(Received for publication, April 17, 1995; and in revised form, July 21, 1995)
From the
Dinitrogenase, the enzyme capable of catalyzing the reduction of
N2, is a heterotetramer (2
2) and contains the iron-molybdenum
cofactor (FeMo-co) at the active site of the enzyme. Mutant strains
unable to synthesize FeMo-co accumulate an apo form of dinitrogenase,
which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain
mutant strains of Azotobacter vinelandii has a subunit
composition of
. The
subunit has been implicated as necessary for the efficient
activation of apodinitrogenase in vitro.
Characterization
of protein in crude extracts and partially pure fractions has
suggested that it is a chaperone-insertase required by apodinitrogenase
for the insertion of FeMo-co. There are three major forms of
protein detectable by Western analysis of native gels. An
apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon
addition of purified FeMo-co. A second form of
protein is
unassociated with other proteins and exists as a homodimer. Both of
these forms of
protein can be converted to a third form by the
addition of purified FeMo-co. This conversion requires the addition of
active FeMo-co and correlates with the incorporation of iron into
protein. Crude extracts that contain this form of
protein are
capable of donating FeMo-co to apodinitrogenase, thereby activating the
apodinitrogenase. These data support a model in which
protein is
able to interact with both FeMo-co and apodinitrogenase, facilitate
FeMo-co insertion into apodinitrogenase, and then dissociate from the
activated dinitrogenase complex.
Nitrogenase is comprised of two components: dinitrogenase (also
known as component I or the MoFe protein) and dinitrogenase reductase
(also known as component II, NifH, or the Fe protein). Nitrogenase
catalyzes the ATP- and reductant-dependent reduction of N and other substrates. Dinitrogenase is a 240-kDa
tetramer encoded by nifKD(1) . Dinitrogenase contains two types of metal
centers: the P-cluster (8Fe-8S)(2, 3) , which bridges
the
and
subunits, and a unique iron-molybdenum cofactor
(FeMo-co)(
)(4) , which is buried within the
subunit (3, 5) and is the site of substrate reduction (6) . The dinitrogenase complex has two of each of these metal
centers.
Dinitrogenase reductase is a 60-kDa dimer encoded by nifH. It specifically reduces dinitrogenase, apparently transferring electrons to the P-cluster, which then channels them to FeMo-co. In addition to this catalytic role, dinitrogenase reductase is also involved in the biosynthesis of FeMo-co and the maturation of dinitrogenase (reviewed in (1) ).
In vivo biosynthesis of FeMo-co depends on the activities of several nif gene products, including nifQ, -B, -V, -N, -E, and -H. Mutations in some of these genes result in
strains that are unable to fix N and accumulate an apo form
of dinitrogenase lacking the active site (reviewed in (1) ).
When isolated from nifB or nifNE strains of either Klebsiella pneumoniae or Azotobacter vinelandii,
apodinitrogenase has an additional associated
subunit(7, 8) . This complex has an
subunit
composition. In K. pneumoniae, the third subunit is encoded by nifY(8, 9) , and the addition of purified
FeMo-co to pure apodinitrogenase is sufficient to yield catalytically
active dinitrogenase(7, 8) . Addition of FeMo-co
effects the dissociation of NifY from the Apo I complex upon formation
of the holoenzyme(8) . In A. vinelandii, the identity
of the gene encoding the third subunit, termed
, is not known (7) although it is known not to be the product of the gene
designated nifY in A. vinelandii(10) .
Further, the third subunits from the K. pneumoniae and A.
vinelandii share no antigenicity and have shown different
dissociation requirements in vitro(7, 8) .
Apodinitrogenase from a nifH strain is biochemically
distinct from the hexameric apodinitrogenase forms described above (11) , requiring dinitrogenase reductase and ATP for in
vitro activation by FeMo-co(12, 13) . Further
analysis revealed that protein was absent from the
apodinitrogenase complex in crude extracts of nifH strains and
that the presence of dinitrogenase reductase and ATP promoted
association of the
subunit, forming the hexameric
apodinitrogenase similar to that found in the nifB and nifNE strains(13) . An implication of this result is
that
protein is essential for the proper in vitro activation of apodinitrogenase by FeMo-co. In this work, we have
further characterized
protein using both in vivo and in vitro techniques. The results suggest a role for
protein in which it interacts with both apodinitrogenase and FeMo-co;
it holds the apodinitrogenase in a conformation that allows access to
the FeMo-co ligand site, and it is also capable of binding FeMo-co and
subsequently donating it to apodinitrogenase. Because
protein
performs both of these functions, we have chosen to describe it as a
chaperone-insertase.
