(Received for publication, June 2, 1995; and in revised form, August 28, 1995 )
From the
NifB-co is an iron- and sulfur-containing precursor to the
iron-molybdenum cofactor (FeMo-co) of dinitrogenase. The synthesis of
NifB-co requires at least the product of the nifB gene.
Incorporation of Fe and
S from NifB-co into
FeMo-co was observed only when all components of the in vitro FeMo-co synthesis system were present. Incorporation of iron and
sulfur from NifB-co into dinitrogenase was not observed in control
experiments in which the apodinitrogenase (lacking FeMo-co) was
initially activated with purified, unlabeled FeMo-co or in assays where
NifB-co was oxygen-inactivated prior to addition to the synthesis
system. These data clearly demonstrate that iron and sulfur from active
NifB-co are specifically incorporated into FeMo-co of dinitrogenase and
provide direct biochemical identification of an iron-sulfur precursor
of FeMo-co.
Under different in vitro FeMo-co synthesis conditions, iron and sulfur from NifB-co were associated with at least two other proteins (NIFNE and gamma) that are involved in the formation of active dinitrogenase. The results presented here suggest that multiple FeMo-co processing steps might occur on NIFNE and that FeMo-co synthesis is most likely completed prior to the association of FeMo-co with gamma.
The conversion of dinitrogen to ammonium by biological systems
is catalyzed by nitrogenase. Nitrogenase is composed of two
oxygen-labile metalloproteins: dinitrogenase (also called MoFe protein
or component I) and dinitrogenase reductase (also called NIFH, Fe
protein, or component II; (1) and (2) ). Dinitrogenase
is an tetramer of the nifD and nifK gene products, and it contains two pairs of
unique metal clusters, known as the iron-molybdenum cofactor (FeMo-co; (
)(3) and (4) ) and the
P-cluster(4, 5) . Dinitrogenase is specifically
reduced by dinitrogenase reductase. Dinitrogenase reductase, which
contains a single Fe
S
cluster, is an
dimer of the nifH gene product (6) .
The electrons transferred to dinitrogenase are ultimately channeled to
FeMo-co, the site of substrate reduction (see (7) for a
concise review).
FeMo-co is composed of molybdenum, iron, sulfur, and homocitrate ((R)-2-hydroxyl-1,2,4-butane tricarboxylic acid) in a ratio of 1:7:9:1(4, 8) . The products of at least six nitrogen fixation (nif) genes, including nifQ, nifV, nifB, nifH, nifN, and nifE, are required for the biosynthesis of FeMo-co(8, 9, 10, 11) . Interestingly, the genes that encode dinitrogenase (nifD and nifK) are not required for FeMo-co biosynthesis, suggesting that FeMo-co is assembled elsewhere in the cell and is then inserted into FeMo-co-deficient dinitrogenase (apodinitrogenase; Refs. 12 and 13). The high degree of sequence similarity between the nifN and nifK sequences and the nifE and nifD sequences suggests that NIFNE might serve as a scaffold for FeMo-co biosynthesis(14) . This hypothesis is supported by the recent observation that the mobility of NIFNE on native (non-denaturing) gels changes specifically upon the addition of NifB-co, a likely FeMo-co precursor (described below; (15) ). An in vitro FeMo-co synthesis system that requires an ATP-regenerating system, molybdate, homocitrate, and at least NIFB, NIFNE, and NIFH has been described(11) . Although use of the in vitro system has yielded significant information concerning FeMo-co biosynthesis, the nature of the iron and sulfur donor(s) for the biosynthesis of FeMo-co remains unknown.
NifB-co is one potential source of iron and sulfur for FeMo-co biosynthesis. In the course of attempting to purify the NIFB protein from Klebsiella pneumoniae, Shah et al.(16) isolated and purified the apparent product of NIFB as a detergent-solubilized, small molecule termed NifB-cofactor (NifB-co). Solutions of NifB-co exhibit certain characteristics that are similar to solutions of purified FeMo-co, including color, stability in N-methylformamide, and oxygen lability. The requirement for NIFB in the in vitro FeMo-co synthesis assay is satisfied by the addition of NifB-co, and the amount of FeMo-co synthesized in vitro is proportional to the amount of NifB-co added to the system in which all other components are present in excess. The stoichiometric requirement of NifB-co is consistent with the hypothesis that NifB-co is an iron-sulfur precursor of FeMo-co. Because a functional nifB gene is also required for the molybdenum-independent nitrogen fixation systems(17) , it has been proposed that NifB-co is the basic iron-sulfur cluster for the synthesis of FeMo-co, the vanadium-containing cofactor (FeV-co) of the vnf-encoded nitrogenase, and the iron-only cofactor of the anf-encoded nitrogenase(16) .
