
dinitrogenase for the subsequent reduction of substrate (3).
Dinitrogenase reductase, an Biochemical and genetic analyses of A. vinelandii and
Klebsiella pneumoniae mutants have revealed that the
biosynthesis of FeMo-co requires the participation of at least seven
different nif gene products: NifQ, V, B, H, X, N, and E
(8-12). Interestingly, the products of the structural genes for
dinitrogenase, nifD and K, are not necessary for
FeMo-co biosynthesis, suggesting that the cofactor is assembled
elsewhere in the cell and then is inserted into the FeMo-co-deficient
apodinitrogenase (13, 14). In the absence of apodinitrogenase (NifKD),
it is known that FeMo-co accumulates on a non-nif protein,
gamma ( Because FeMo-co is unstable in aqueous solutions, intermediates in the
FeMo-co biosynthetic pathway are believed to be present as
protein-bound species. The accumulation of molybdenum in the FeMo-co
biosynthetic pathway has been studied in various nif mutants of K. pneumoniae (13) and A. vinelandii (16) as
an approach to identify the sequence of steps in the FeMo-co
biosynthetic pathway. Previous studies have identified the metabolic
product of NifB, NifB-co, as a potential iron and sulfur source for
FeMo-co biosynthesis (17). Studies with purified
55Fe-labeled NifB-co have conclusively shown that NifB-co
serves as the specific iron and sulfur donor to FeMo-co (18).
Furthermore, nifB is one of the nif genes that is
required for the full activity of the Mo-independent nitrogen fixation
systems (19). Thus, NifB-co is proposed to function as the fundamental
iron-sulfur cluster for the synthesis of FeMo-co of Mo-nitrogenase,
FeV-co of V-nitrogenase, and FeFe-co of Fe-only-nitrogenase.
To identify proteins that play a role in FeMo-co biosynthesis, we have
used 55Fe-labeled NifB-co in the in vitro
FeMo-co synthesis reaction. We have followed the incorporation of the
radiolabel into FeMo-co and its precursors by electrophorescing the
reaction mixtures on non-denaturing polyacrylamide gels and detecting
the radiolabeled protein bands by phosphorimaging analysis. Here, we
report the accumulation of a radiolabeled species on NifH under
conditions in which FeMo-co synthesis is allowed to take place. Another
protein, NifX, also accumulates the radiolabel from
55Fe-labeled NifB-co under similar conditions.
Materials--
All materials used for growth medium preparation
were of analytical grade. Sodium dithionite (DTH) was from Fluka.
Leupeptin, phenylmethylsulfonyl fluoride, phosphocreatine, creatine
phosphokinase, and homocitrate lactone were from Sigma Chemical Co. ATP
was purchased as a disodium salt from Sigma. Tris base and glycine were
from Fisher Scientific Co. Nitrocellulose membrane was from Millipore, and the polyvinylidene difluoride membrane (Problott) was from Applied
Biosystems. Acrylamide/bis acrylamide solution (37.5%:1%) and the
equipment for SDS-PAGE were from Bio-Rad. Sephacryl S-200, Sephadex
G-75, Q-Sepharose, and phenyl-Sepharose were from Amersham Pharmacia
Biotech Inc. DE52 was a Whatman product.
55FeCl3 was from PerkinElmer Life Sciences.
Strains and Growth Conditions--
All strains were grown in the
presence of molybdenum and were nif-derepressed. A list of
the A. vinelandii mutant strains used in this study is
presented in Table I. The procedure for the preparation of cell-free extracts by osmotic shock has been described previously (20).
A. vinelandii strain CA11.3
( Conditions for the Growth of A. vinelandii Strain CA11.1 in the
Presence of 55FeCl3--
All glassware was
rinsed with 4 N HCl overnight to remove traces of
contaminating iron and then rinsed thoroughly with deionized water. A
25-ml starter culture of strain CA11.1 was grown in Burk's modified
medium (22) in the presence of 18 µM unlabeled iron and
400 µg of nitrogen/ml in the form of ammonium acetate at
30 °C for 20-24 h in a rotary shaker. One milliliter of this
culture was used as the inoculum for each of six 250-ml cultures, each containing 9 µM iron and 400 µg fixed nitrogen/ml. The
cultures were grown for 20-24 h, and the cells were then collected by
centrifugation. The supernatant was discarded, and the cell pellets
were resuspended in fresh medium containing 9 µM iron, 2 mCi of 55FeCl3 (18.5-20 mCi/ml) and with no
source of fixed nitrogen. 55Fe was added to the cultures as
ferric citrate, prepared according to the procedure described
previously (18). The cultures were derepressed for 4 h after which
the cells were harvested by centrifugation. The cell pellets were
stored at Buffer Preparation--
All buffers were sparged with nitrogen
for 20-30 min and degassed on a gassing manifold three times, with
alternating cycles of vacuum and flushing with argon gas. Sodium
dithionite (DTH) was added to a final concentration of 1.7 mM. Buffers used for protein purification contained 0.5 µg/ml leupeptin and 0.2 mM phenylmethylsulfonyl fluoride.
25 mM Tris-HCl (pH 7.5) was used throughout as the buffer unless
otherwise stated.
NifB-co Purification--
NifB-co was purified from the
cell-free extracts of unlabeled and 55Fe-grown A. vinelandii mutant CA11.1
( In Vitro FeMo-co Synthesis Assay--
FeMo-co synthesis
reactions were carried out as described by Shah et al. (11).