Figure 1:
The forms of protein as shown by
a Western blot of an anoxic native gel developed with antibody to
protein (
150 ng of protein/lane). Arrows denote the
various forms of
protein defined in the beginning of
``Results and Discussion.'' Lane 1, extract from nif-derepressed UW45 (nifB); lane 2, extract
from ammonium-grown cells; lane 3: extract from nif-derepressed UW45 with added
FeMo-co.
Figure 2:
Dissociation of protein from
apodinitrogenase. Western blots of identical anoxic native gels. Panel A was developed with antibody to
protein, and panel B was developed with antibody to dinitrogenase. Arrows denote the forms of
protein in panel A and apodinitrogenase (a) and holodinitrogenase (h) in panel B. Lane 1, extract from nif-derepressed UW (wild type) (
150 ng); lane 2,
extract from UW45 (nifB) (
150 ng); lane 3,
extract from UW45 (nifB) + 5 µl FeMo-co; lane
4, partially purified apodinitrogenase/
protein (
0.75
ng); lane 5, partially purified apodinitrogenase/
protein
+ 5 µl FeMo-co; lane 6, partially purified
apodinitrogenase/
protein + 5 µl FeMo-co + extract
from ammonium-grown UW (wild type) (
50
ng).
To study the dissociation of protein from apodinitrogenase in
crude extract from A. vinelandii, crude extract from UW45 (nifB) was used. It contains apodinitrogenase (Fig. 2B, lane 2) and both the A form
(apodinitrogenase-associated) and the B form (unassociated) of
protein (Fig. 2A, lane 2). The faint band
detected above the B form and below the A form is thought to be a
proteolytic product due to high protease contamination in the DNase
added during cell breakage (data not shown) and the faint band detected
immediately below the B form is unidentified and does not appear
reproducibly. The addition of FeMo-co completely activated the
apodinitrogenase, as demonstrated by an acetylene reduction assay (Table 2, lines 1 and 2), and caused the dinitrogenase to migrate
at the position of holodinitrogenase (Fig. 2B, lane
3), both indicating that the apodinitrogenase had been activated
to the holo form. There was no longer any
protein associated with
nitrogenase (Fig. 2A, lane 3) indicating that,
at least in crude extract, activation of apodinitrogenase by FeMo-co
was sufficient to cause dissociation of
protein.
Purified
apodinitrogenase from UW45 (nifB) is associated with
protein (Fig. 3, lane 1), and in contrast to the above
result, addition of a saturating amount of FeMo-co (as defined by
apodinitrogenase activation measured by an acetylene reduction assay)
promoted incomplete dissociation of
protein (Fig. 3, lane 2). For unknown reasons, when twice as much FeMo-co was
added to an identical sample, nearly all of the
protein
dissociated (Fig. 3, lane 3). The observation that a
saturating amount of FeMo-co (saturating for activation of
apodinitrogenase as determined by acetylene reduction) causes less
dissociation of
protein from the purified apodinitrogenase than
in crude extracts suggests the involvement of other proteins or factors
in this process in crude extract. A partially purified fraction
containing apodinitrogenase and
protein (see ``Experimental
Procedures'') from UW45 (nifB) was examined and showed
dissociation properties similar to that of the highly purified sample
above. There are two major species of
protein in the partially
purified sample: the A form and the B form (Fig. 2, A and B, lane 4). The band detected immediately
below the A form is the proteolyzed form of
protein associated
with apodinitrogenase (data not shown). When saturating levels of
FeMo-co (as defined by apodinitrogenase activation measured by an
acetylene reduction assay) were added to this sample, the
apodinitrogenase was activated (Table 2, lines 3 and 4) and
showed an appropriate shift in gel migration (Fig. 2B, lane 5), but not all of the
protein dissociated (Fig. 2A, lane 5). This distinction between
the activation of apodinitrogenase and the dissociation of
protein is consistent with previous results (7) where the
purified apodinitrogenase could be activated without dissociation of
protein.
Figure 3:
Dissociation of protein from
purified apodinitrogenase. Western blot of anoxic native gel developed
with antibody to
protein. Lane 1, purified
apodinitrogenase (
2 ng); lane 2, purified
apodinitrogenase + 5 µl FeMo-co; lane 3, purified
apodinitrogenase + 10 µl FeMo-co.