In vitro activation of apodinitrogenase by FeMo-co apparently requires the presence of a protein designated as gamma(18) . Recent studies show that addition of purified FeMo-co to crude extracts and partially purified fractions containing gamma results in a shift in the electrophoretic mobility of gamma on native gels. The mobility change correlates with the incorporation of iron into gamma(19) . In addition, crude extracts that contain this faster migrating form of gamma (with associated FeMo-co) are capable of activating apodinitrogenase in vitro(19) .
To date there has been no direct evidence for the incorporation of iron and sulfur from NifB-co into FeMo-co or for the flow of iron and sulfur from NifB-co through the NIFNE and gamma proteins. This report directly demonstrates that iron and sulfur from NifB-co can become associated with NIFNE and gamma, and ultimately become incorporated into apodinitrogenase as FeMo-co.
Strain UW45 was grown and derepressed in tungsten-containing medium (molybdenum-free) as described previously(11) . When necessary, small molecules (i.e. homocitrate) were removed from the cell extracts by Sephadex G-25 column chromatography.
For sulfur
donation studies, a starter culture of strain UN1217 was grown
aerobically on a rotary shaker at 30 °C for 20-24 h in 275 ml
of medium (minimal medium as described previously; (16) )
containing 15.6 mM ammonium acetate (filter-sterilized). One
ml of this culture (stationary phase) was transferred to 250 ml of
fresh medium containing 0.2 mM sulfur added as
NaSO
. Approximately 15 h later, this starter
culture was used to inoculate a larger culture that would be
derepressed. A 5-liter Pyrex carboy containing 4.5 liter of the medium
plus 3 ml of a 22% ammonium acetate solution (approximately 1.9 mM final concentration) and 28.7 ml of a 10 mM
Na
SO
solution (approximately 0.063 mM sulfur final concentration) was inoculated with 105 ml of the
starter culture. The large culture was grown at 30 °C and sparged
vigorously with compressed air (filter-sterilized). The culture was
spot tested for ammonium using Nessler's reagent. Thirty min
after exhaustion of the ammonium (which occurred approximately 4 h post
inoculation), derepression of the Nif proteins was initiated by
switching the gas from air to 95% N
+ 5% CO
and by adding 4.4 ml of a sterile 10% L-serine solution.
At this time, approximately 4 mCi of
Na
SO
(specific activity
561-572 mCi/mmol) were added to the culture. Six hours after the
start of derepression, the cells were harvested (approximately 5 g, wet
weight) by centrifugation and stored at -80 °C. Approximately
60% of the radiolabel added to the medium was incorporated into the
cells.
Growth and derepression of strain UN1217 in the presence of FeCl
were similar to that described above,
with the following modifications. The starter cultures were not
supplemented with any iron. The medium in the 5-liter carboy was
supplemented with FeCl
to a final concentration of
approximately 10 µM. At the start of derepression, 2 mCi
of
FeCl
(specific activity 26.7-29.2
mCi/mg) were added.
Fe-Labeled ferric citrate was prepared
by diluting the
FeCl
into 1 ml of distilled
H
O containing 5 mg of sodium citrate (pH approximately 7).
The solution was heated and the entire volume was added to the carboy.
Approximately 50-70% of the radiolabel added to the medium was
incorporated into the cells.
Approximately 4.5 ml of the cell
suspension were transferred to 10-ml polycarbonate centrifuge tubes (16
76 mm) that contained 9.5 g of 0.1-0.2 mm glass beads.
The beads had been equilibrated overnight with anoxic 0.1 M Tris-HCl buffer (pH 7.4). The tubes were degassed, and cell
breakage was initiated by vortexing (standard bench-top vortexer on
high setting) each tube for 1 min. Each tube was placed on ice for at
least 2 min following vortexing. Seven vortex/ice cycles were used for K. pneumoniae cell breakage. N-Lauroylsarcosine was
added to a final concentration of 2% (from a 20% anoxic stock solution
in 0.1 M Tris-HCl (pH 7.4)). After 10 min at room temperature,
the tubes were centrifuged at 27,200
g for 10 min. The
temperature during centrifugation was maintained at approximately 20
°C to avoid precipitation of the detergent. The supernatants
containing NifB-co were anoxically transferred and combined. To ensure
that all of the NifB-co was collected, 2 ml of 0.1 M Tris-HCl
(pH 7.4) and 0.2 ml of 20% N-lauroylsarcosine stock solution
were added to each tube immediately after the supernatant was removed.