To 9-ml serum vials flushed with purified argon and rinsed with
anaerobic Tris-HCl buffer were added the following: 100 µl of
anaerobic Tris-HCl, 10 nmol of sodium molybdate, 100 nmol of
homocitrate, 200 µl of an ATP-regenerating mixture (containing 3.6 mM ATP, 6.3 mM MgCl2, 51 mM creatine phosphate, 20 units/ml creatine phosphokinase,
and 6.3 mM DTH), 200 µl of the appropriate A. vinelandii extract, and 10 µl of a solution containing purified
55Fe-labeled NifB-co (60,000 cpm). The total volume of the
reaction mixture was 555 µl. The reaction mixtures were incubated for
30 min at 30 °C. After incubation, the samples to be applied to
native polyacrylamide gels were placed on ice. Purified
apodinitrogenase (the Purification of Upper and Lower 55Fe-Labeled
Bands--
A 1200-fold scale-up of the in vitro FeMo-co
synthesis reaction containing the extract of strain CA117.3 and
55Fe-NifB-co was performed as follows. Thirty 100-ml vials
were stoppered and degassed thoroughly on a gassing manifold four times and rinsed with anoxic Tris-HCl buffer. To each vial were added the
following: 2 ml of anoxic Tris-HCl containing 1 mM DTH, 0.4 ml of 5 mM homocitrate, 0.2 ml of 1 mM sodium
molybdate, 8 ml of an ATP-regenerating mixture (as described earlier),
8 ml of extract of strain CA117.3
(
The DEAE-cellulose fractions containing the upper band were separated
and pooled from those containing the lower band. The upper band- and
lower band-containing pools were separately concentrated down to 12 ml
using an Amicon system fitted with a PM-10 membrane and subjected to
Sephadex G-75 column (2.5 × 95 cm) chromatography. The column was
equilibrated with anoxic Tris-HCl containing 0.05 M NaCl
and 10% glycerol. Ten-milliliter fractions were collected, and those
containing the upper band, as determined by anoxic gel electrophoresis
and phosphorimaging analysis, were pooled and brought to 1 M (NH4)2SO4. This was
applied to a 1- × 10-cm phenyl-Sepharose column equilibrated with 1 M (NH4)2SO4 in
Tris-HCl, washed with one column volume of the same buffer, and protein
was eluted stepwise with two column volumes each of 0.75, 0.5, 0.25, 0.025, and 0.0 M
(NH4)2SO4. The upper
band-containing fractions (0.25 M
(NH4)2SO4)) were pooled, diluted
2-fold with anoxic Tris-HCl, and concentrated by ultrafiltration using
the same membrane as stated above. The sample produced in this manner
was not homogeneously pure, so ~1 ml of this sample was subjected to
preparative anoxic native gel electrophoresis; the region of the gel
containing radioactivity was sliced, and the protein was then eluted
from the acrylamide, as described below.
The Sephadex G-75 fractions containing the 55Fe-labeled
lower band were pooled and subjected to phenyl-Sepharose column
chromatography, as described above. The lower band containing fractions
(0.5-0.25 M (NH4)2SO4)
were then pooled and concentrated using an Amicon system fitted with a
PM-10 membrane. One milliliter of this concentrate was applied to a
preparative anoxic native gel, and the region of the gel containing
radioactivity was sliced. The protein from the gel was eluted as
described below. A SDS gel of the elute showed an 18-kDa protein. The
protein was transferred to a polyvinylidene difluoride membrane, and
N-terminal sequencing was performed at the Protein/Peptide Micro
Analytical Laboratory at the California Institute of Technology
(Pasadena, CA).
Purification of NifX from A. vinelandii Strain CA11.3--
NifX
was partially purified from an A. vinelandii nifB strain,
CA11.3
( Assaying Activity of Partially Purified NifX--
The activity
of the partially purified NifX was followed by use of the in
vitro FeMo-co synthesis assay using the extract of the strain
DJ42.48 ( Purification of Other Components--
FeMo-co was purified
as described by Shah and Brill (7). Dinitrogenase and NifH were
purified as previously described (23). Apodinitrogenase was purified
from strain UW45 (nifB) as described by Paustian et
al. (24), and NifNE was purified from strain CA117.3
( O2 Denaturation of Components--
O2
treatment of cell-free extracts and various purified components was
performed by exposure of the samples to air for 10 min at room
temperature with gentle shaking. Following this, the samples were made
anaerobic by thoroughly degassing the stoppered vials on a gassing
manifold with alternating cycles of vacuum and flushing with Ar gas.
DTH was added to a final concentration of 1.7 mM.
Incubation of 55Fe-NifX with
Apodinitrogenase--
Apodinitrogenase (25 µg of total protein)
purified from A. vinelandii strain UW45
(nifB Elution of Proteins from Acrylamide Gels--
The gel slices
containing either 55Fe-labeled upper or lower bands were
placed in clean 10-ml culture tubes to which 2 ml of the elution buffer
(0.05 M Tris-HCl, pH 7.5, containing 0.1 mM EDTA and 150 mM NaCl) were added. The gel slices were
crushed with a clean pestle, and the tubes were shaken in a rotary
shaker at 30 °C overnight. The solutions were centrifuged at 10,000 rpm for 10 min, and the supernatants were removed into Eppendorf tubes, to which an equal volume of SDS-sample buffer was added. The entire procedure was performed under aerobic conditions.
Electrophoresis--
SDS-PAGE was performed as described (12)
with a 2.8% stacking gel and a 12% resolving gel. The proteins were
electrophoresed at 100 V until the bromphenol blue entered the
resolving gel and then electrophoresed at 200 V until the dye
front reached the bottom of the gel. Low molecular weight markers
(Bio-Rad) were used. Proteins were separated on anaerobic native gels
with a 7-20% acrylamide and 0-20% sucrose gradient as previously
described (26).
Iron Stain--
Native gels were stained for iron as
described previously (27).
Antibodies and Immunoblot Analysis--
Antibodies to the
various proteins were prepared at the University of Wisconsin antibody
facility. The protocols for immunoblotting and developing with
modifications by Brandner et al. (28) have been described.
Visualization of Radioactivity--
Gels were exposed to a
phosphor screen for 1-2 days and were scanned using a Molecular
Dynamics Model 425e PhosphorImager.
Incorporation of 55Fe-Label from
55Fe-NifB-co into Extracts of Various A. vinelandii
Mutants--
FeMo-co is unstable in aqueous solutions; thus it is
likely that precursors of FeMo-co accumulate on proteins during the
course of cofactor biosynthesis. Though use of the in vitro
FeMo-co biosynthesis system has yielded considerable information
regarding the biosynthesis of FeMo-co, not much is known about the
proteins that are associated with precursors of the cofactor. Previous
studies with 55Fe- and 35S-labeled NifB-co have
shown that NifB-co serves as a specific iron and sulfur donor to
FeMo-co (18). The in vitro FeMo-co synthesis system,
together with native anoxic gel electrophoresis, was employed to
monitor the incorporation of radiolabel from purified 55Fe-labeled NifB-co into FeMo-co and its precursors.
Our strategy for the identification of proteins that accumulate FeMo-co
precursors was as follows: (i) perform in vitro FeMo-co biosynthesis in the presence of purified 55Fe-NifB-co, (ii)
separate the proteins on anoxic native gels, and (iii) visualize
proteins accumulating radiolabel by phosphorimaging analyses of these
gels. One limitation in this procedure is that only precursor·protein
complexes that are sufficiently stable to survive non-denaturing
gel electrophoresis can be visualized. The in vitro FeMo-co
synthesis reaction was performed as described under "Experimental
Procedures" by the addition of purified 55Fe-labeled
NifB-co to extracts of A. vinelandii strains carrying deletion of one or more genes that are required for FeMo-co synthesis. The results of this study are illustrated in Fig.