To determine if some factor is missing from the
partially purified apodinitrogenase fraction that might assist in
dissociation of protein, crude extracts from various strains were
added to this preparation. Addition of crude extract from
ammonium-grown UW (wild type, Fig. 2A, lane 6)
or from DJ40 (a deletion of most of the main cluster of nif genes, data not shown) was sufficient to promote complete
dissociation of
protein from apodinitrogenase upon activation
with the same FeMo-co levels without affecting dinitrogenase activity (Table 2, line 5), suggesting that another protein may be
involved in dissociation. The presence of the putative dissociation
factor in these particular extracts suggests it is neither nif-encoded nor nif-co-regulated. Further, this
enhancement of dissociation of
protein from apodinitrogenase was
probably due to a specific protein in A. vinelandii, since the
addition of either bovine serum albumin or crude extract from a K.
pneumoniae nif deletion strain (UN1978, (25) ) showed no
effect on activity or dissociation (data not shown). Appropriate
solvent controls for FeMo-co addition showed that addition of N-methylformamide alone had no effect on the dissociation of
protein (data not shown). It seems likely that the putative
``dissociation factor'' is a specific A. vinelandii protein that increases the efficiency of the dissociation process
but is not required for activation.
Figure 4:
Two-dimensional electrophoresis of
partially purified apodinitrogenase/ protein. Panels A and B are Coomassie-stained, while panels C and D are Western blots developed with antibody to
protein. Panels A and C display samples of partially purified
apodinitrogenase/
protein (
125 ng). Panels B and D are the identical samples to which FeMo-co has been added. A, B, and C forms of
protein are circled in the panels. The first dimension (native gel) was
run left to right, and the second dimension
(SDS-PAGE) was run from top to bottom as
pictured.
Should either the B or C forms of protein form a complex with
another protein, that protein should either shift with
protein in
the horizontal dimension or shift to a different position upon its
dissociation from
protein. The failure to see such a shift in the
horizontal position of any other protein argues that neither the B nor
C forms of
protein form such a complex (Fig. 4, panels
A and B). As demonstrated below, the B and C forms appear
to be dimeric and monomeric, respectively, so that any putative
associated protein would necessarily be at a 1:1 or 1:2 molar ratio and
therefore be easily detected in this analysis.
Figure 5:
Gel filtration analysis of protein
without (peak A) and with FeMo-co added (peak B) in
separate experiments. The elution profile of
protein was
determined by a densitometry scan of both a Coomassie-stained SDS gel
of the Sephacryl S-200 fractions and a Western blot of an SDS gel of
the fractions developed with antibody to
protein. The calibration
curve was made using bovine serum albumin, dinitrogenase reductase,
carbonic anhydrase, and cytochrome c as
standards.
Figure 6:
protein responds specifically to
FeMo-co. Western blots of anoxic native gels developed with antibody to
protein. Panel A: lane 1, extract from
ammonium-grown UW (wild type) (
150 ng); lane 2, same
extract as in lane 1 + 5 µl of purified FeMo-co; lane 3, same extract as in lane 1 + 5 µl of
oxidized FeMo-co; lane 4, same extract as in lane 1 + 5 µl of N-methylformamide (NMF); lane 5, same extract as in lane 1 + 20 nmol
MoS
; lane 6, same extract as in lane 1 + 10 µl NifB-co; lane 7, same extract as in lane 1 + 10 µl of 1 mM
Na
MoO
solution; lane 8, same mixture
as in lane 6 followed by 5 µl of purified FeMo-co; lane 9, same mixture as in lane 7 followed by 5
µl of purified FeMo-co.
The shift from B form to C form strongly
suggests that protein interacts with FeMo-co. The following
experiment also demonstrates that
protein becomes associated with
iron upon FeMo-co addition. Samples of the partially purified
apodinitrogenase/
protein fraction (see ``Experimental
Procedures'') were analyzed on a native gel with and without
FeMo-co addition and then stained for iron. Fig. 7shows that
the apodinitrogenase is the only major iron-containing protein present
in the sample before FeMo-co addition; form B of
protein contains
no detectable iron. Following FeMo-co addition, there is a major
iron-containing band that co-migrates with the C form of
protein.
It was demonstrated in Fig. 4that no other protein in this
sample responds detectably to the addition of FeMo-co (except
apodinitrogenase), and it is therefore highly probable that
protein has incorporated the iron. Negative controls utilizing either
oxidized FeMo-co or NifB-co did not cause incorporation of detectable
iron in the gel (data not shown). This result is consistent with the
hypothesis that the electrophoretic shift of
protein is due to
its binding of FeMo-co.