1. Upon the addition of
55Fe-labeled NifB-co to an in vitro FeMo-co
synthesis reaction mixture containing the extract of an A. vinelandii strain carrying a lesion of nifB (strain
UW45), FeMo-co is synthesized and inserted into apodinitrogenase
forming dinitrogenase (Fig. 1, lane 1), the migration of
which was identified by immunoblot analysis (data not shown). A
C2H2 reduction activity of 5.0 nmol/min/mg of
protein was obtained for this reaction, consistent with the formation
of holodinitrogenase. We estimate that this activity is equivalent to
greater than 10% of the specific activity of a crude extract of
wild-type A. vinelandii. Furthermore, this represents an
activity equivalent to our best in vitro FeMo-co
synthesis.
A reaction mixture containing the extract of strain CA117.3
(
Strain CA11.3
(
When the strain CA142
(
A reaction mixture containing the extract of the wild-type UW strain
grown in presence of excess fixed nitrogen (nif-repressed) did not show either the upper or lower bands,
suggesting that nif proteins were required for the
incorporation of radiolabel from 55Fe-NifB-co into the
above-mentioned bands (Fig. 1, lane 6). A slow-migrating
protein that incorporated 55Fe-label from radiolabeled
NifB-co was observed in extracts of several A. vinelandii
strains. This band, labeled "? " in Fig. 1, was observed
even under conditions when FeMo-co synthesis was not allowed to occur
and thus represents a protein that is most likely not involved in
FeMo-co synthesis.
At least four lines of evidence suggest that the
55Fe-labeled upper and lower bands
contain proteins that are bound to precursors of FeMo-co: (i) The
accumulation of radiolabel from 55Fe-NifB-co on these bands
was seen only under conditions when FeMo-co synthesis occurred. For
example, these bands were not observed when air-oxidized cell-free
extracts of A. vinelandii mutants were used in the in
vitro FeMo-co synthesis reaction (data not shown). As shown in
Fig. 2, the incorporation of radiolabel from 55Fe-NifB-co into these bands did not occur; (ii) when
O2-denatured 55Fe-NifB-co was used in the
in vitro FeMo-co synthesis reaction, suggesting that a
native form of NifB-co was necessary for the formation of the
55Fe-labeled upper and lower bands (Fig. 2, lane
4); nor did it occur (iii) when 55Fe-labeled
FeCl3 was used in place of 55Fe-labeled NifB-co
(Fig. 2, lane 5). This suggests that the radiolabel on these
bands was not adventitiously bound 55Fe. (iv) The
radiolabel could be transferred to apodinitrogenase from these bands
upon the addition of purified apodinitrogenase to an in
vitro FeMo-co synthesis reaction mixture lacking NifKD (compare
lanes 2 and 3 of Fig. 1). This not only suggests
the association of FeMo-co precursors with the 55Fe-labeled
upper and lower bands, but also indicates that
when purified apodinitrogenase was made available to the components of
the in vitro FeMo-co synthesis reaction mixture, the newly synthesized FeMo-co was inserted into the added apodinitrogenase.
Purification and Identification of the 55Fe-Labeled
Upper Band--
A 1200-fold scale up of the in vitro
FeMo-co synthesis reaction containing the extract of strain CA117.3
(
The involvement of NifH in FeMo-co biosynthesis was examined using the
extract of CA11.3
(
The radioactive species accumulating on NifH was O2-labile,
because exposure of the identical sample to air eliminated the detection of this band (data not shown). The amount of radiolabel on
NifH diminished during the course of purification, suggesting a
transient association of a FeMo-co precursor with NifH. The continual
loss of radiolabel has so far prevented the definitive identification
and the characterization of the cluster on NifH. However, at least
three lines of evidence support the hypothesis that the label
associated with NifH is in the form of a FeMo-co precursor and is not
adventitiously bound Fe: 1) No accumulation of radiolabel on NifH was
observed upon the addition of radiolabeled NifB-co to strains
containing NifH, such as UW45 (nifB) and CA142 ( Purification and Identification of the 55Fe-Labeled
Lower Band--
The radiolabeled lower band that formed
upon the incubation of 55Fe-labeled NifB-co with the
extract of CA117.3
( Transfer of Radiolabel from 55Fe-NifX to
Apodinitrogenase--
To determine if the radiolabeled species on NifX
can be donated to apodinitrogenase, purified NifX bound to the
radiolabeled FeMo-co precursor (55Fe-NifX) was incubated
with purified apodinitrogenase
( NifB-co Binding Perturbs the Migration of NifX--
Immunoblot
analysis of anoxic native gels containing the extract of the A. vinelandii strain CA11.1
(
To characterize the two electrophoretically distinct NifX species, NifX
that is not bound to the FeMo-co precursor was purified partially from
the extract of an A. vinelandii strain carrying a deletion
of nifB, strain CA117.3
(
By use of the in vitro FeMo-co synthesis system, NifX has
been shown to be required for FeMo-co synthesis (12). Surprisingly, NifX is not absolutely required for the synthesis of the cofactor in vivo, because strains carrying a deletion of
nifX are capable of diazotrophic growth (29) exhibiting
total nitrogenase activity similar to that of wild-type. This suggests
that some other gene product can replace NifX in the in vivo
synthesis of FeMo-co.
Results similar to those presented here were obtained for VnfX, an NifX
homolog in the vnf system (26). VnfX has been purified with
a V-Fe cluster bound from extracts of strain CA11.1
( The major observations made in the present study are: 1) NifH and
NifX accumulate FeMo-co precursors during the course of FeMo-co
biosynthesis; 2) radiolabel from NifX can be transferred to
apodinitrogenase, and the corresponding C2H2
reduction activity strongly suggests that NifX may be associated with
mature FeMo-co; and 3) NifX can bind NifB-co and consequently migrates
at a different position on anoxic native gels than does free NifX.
Based on these results and on other observations from previous studies,
our working model for the biosynthesis of FeMo-co is as follows: the
binding of NifB-co to NifNE occurs as the first step in the FeMo-co
biosynthetic pathway. The FeMo-co precursor is then transferred from
NifNE to NifH. We propose that the heterometal addition to the FeMo-co
precursor occurs either on NifH or on NifX. The addition of homocitrate
might occur as the last step during cofactor biosynthesis on NifX. The
mature FeMo-co is bound by gamma, which delivers the cofactor to
apodinitrogenase to form holodinitrogenase.
The binding of NifB-co to NifNE has been observed previously both
in vivo as well as in vitro (25). That this
binding is not dependent on the presence of MgATP, NifH, or NifX
in vitro suggests that NifB-co binding to NifNE occurs as
one of the early events in the FeMo-co biosynthetic pathway (25). In
the present study, we have shown that, like NifNE, NifX also is capable
of binding NifB-co and that this binding is not dependent on the presence of NifNE. However, whether the binding of NifB-co to NifX is
physiologically relevant remains to be determined. Also, it is not yet
known if either NifNE or NifX catalyze any modification of NifB-co upon
binding to the cofactor. The structure of NifB-co has not yet been
established, but recent results suggest that NifB-co may be a cluster
of complex Fe-S
species.3 The binding of
NifB-co to NifNE and NifX might be better understood when the structure
of NifB-co becomes available.
Our results with the addition of 55Fe-NifB-co and purified
NifH to the extract of a strain lacking NifH showed that the radiolabel associated with NifNE diminished with the concomitant appearance of
radiolabel on NifH, although there was no change in the radiolabel associated with NifX. This suggests that the step catalyzed by NifH
occurs after the one catalyzed by NifNE. These results also suggest
that the binding of NifB-co to NifX is most probably due to the
presence of a FeMo-co precursor binding site on NifX. In support of the
hypothesis that the action of NifH occurs after the reaction catalyzed
by NifNE, we have shown the interaction of NifH with the
NifNE·NifB-co complex (31). We propose that NifH may lose its
affinity to the [Fe-Mo-S] species upon the addition of molybdenum to
the cofactor precursor. This might explain the transient association of
the FeMo-co precursor with NifH. We hypothesize that the addition of
homocitrate occurs on NifX as the last step during cofactor
biosynthesis. In this context, we note the presence of completed
FeMo-co on NifX. In vivo, the finished FeMo-co is bound by
gamma, which might possess greater affinity for FeMo-co than does NifX.
FeMo-co is then delivered to apodinitrogenase to form holodinitrogenase.
One of the central claims in this study is that a FeMo-co precursor
accumulates on NifH. Apart from its role in nitrogenase turnover, NifH
has been known to function as a required participant in the
biosynthesis of FeMo-co (9, 10, 32). NifH is thus a "moonlighting
protein" (33) that serves as the electron donor to dinitrogenase and
as a biosynthetic enzyme in FeMo-co biosynthesis. It has also been
known that the properties of NifH required for its function in FeMo-co
biosynthesis are different from those required for its function in
electron transfer to dinitrogenase (34-36). For example, we have shown
in a separate study that a redox-active 4Fe-4S cluster in NifH is not
necessary for its function in FeMo-co biosynthesis (37). Nevertheless,
the exact role played by NifH in cofactor biosynthesis remains unclear.
In the present study, we have shown the accumulation of a radiolabeled
FeMo-co precursor on NifH. However, this association seems weaker than that of NifX and NifB-co due to the loss of radiolabel during the
course of purification of NifH bound to the FeMo-co precursor. Studies
involving 99Mo might elucidate the role of NifH in FeMo-co
biosynthesis and determine whether the cluster on NifH contains
molybdenum. Besides its role in substrate reduction, FeMo-co synthesis
and apodinitrogenase maturation, NifH has been implicated in the
expression of the alternative nitrogenases in A. vinelandii
(38). Our results help support the idea that the role played by NifH in
the regulation of expression of alternative nitrogenases might be as an
indicator of molybdenum sufficiency. Specifically, NifH bound to the
Fe-Mo-S species might serve to signal the proper functioning of the
nif system. Moreover, it has been shown that NifH is
required for the expression of anf-nitrogenase in Mo- and
V-deficient conditions (38). Under these conditions, it is expected
that NifH would not be bound to the FeMo-co precursor and thus could
serve to indicate the impairment of nif-nitrogenase, in turn
promoting the expression of anf-nitrogenase.
Finally, there remain a number of gaps in our understanding of the
biosynthesis of FeMo-co. It is not known whether the NifNE proteins
perform any modification(s) to NifB-co. The identity of the molybdenum
donor to the FeMo-co biosynthetic pathway and the chemical form of
molybdenum as it enters the pathway are unknown. In addition, the exact
roles played by NifH and NifX are not yet clear. We believe in
vitro FeMo-co synthesis reactions, including 99Mo, the
radioisotope of molybdenum, will clarify the roles played by NifH and
NifX in FeMo-co biosynthesis and will reveal the step at which
molybdenum is incorporated into the FeMo-co precursor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 dimer of the nifH gene product, contains one 4Fe-4S cluster which is
symmetrically coordinated by the thiol groups of Cys-97 and
Cys-132 from each subunit of the homodimer (4, 5). Dinitrogenase
is an
2
2 tetramer of the nifD
and K gene products and is associated with two unique metal
clusters: the P-cluster (6) and the iron-molybdenum cofactor (FeMo-co)
(6, 7). FeMo-co is the active site in dinitrogenase where substrate
reduction is believed to occur and is composed of a
MoFe3-S3 cluster bridged to a
Fe4-S3 cluster by three sulfur ligands. The
molybdenum atom is also coordinated to the C-2 carboxyl and hydroxyl
groups of the organic acid R-homocitrate.
), that serves as a chaperone-insertase during FeMo-co
biosynthesis and its insertion into NifKD (15). An in vitro
reaction for the biosynthesis of FeMo-co has been described that
requires the presence of an ATP-regenerating system,
MoO
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A. vinelandii strains
nifHDK
nifB::kan) was
constructed in the laboratory of P. E. Bishop by transforming
A. vinelandii strain CA11 with genomic DNA from CA30 (19)
and selecting for kanamycin-resistant transformants. A. vinelandii strain CA142 (
nifDKY
nifENX::kan)
was also constructed in P. E. Bishop's laboratory by transforming
A. vinelandii strain CA117 (21) with genomic DNA from strain
DJ678 (18); transformants were selected for kanamycin resistance.
80 °C. Typically about 60-75% of the radioactivity was
incorporated into the cells.
nifHDK
vnfDGK::spc)
according to published procedures (17, 18).
2
2
2
form, 50 µg of total protein) was added to certain reaction mixtures.
Acetylene reduction was initiated in the remainder of the vials by the
addition of excess MgATP, purified NifH (0.1 mg of protein), and 0.5 ml
of acetylene. Nitrogenase activity was then quantitated by acetylene
reduction as described elsewhere (11).
nifDK
nifB), 0.4 ml of purified unlabeled NifB-co solution (equivalent to 200 nmol of Fe), and 0.1 ml of purified
55Fe-NifB-co solution (6 × 106 cpm/ml).