Figure 7:
Detection of iron-containing proteins on
native gels. Panel A, Western blot of anoxic native gel
developed with antibody to protein. Panel B, anoxic
native gel stained for iron with 3,5-diaminobenzoic acid. Lane
1, partially purified apodinitrogenase/
protein (
125
ng); lane 2, partially purified apodinitrogenase/
protein
+ 10 µl of FeMo-co.
Addition of the Sephadex G-25 eluate containing the
presumed -FeMo-co complex to the extract from DJ1030 (Table 3, lines 4 and 7) yielded a specific activity similar to
that of adding purified FeMo-co (Table 3, lines 5 and 6). The
Sephadex G-25 eluate that contains the C form is therefore capable of
activating the tetrameric apodinitrogenase. To be sure that association
of the C form of
protein with apodinitrogenase could occur, the
assays were performed in the presence of dinitrogenase reductase and
nucleotides.
The Sephadex G-25 eluate containing the C form was also
added to the extract from strain UW45, which contains the
apodinitrogenase hexamer. The Sephadex G-25 eluate that contains the C
form is capable of activating the hexameric apodinitrogenase (Table 3, lines 1 and 3) to levels comparable with adding
purified FeMo-co directly (Table 3, line 2). Sephadex G-25
eluates containing the C form of protein are therefore capable of
activating the apodinitrogenase hexamer as well.
This result can be
explained by either of two possibilities; either the C form donates the
FeMo-co to protein already associated with the apodinitrogenase
complex, or the C form replaces the associated
protein in the
apodinitrogenase complex. Because both forms of apodinitrogenase were
activable by the Sephadex G-25 eluate containing the C form, a
distinction between the two possibilities cannot be made, and this will
be discussed below.
The volume of Sephadex G-25 eluate required to
generate the same levels of nitrogenase activity is much greater than
that necessary with purified FeMo-co because the Sephadex G-25 column
dilutes the protein eluate. We cannot exclude the possibility of other
proteins in the crude extracts binding and donating FeMo-co; however,
the results described above suggest that this is unlikely. Purification
of protein and subsequent activation assays with the purified
protein will be essential to demonstrate specificity of the insertion
process.
Although protein responds specifically to and most
likely binds FeMo-co in vitro, it was of particular interest
to see if such a FeMo-co-bound
protein species could accumulate in vivo. FeMo-co can be synthesized in vivo in the
absence of the structural components of
dinitrogenase(26, 27) , and it would be under these
conditions that one might expect a
-bound FeMo-co species to
accumulate. When crude extracts from A. vinelandii strains UW6 (nifK) and UW10 (nifD) were examined by a Western
blot of a native gel (data not shown),
50% of the detectable
protein was in the C form, consistent with the idea that the FeMo-co
that is synthesized in these strains accumulates on
protein. When
crude extract from DJ33 extract was examined, however, the detectable
levels of FeMo-co were consistently much lower than those seen in UW6
and UW10, and none of the C form was detected; it is possible that the
levels of the C form are lower than the detection limit of our Western
blots of native gels. While the reason for these differences is
unresolved, it may be dependent on the observation that UW6 and UW10
are capable of accumulating inactive NifKD peptides, whereas DJ33 is a
complete nifKD deletion. Regardless, this data indicates that
the C form of
protein is of physiological relevance and that this
response to FeMo-co does occur in vivo.
These results are
reminiscent of previous observations of a protein estimated to be 65
kDa in strains with mutations in the structural genes for
nitrogenase(26, 27) ; this protein accumulates
significant amounts of molybdenum and is capable of activating
apodinitrogenase-containing extracts(26) . The presence of the
C form in UW6 and UW10 (the two strains used in the analysis of the
65-kDa protein) is consistent with the possibility that protein
and the 65-kDa protein are the same.
Figure 8:
protein's response to FeMo-co
is reversible. Western blot of anoxic native gel developed with
antibody to
protein. Arrows denote the forms of
protein, including the intermediate (int). Lane 1,
extract from ammonium-grown UW (
150 ng); lane 2, extract
from ammonium-grown UW + 5 µl of FeMo-co; lane 3,
extract from ammonium-grown UW + FeMo-co, exposed to air for 60
min; lane 4, extract from ammonium-grown UW + FeMo-co,
exposed to air for 30 min; lane 5, extract from ammonium-grown
UW + FeMo-co, exposed to air for 15 min; lane 6, extract
from ammonium-grown UW + FeMo-co, exposed to air for 60 min,
re-reduced, and + 5 µl of FeMo-co
again.