The vials were placed in a rotary shaker and were gently shaken at
30 °C for 30 min. The reaction mixtures were pooled after the
incubation period and applied to a 93-ml DEAE-cellulose column
(2.5 × 19 cm) equilibrated with anoxic Tris-HCl containing 0.1 M NaCl. The column was washed with 100 ml of the same
buffer, and proteins were eluted from the column using a step gradient of 0.15, 0.25, 0.35, and 0.5 M NaCl in the above
buffer containing 20% glycerol. The presence of upper and lower
bands was determined by subjecting aliquots of the DEAE-cellulose
column fractions to anoxic, native gel electrophoresis, and
phosphorimaging analyses. The upper band was found to elute with buffer
containing 0.25 M NaCl, whereas the lower band eluted with
buffer containing 0.35 M NaCl.
nifHDK
nifB::kan). The
cell-free extract of 100 g of cells was applied to a
DEAE-cellulose column (4 × 15 cm), equilibrated with anoxic
Tris-HCl containing 0.1 M NaCl and 20% glycerol. The column was washed with 1 column volume of the same buffer, and proteins
were eluted stepwise with 0.15, 0.25, 0.35, and 0.5 M NaCl
contained in the buffer described above. The presence of NifX was
determined by SDS-PAGE electrophoresis followed by immunoblot analysis
of the fractions using anti-NifX antibody. The fractions containing
NifX (0.15 M NaCl elute) were pooled and concentrated using
an Amicon system fitted with a XM100A membrane. The concentrate (15 ml)
was then chromatographed on a Sephacryl S-100 column (2.5 × 96 cm), equilibrated with buffer containing anoxic Tris-HCl, 0.05 M NaCl, and 10% glycerol. The fractions containing NifX, as determined by immunoblot analysis, were pooled and applied to a
Q-Sepharose column (1.5 × 12.5 cm), equilibrated with buffer containing anoxic Tris-HCl and 0.1 M NaCl. The column was
washed with 25 ml of the above buffer, and protein was eluted stepwise with one column volume each of 0.15, 0.35, and 0.5 M of
NaCl in Tris-HCl. The NifX-containing fractions (0.35 M
elute) were pooled, brought to 1 M in
(NH4)2SO4, and applied to a
phenyl-Sepharose column (1 × 10 cm) equilibrated with 1 M (NH4)2SO4 in
Tris-HCl. The column was washed with 12 ml of the same buffer, and
protein was eluted stepwise with 0.5, 0.25, 0.125, and 0.0 M (NH4)2SO4 in
Tris-HCl. The fractions containing NifX (0.5 and 0.25 M
(NH4)2SO4) were pooled and frozen
as pellets in liquid nitrogen.
nifENX
vnfX), as described
previously (12).
nifDK
nifB) as described by Roll et
al. (25).
) was incubated with the partially
purified 55Fe-NifX (5000 cpm) and purified NifH (4.5 µg
of protein) in an anoxic, stoppered 9-ml vial. The total volume of the
reaction mixture was 170 µl. The reaction mixture was incubated for
15 min at 30 °C, after which 100 µl was subjected to anoxic,
native gel electrophoresis. For the C2H2
reduction assay, 800 µl of ATP-regenerating mixture and 0.5 ml of
C2H2 were added to the reaction mixture (as
described above), and nitrogenase activity was then quantitated as
described elsewhere (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (84K):
[in a new window]
Fig. 1.
PhosphorImager analysis of anoxic, native gel
illustrating the incorporation of 55Fe label from
55Fe-NifB-co into extracts of various A. vinelandii strains. In vitro FeMo-co
synthesis reactions, including 55Fe-labeled NifB-co
and extracts of various A. vinelandii strains were performed
as described under "Experimental Procedures." Lane 1,
reaction including UW45 (nifB ); lane
2, including CA117.3 (
nifDK
nifB);
lane 3, including CA117.3 plus purified apodinitrogenase (50 µg of protein); lane 4, including CA11.3
(
nifHDK
nifB::kan);
lane 5, including CA142
(
nifDKY
nifENX::kan);
and lane 6, including UW
(NH
, refers to less than 0.1 nmol/min/mg of
protein.
nifDK
nifB) showed the incorporation of the
radiolabel from 55Fe-NifB-co into a faster-migrating
species (Fig. 1, lane 2), identified as NifNE by immunoblot
analysis (data not shown). Two other faster-migrating radiolabeled
bands were observed apart from the radiolabel associated with NifNE,
which were named as 55Fe-labeled upper and
lower bands, as shown in Fig. 1 (lane 2). It is
essential to note that strain CA117.3 is able to support FeMo-co
synthesis upon the addition of NifB-co alone, because NifKD proteins
are not required for cofactor biosynthesis (13, 14). Results from
previous studies indicate that completed FeMo-co is bound by the gamma
protein (15). Thus, the presence of FeMo-co bound to gamma
(
·FeMo-co) is expected when the in vitro FeMo-co synthesis reaction is carried out with the extract of strain CA117.3. However, under the electrophoretic conditions used in this study, completed FeMo-co bound to
comigrated with the upper
band and was separated during the course of purification of the
upper band (data not shown). Upon the addition of purified
apodinitrogenase (
2
2
2) to
the reaction mixture containing the extract of strain CA117.3, the
incorporation of the radiolabel into dinitrogenase was observed (Fig.
1, lane 3), suggesting that FeMo-co had been synthesized and
inserted into the added apodinitrogenase. This is supported by a
C2H2 reduction activity of 4.7 nmol/min/mg of protein for this reaction.
nifHDK
nifB::kan)
showed the incorporation of the radiolabel from
55Fe-NifB-co into NifNE and into one of the
faster-migrating bands termed lower band (Fig. 1, lane
4) but not into the upper band. This suggested that the
presence of NifH was required for the accumulation of the radiolabel on
the band termed upper band. Note that the only difference
between the CA117.3 and CA11.3 genotypes is the presence or absence of
nifH; the nifH gene is present in strain CA117.3
and is deleted in strain CA11.3.
nifDKY
nifENX::kan)
was used in the in vitro FeMo-co synthesis reaction, no
incorporation of the radiolabel from 55Fe-NifB-co into
either the upper or the lower bands could be
observed, suggesting that NifNE or NifX or both may be required for the incorporation of radiolabel into these bands (Fig. 1, lane
5).
View larger version (75K):
[in a new window]
Fig. 2.
PhosphorImager analysis of an anoxic native
gel illustrating the transfer of radiolabel from NifNE to NifH.
In vitro FeMo-co synthesis reactions, including
55Fe-labeled NifB-co and extract of strain CA11.3
( nifHDK
nifB::kan) were
performed as described under "Experimental Procedures." Three
anoxic vials containing the extract of strain CA11.3 were used.
Purified NifH (45 µg of protein) was added to the second and the
third vials. The vials were incubated for 30 min for in
vitro FeMo-co synthesis. At the end of 30 min, purified
apodinitrogenase (50 µg of protein) was added to the third vial. The
vials were incubated for an additional 10 min before application onto
the anoxic native gel. Reaction mixtures in lanes 4 and
5 contained the extract of strain CA11.3, purified NifH (45 µg of protein), and purified apodinitrogenase (50 µg of protein)
but not 55Fe-labeled NifB-co. Lane 1, reaction
including extract of strain CA11.3; lane 2, same as in
lane 1 plus purified NifH; lane 3, same as in
lane 2 plus purified apodinitrogenase; lane 4,
same as in lane 3 minus 55Fe-labeled NifB-co but
plus anoxic O2-denatured NifB-co; and lane 5,
same as in lane 3 minus 55Fe-labeled NifB-co but
plus anoxic 55FeCl3. The table below the
figure indicates the components known to be present in the
reaction mixture. The C2H2 reduction activities
(nmol/min/mg of protein) of the assays are reported where appropriate.
, refers to less than 0.1 nmol/min/mg of protein. The position of
dinitrogenase, NifNE, NifH, and 55Fe-lower band are
indicated by arrows.
nifDK
nifB) and 55Fe-NifB-co
was carried out to identify the protein in the upper 55Fe-labeled band, as described under "Experimental
Procedures." The 55Fe-labeled upper band was
purified by following the radioactivity associated with it.
Purification steps included DEAE-cellulose, Sephadex G-75,
phenyl-Sepharose chromatography, and preparative native gel
electrophoresis. The region of the gel containing radioactivity was
sliced, and the protein was eluted from the acrylamide. The protein was
identified as NifH based on its migration on a SDS-gel (Fig.
3, lanes 1 and 2)
and its cross-reactivity with anti-NifH antibody (Fig. 3, lanes
3 and 4). These results suggest that a FeMo-co
precursor actually accumulated on NifH during cofactor biosynthesis.
View larger version (69K):
[in a new window]
Fig. 3.
SDS-PAGE of 55Fe-upper band.
Coomassie Blue-stained SDS-gel. Lane 1, purified NifH (10 µg of protein); and lane 2, 55Fe-upper band
purified by preparative native gel electrophoresis. Anti-NifH
immunoblot. Lane 3, purified NifH (2 µg of protein); and
lane 4, 55Fe-upper band purified by preparative
native gel electrophoresis. Numbers at the left show
molecular masses in kilodaltons.
nifHDK
nifB::kan). As
seen in Fig. 2 (lane 1), the extract of this strain showed
the accumulation of radiolabel on NifNE and on the lower
band when the in vitro FeMo-co biosynthesis reaction
was performed with 55Fe-NifB-co, as described under
"Experimental Procedures." Upon the addition of purified NifH to
the above reaction (Fig. 2, lane 2), the accumulation of
radiolabel on NifH was observed with the concomitant decrease in the
amount of radiolabel accumulating on NifNE. These results suggest that
FeMo-co biosynthesis was blocked before the addition of NifH and that
the addition of NifH to the reaction mixture allowed cofactor
biosynthesis to proceed beyond NifNE. The addition of purified NifH and
purified apodinitrogenase to the extract of CA11.3 allowed the
accumulation of the radiolabel at the dinitrogenase position with the
concomitant decrease in the radiolabel on NifH and on the lower
band (Fig. 2, lane 3). This demonstrates that the
radiolabel associated with NifH and the lower band can be
transferred to apodinitrogenase. The C2H2 reduction activity (4.7 nmol/min/mg of protein) of this reaction mix
was also consistent with the formation of holodinitrogenase.
nifDKY
nifENX) (Fig. 1, lanes 1 and 5). If the radiolabel on NifH were adventitiously bound
iron, then the presence of radiolabel on NifH in all strains containing
NifH would be expected. 2) The radiolabel on NifH was transferred to
apodinitrogenase upon the addition of purified apodinitrogenase to an
extract lacking nifDK (compare lanes 2 and
3 in both Figs. 1 and 2) strongly suggesting the association
of NifH with a FeMo-co precursor. 3) The absence of a band that
co-migrated with NifH in samples where O2-inactivated 55Fe-NifB-co or 55FeCl3 was
utilized demonstrates that active NifB-co was required for
incorporation of 55Fe into NifH (Fig. 2, lanes 4 and 5).
nifDK
nifB::kan) in a
FeMo-co synthesis reaction was purified by following the radioactivity
associated with it. Purification steps of the 55Fe-labeled
lower band from a 1200-fold scale-up of the in
vitro FeMo-co synthesis reaction included DEAE-cellulose, Sephadex
G-75, phenyl-Sepharose chromatography, and preparative native gel
electrophoresis. The portion of the gel containing radioactivity was
sliced, and protein was eluted from the acrylamide, as described under
"Experimental Procedures." The protein was identified as NifX,
based on the N-terminal sequence obtained and on its identity to the
sequence predicted from nifX (MSSPTRQLQVLD). The
nifX gene is located downstream of nifE and
N, in the nifENX operon of A. vinelandii. We have shown, in a separate study, the requirement of
NifX for cofactor biosynthesis by the in vitro FeMo-co
synthesis reaction (12). The data presented here suggest the
association of NifX with a FeMo-co precursor during the course of
cofactor biosynthesis. This species will be termed
55Fe-NifX for the remainder of the manuscript.
2
2
2), as described under
"Experimental Procedures." A solution substantially enriched in
55Fe-NifX obtained from a 1200-fold scale-up of the
in vitro FeMo-co synthesis reaction was used for this study.
The results, presented in Fig. 4, show
that the radiolabeled species on NifX could be transferred to
apodinitrogenase, although not completely (Fig. 4, lane 4).
The transfer of the radiolabel from 55Fe-NifX to
apodinitrogenase correlated with C2H2 reduction
activity of 224 nmol/min/mg of 55Fe-NifX, strongly
suggesting the presence of completed FeMo-co on NifX. In support of
this hypothesis, the binding of purified FeMo-co to purified NifX has
been observed in
vitro.2 The partial
transfer of radio label from 55Fe-NifX to apodinitrogenase
suggested that 55Fe-NifX is comprised of a mixture of
either NifX bound to finished FeMo-co or to NifX bound to a FeMo-co
precursor that is yet to be completed. Increasing the concentration of
purified apodinitrogenase in the reaction mixture did not increase the
amount of transferred label from 55Fe-NifX, suggesting that
apodinitrogenase is not limiting in the reaction mix (data not shown).
A matter of concern here is that the C2H2
reduction activity observed might result from the presence of
·FeMo-co in the partially purified 55Fe-NifX sample;
has been shown to bind FeMo-co (15). However, no
was observed,
as judged by anti-
immunoblot analysis of the 55Fe-NifX
solution. That the radiolabel at the apodinitrogenase position is not
adventitiously bound iron is evidenced by the lack of transfer of the
radiolabel when O2-denatured 55Fe-NifX was used
in the reaction (Fig. 4, lane 5).
View larger version (110K):
[in a new window]
Fig. 4.