When the mixture of FeMo-co and
crude extract was only exposed for 15 min, protein migrated at a
position between the B and C forms (lane 5). This intermediate
form might represent either the binding of a single FeMo-co to the
dimer or a
monomer free of any metals. When FeMo-co was
titrated against the B form of
protein, no such intermediate was
observed;
protein migrated at the B position, the C position, or
a combination of both (data not shown). This result supports the latter
model, where the intermediate is monomerized
protein having no
metals bound.
It is interesting to note that FeMo-co in aqueous
solution is destroyed upon exposure to air in less than 1
min(4) , whereas the C form of protein is still present
after 5 min of exposure to air (data not shown). While it is not known
whether the metal species remaining bound to
protein after 5 min
is still capable of activating dinitrogenase, this result suggests that
protein might provide some degree of oxygen stability or
protection to FeMo-co.
In an
effort to identify the gene encoding protein and gain some
insight into its regulation, crude extracts from various strains grown
under a range of conditions were examined by Western blots of native
gels and SDS-PAGE to see if any of the resulting extracts lacked
protein.
protein was present in similar amounts in all of the
strains and under all of the conditions we examined: ammonium-grown,
urea-grown, and nitrate-grown, derepressed and heat-shocked UW (wild
type), and ammonium-grown and derepressed DJ40 (a deletion of the
entire major nif gene cluster; (10) ). The similar
amounts of
protein present indicate that
protein is not
encoded by a nif gene nor is it co-regulated. The presence of
protein under a range of physiological conditions suggests it has
other roles in the cell besides maturation of dinitrogenase. It is
possible, for example, that it is a processing protein for some other
metal-containing proteins.
Figure 9:
Models for the maturation of
dinitrogenase. Scheme 1 shows the protein binding
FeMo-co first and simultaneously monomerizing. With the addition of ATP
and dinitrogenase reductase, the
-FeMo-co complex can associate
with the apodinitrogenase tetramer. The
protein then inserts the
FeMo-co into apodinitrogenase and subsequently dissociates. Scheme
2 shows the
protein binding to the apodinitrogenase tetramer
first, in the presence of ATP and dinitrogenase reductase. FeMo-co then
binds to the apodinitrogenase-bound
protein. The
protein
then inserts the FeMo-co into apodinitrogenase and subsequently
dissociates. The darker subunits of dinitrogenase represent the
subunit, while the lighter subunits represent the
subunit in
which FeMo-co is placed. Only a single FeMo-co and P-cluster for each
dinitrogenase tetramer are shown.
A remaining
issue in these studies is where protein binds the
apodinitrogenase complex.
protein's role in promoting
insertion and actually binding FeMo-co necessitates its binding to the
subunit of nitrogenase, proximal to the FeMo-co binding site.
Analysis of crystallographic data of dinitrogenase (5) and
chemical modification data of the apodinitrogenase hexamer (
)indicate that the hexameric apodinitrogenase, in contrast
to the holodinitrogenase, has an exposed cysteine (Cys-275
)
involved in FeMo-co binding and an exposed non-ligand cysteine
(Cys-45
).
This implies a structural change in domain
III (5) during the maturation of dinitrogenase; the proposed
binding region for
protein is a potential ``hinge''
region in domain III of the
subunit that potentially has a degree
of flexibility and could expose the FeMo-co site if held in the right
conformation.
It has been suggested that protein
and its K. pneumoniae counterpart NifY are chaperone-like
proteins, whose function is to hold the dinitrogenase subunits in a
conformation that is competent for FeMo-co insertion(8) . The
tight association of
protein with the apodinitrogenase complex
and its dissociation upon addition of required metals are reminiscent
of proteins in two other metalloenzyme systems, urease (Ni) and
tyrosinase (Cu)(28, 29, 30) . These proteins
are also believed to be chaperone/accessory proteins required to
stabilize the apoproteins; however, neither has been shown to bind
metals themselves, so it is unclear if these have the insertase
activity that
protein appears to have.
In conclusion, data
presented here show that the A. vinelandii protein is
involved in the maturation of dinitrogenase. Its association with the
dinitrogenase complex is required for proper insertion of FeMo-co,
perhaps by binding to the
subunit such that the FeMo-co binding
site remains accessible. Further,
protein binds FeMo-co itself
and probably inserts the co-factor into its proper position within the
subunit.
protein therefore may have properties of both
metal co-factor insertases and chaperone proteins.