PhosphorImager analysis of an anoxic native
gel illustrating the transfer of radiolabel from 55Fe-NifX
to apodinitrogenase. The position of apodinitrogenase and
55Fe-NifX are indicated. Incubations were carried out as
described under "Experimental Procedures." Lane 1,
partially purified 55Fe-NifX (5,000 cpm); lane
2, anoxic O2-denatured 55Fe-NifX (5000 cpm); lane 3, purified apodinitrogenase (25 µg of total
protein); lane 4, 55Fe-NifX (5000 cpm) incubated
with apodinitrogenase; lane 5, anoxic
O2-denatured 55Fe-NifX (5000 cpm) incubated
with apodinitrogenase. The table below the figure indicates
the components known to be present in the reaction mixture.
nifHDK
vnfDGK::spc) using anti-NifX antibody showed the presence of a slow-migrating and a
fast-migrating NifX species (Fig. 5,
lane 3). The fast-migrating NifX species comigrated with
55Fe-NifX (Fig. 5, lane 1) and was not observed
upon exposure of the sample to air (Fig. 5, lanes 2 and
4). This is presumably due to the loss of the FeMo-co
precursor bound to NifX upon O2 exposure. The extract of
NifB-co-deficient strain CA117.3
(
nifDK
nifB::kan), on
the other hand, showed the presence of only the slow-migrating species
of NifX (Fig. 5, lane 5). Upon the addition of purified NifB-co to the extract of strain CA117.3, a shift in the
electrophoretic mobility of NifX was observed (Fig. 5, lane
6). These data suggest that the electrophoretic mobility of NifX
on anoxic, native gels is dependent upon the presence or absence of
NifB-co. Roll et al. (25) have observed similar shifts on
anoxic native gels for NifNE upon addition of purified NifB-co to
extracts lacking nifB.
View larger version (28K):
[in a new window]
Fig. 5.
Analysis of NifX by anoxic native gel
electrophoresis. PhosphorImager analysis of an anoxic native gel.
Lane 1, purified 55Fe-NifX (5000 cpm);
lane 2, anoxic O2-denatured
55Fe-NifX (5000 cpm). Anti-NifX immunoblot of an anoxic,
native gel. Lane 3, extract of A. vinelandii
strain CA11.1
( nifHDK
vnfDGK::spc;
260 µg of total protein); lane 4, anoxic
O2-denatured extract of strain CA11.1 (260 µg of total
protein); lane 5, extract of strain CA117.3
(
nifDK
nifB; 260 µg of total protein); and
lane 6, extract of strain CA117.3 (260 µg of total
protein) incubated with NifB-co (1.5 nmol of Fe).
nifDK
nifB::kan). The
purification of NifX was performed as described under "Experimental
Procedures." NifX, purified in this manner, was ~60% pure as
judged by scanning densitometry and showed a 6-fold stimulation of
in vitro FeMo-co synthesis assays involving the extract of
strain DJ42.48 (
nifENX
vnfE), as reported
previously by Shah et al. (12). The results presented in
Fig. 6 show that the purified NifX
comigrated with the slow-migrating species of NifX (compare lanes
1 and 2). The addition of purified NifB-co caused a
shift in the migration of NifX on anoxic, native gels; NifX now
comigrated with the fast-migrating species (Fig. 6, compare lanes
2 and 3). These results suggest the binding of NifX
with NifB-co. Fig. 6 also shows the incorporation of radiolabel from
55Fe-labeled NifB-co into NifX as judged by phosphorimaging
(lane 6) and the incorporation of iron from unlabeled
NifB-co as judged by iron staining (lane 7). These results
confirm the binding of NifB-co to NifX. O2-inactivated
NifB-co did not cause a shift in the migration of NifX; nor did a
solution of SB-12, the detergent used in the extraction of NifB-co
(data not shown). On the other hand, O2-exposed NifX (made
anoxic after O2 exposure by degassing and flushing with
argon gas and by the addition of DTH) still retained its ability to
bind NifB-co (Fig. 6, lane 4). That NifNE is not required
for the binding of NifB-co to NifX was determined by examination of the
electrophoretic mobility of NifX in the extract of a nifE
strain (DJ35), where in a portion of NifX migrated as the
fast-migrating species (Fig. 6, lane 5).
View larger version (31K):
[in a new window]
Fig. 6.
NifX can bind NifB-co. Anti-NifX
immunoblot of anoxic, native gel. Lane 1, extract of
A. vinelandii strain CA11.1
( nifHDK
nifDGK; 260 µg of total protein);
lane 2, NifX partially purified from strain CA117.3
(
nifDK
nifB; 25 µg of total protein);
lane 3, NifX partially purified from strain CA117.3 (25 µg
of total protein) incubated with NifB-co (1.5 nmol of Fe); lane
4, anoxic O2-exposed NifX partially purified from
strain CA117.3 (25 µg of total protein) incubated with NifB-co (1.5 nmol of Fe); and lane 5, extract of strain DJ35
(
nifE; 260 µg of total protein). PhosphorImager
analysis of anoxic, native gel. Lane 6, NifX partially
purified from strain CA117.3 (500 µg of total protein) incubated with
55Fe-NifB-co (10,000 cpm). Iron stain of anoxic, native
gel. Lane 7, NifX partially purified from strain CA117.3
(500 µg of total protein) incubated with NifB-co (1.5 nmol of
Fe).
nifHDK
vnfDGK::spc) grown in the presence of vanadium, thus suggesting a role for the
protein in FeV-co synthesis (26). VnfX purified without the cluster
bound has been shown to bind NifB-co specifically, and NifB-co binding
caused a shift in its mobility on anoxic, native gels. Like NifX, VnfX
is not absolutely required for FeV-co synthesis in vivo,
suggesting that it can be replaced by some other gene product.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Gary Roberts, Dr. Chris
Staples, and Dr. Dennis Dean for helpful discussions, Dr. Mary Homer
for providing anti- antibody and Dr. Jon Roll for providing
anti-NifNE antibody. Dr. Gary Hathaway (Caltech) is acknowledged for
N-terminal sequencing of the lower band. We thank Dr. Paul Bishop and
Dr. Dennis Dean for sharing strains.
![]() |
FOOTNOTES |
---|
* This work was supported by NIGMS, National Institutes of Health Grant 35332 (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: Department of
Biochemistry, 433 Babcock Ave., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-6859; Fax: 608-262-3453; E-mail: ludden@biochem.wisc.edu.
Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M100907200
2 P. J. Goodwin and D. R. Dean, personal communication.
3 C. R. Staples, P. Rangaraj, C. Rüttimann-Johnson, S. J. Yoo, E. Munck, and P. W. Ludden, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NifKD, dinitrogenase (also called MoFe protein); FeMo-co, iron-molybdenum cofactor; NifB-co, NifB-cofactor; DTH, sodium dithionite; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bulen, W. A., and LeComte, J. R. (1966) Proc. Natl. Acad. Sci. U. S. A. 56, 979-986[Medline] [Order article via Infotrieve] |
2. | Hageman, R. V., and Burris, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2699-2702[Abstract] |
3. | Burris, R. H., and Roberts, G. P. (1993) Annu. Rev. Nutr. 13, 317-335[CrossRef][Medline] [Order article via Infotrieve] |
4. | Georgiadis, M. M., Komiya, H., Chakrabarti, P., Woo, D., Kornuc, J. J., and Rees, D. C. (1992) Science 257, 1653-1659[Medline] [Order article via Infotrieve] |
5. |
Hausinger, R. P.,
and Howard, J.
(1983)
J. Biol. Chem.
258,
13486-13492 |
6. | Kim, J., and Rees, D. C. (1992) Science 257, 1677-1682[Medline] [Order article via Infotrieve] |
7. | Shah, V. K., and Brill, W. J. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3249-3253[Abstract] |
8. | Hoover, T. R., Imperial, J., Ludden, P. W., and Shah, V. K. (1989) Biochemistry 28, 2768-2771[Medline] [Order article via Infotrieve] |
9. | Filler, W. A., Kemp, R. M., Ng, J. C., Hawkes, T. R., Dixon, R. A., and Smith, B. E. (1986) Eur. J. Biochem. 160, 371-377[Abstract] |
10. |
Robinson, A. C.,
Dean, D. R.,
and Burgess, B. K.
(1987)
J. Biol. Chem.
262,
14327-14332 |
11. | Shah, V. K., Imperial, J., Ugalde, R. A., Ludden, P. W., and Brill, W. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1636-1640[Abstract] |
12. |
Shah, V. K.,
Rangaraj, P.,
Chatterjee, R.,
Allen, R. M.,
Roll, J. T.,
Roberts, G. P.,
and Ludden, P. W.
(1999)
J. Bacteriol.
181,
2797-2801 |
13. | Ugalde, R. A., Imperial, J., Shah, V. K., and Brill, W. J. (1984) J. Bacteriol. 159, 888-893[Medline] [Order article via Infotrieve] |
14. | Imperial, J., Shah, V. K., Ugalde, R. A., Ludden, P. W., and Brill, W. J. (1987) J. Bacteriol. 169, 1784-1786[Medline] [Order article via Infotrieve] |
15. |
Homer, M. J.,
Dean, D. R.,
and Roberts, G. P.
(1995)
J. Biol. Chem.
270,
24745-24752 |
16. |
Allen, R. M.,
Roll, J. T.,
Rangaraj, P.,
Shah, V. K.,
Roberts, G. P.,
and Ludden, P. W.
(1999)
J. Biol. Chem.
274,
15869-15874 |
17. |
Shah, V. K.,
Allen, J. R.,
Spangler, N. J.,
and Ludden, P. W.
(1994)
J. Biol. Chem.
269,
1154-1158 |
18. |
Allen, R. M.,
Chatterjee, R.,
Ludden, P. W.,
and Shah, V. K.
(1995)
J. Biol. Chem.
270,
26890-26896 |
19. | Joerger, R. D., and Bishop, P. E. (1988) J. Bacteriol. 170, 1475-1487[Medline] [Order article via Infotrieve] |
20. | Shah, V. K., Davis, L. C., and Brill, W. J. (1972) Biochim. Biophys. Acta 256, 498-511[Medline] [Order article via Infotrieve] |
21. |
Chatterjee, R.,
Allen, R. M.,
Ludden, P. W.,
and Shah, V. K.
(1996)
J. Biol. Chem.
271,
6819-6826 |
22. | Strandberg, G. W., and Wilson, P. W. (1968) Can. J. Microbiol. 14, 14-25 |
23. | Shah, V. K., and Brill, W. J. (1973) Biochim. Biophys. Acta 305, 445-454[Medline] [Order article via Infotrieve] |
24. | Paustian, T. D., Shah, V. K., and Roberts, G. P. (1990) Biochemistry 29, 3515-3522[Medline] [Order article via Infotrieve] |
25. |
Roll, J. T.,
Shah, V. K.,
Dean, D. R.,
and Roberts, G. P.
(1995)
J. Biol. Chem.
270,
4432-4437 |
26. |
Rüttimann-Johnson, C.,
Staples, C. R.,
Rangaraj, P.,
Shah, V. K.,
and Ludden, P. W.
(1999)
J. Biol. Chem.
274,
18087-18092 |
27. | Kuo, C.-F., and Fridovich, I. (1988) Anal. Biochem. 170, 183-185[Medline] [Order article via Infotrieve] |
28. | Brandner, J. P., McEwan, A. G., Kaplan, S., and Donohue, T. (1989) J. Bacteriol. 171, 360-368[Medline] [Order article via Infotrieve] |
29. | Jacobson, M. R., Brigle, K. E., Bennett, L. T., Setterquist, R. A., Wilson, M. S., Cash, V. L., Beynon, J., Newton, W. E., and Dean, D. R. (1989) J. Bacteriol. 171, 1017-1027[Medline] [Order article via Infotrieve] |
30. | Wolfinger, E. D., and Bishop, P. E. (1991) J. Bacteriol. 173, 7565-7572[Medline] [Order article via Infotrieve] |
31. |
Rangaraj, P.,
Ryle, M. J.,
Lanzilotta, W. N.,
Goodwin, P. J.,
Dean, D. R.,
Shah, V. K.,
and Ludden, P. W.
(1999)
J. Biol. Chem.
274,
29413-29419 |
32. | Shah, V. K., Hoover, T. R., Imperial, J., Paustian, T. D., Roberts, G. P., and Ludden, P. W. (1988) in Nitrogen Fixation: Hundred Years After (Bothe, H. , de Bruijn, F. J. , and Newton, W. E., eds) , pp. 115-120, Gustav Fischer, Cologne |
33. | Jeffery, C. J. (1999) Trends Biochem. Sci. 24, 8-11[CrossRef][Medline] [Order article via Infotrieve] |
34. | Wolle, D., Dean, D. R., and Howard, J. B. (1992) Science 258, 992-995[Medline] [Order article via Infotrieve] |
35. |
Gavini, N.,
and Burgess, B. K.
(1992)
J. Biol. Chem.
267,
21179-21186 |
36. |
Rangaraj, P.,
Ryle, M. J.,
Lanzilotta, W. N.,
Ludden, P. W.,
and Shah, V. K.
(1999)
J. Biol. Chem.
274,
19778-19784 |
37. |
Rangaraj, P.,
Shah, V. K.,
and Ludden, P. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11250-11255 |
38. | Joerger, R. D., Wolfinger, E. D., and Bishop, P. E. (1991) J. Bacteriol. 173, 4440-4446[Medline] [Order article via Infotrieve] |
39. | Waugh, S. I., Paulsen, D. M., Mylona, P. V., Maynard, R. H., Premakumar, R., and Bishop, P. E. (1995) J. Bacteriol. 177, 1505-1510[Abstract] |
40. | Brigle, K. E., Weiss, M. C., Newton, W. E., and Dean, D. R. (1987) J. Bacteriol. 169, 1547-1553[Medline] [Order article via Infotrieve] |