The tubes were degassed, vortexed for 1 min, and centrifuged as
described above. The supernatants were anoxically transferred and
combined (but kept separately from the first supernatant). Both
supernatants were assayed for NifB-co activity using the in vitro FeMo-co synthesis assay (described below). The supernatants were
stored at -20 °C.
In control experiments, various components of the complete reaction mixture were excluded as indicated under ``Results and Discussion.'' Where indicated, apodinitrogenase in 200 µl of the appropriate extract was activated by incubation with an excess of purified, unlabeled FeMo-co for 10 min before performing the in vitro FeMo-co synthesis reaction with the labeled NifB-co.
To
investigate the role of NifB-co as an iron-sulfur donor for FeMo-co
biosynthesis, NifB-co was independently labeled in vivo with Fe or
S. The labeled cofactor was purified as
described under ``Experimental Procedures.'' The
Fe- and
S-labeled NifB-co fractions contained
an average of 2,200,000 and 300,000 cpm/ml, respectively. The average
activities of the
Fe and
S-labeled NifB-co
fractions were 770 and 400 nmol of C
H
reduced/min by dinitrogenase formed/ml of the NifB-co-containing
fraction, respectively.
The in vitro FeMo-co synthesis
system together with anoxic, native gel electrophoresis was employed to
monitor the incorporation of iron and/or sulfur from NifB-co into the
FeMo-co of dinitrogenase. A complete reaction mixture that included all
of the components known to be required for FeMo-co biosynthesis was
used to monitor donation of iron and sulfur from NifB-co to FeMo-co.
The complete reaction mixtures contained molybdenum, homocitrate, an
ATP-regenerating system, and cell-free extract from strain UW45 (nifB), which served as a source of NIFNE, dinitrogenase
reductase, apodinitrogenase, and any other unidentified factors
required for in vitro FeMo-co biosynthesis. Purified Fe- or
S-labeled NifB-co was added to
complete the reaction mixture. A number of control reactions (in which
iron and sulfur from NifB-co were not expected to be incorporated into
dinitrogenase) were performed to demonstrate the specificity of
incorporation of radiolabel from active NifB-co into dinitrogenase.
Homocitrate, molybdenum, and MgATP were omitted from some in vitro FeMo-co synthesis reaction mixtures to prevent FeMo-co synthesis.
In other control reaction mixtures, the oxygen-labile, labeled NifB-co
was inactivated by exposure to air prior to addition to the reactions.
In other control assays, all of the apodinitrogenase present in the
UW45 extracts was activated in vitro with unlabeled, purified
FeMo-co prior to the addition of labeled NifB-co to the complete
reaction mixture. In this system, all of the available FeMo-co binding
sites on the apodinitrogenase should be occupied by unlabeled FeMo-co,
and therefore iron and sulfur from labeled NifB-co were not expected to
be associated with dinitrogenase.
To investigate the incorporation
of Fe and
S from NifB-co into FeMo-co, the
proteins in the various in vitro FeMo-co synthesis reaction
mixtures were separated on anoxic native gels. The position to which
dinitrogenase migrated was determined by immunoblot analysis (data not
shown). The data in Fig. 1A revealed that incorporation
of
Fe from labeled NifB-co into dinitrogenase required the
presence of all of the components known to be required for in vitro FeMo-co synthesis. A prominently labeled band that comigrated with
dinitrogenase was only detected in the lanes to which the complete
FeMo-co synthesis reaction mixture (plus apodinitrogenase) was applied (Fig. 1A, lanes 7 and 8). The species
that migrated slightly faster than dinitrogenase has been identified as
NIFNE and is discussed in detail below. At least five lines of evidence
suggested that in the complete in vitro FeMo-co synthesis
reaction mixture, iron from NifB-co was specifically incorporated into
the FeMo-co of dinitrogenase. (i) Only very low levels of iron were
associated with dinitrogenase when FeMo-co synthesis was prevented in
the reaction mixtures by omission of MgATP, molybdenum, or homocitrate
(each of which is a known requirement for in vitro FeMo-co
synthesis; see Fig. 1A, lanes 2, 6,
and 9, respectively). (ii) The absence of a band that
co-migrated with dinitrogenase in samples where the air-inactivated
Fe-NifB-co was utilized demonstrated that active NifB-co
was required for incorporation of the
Fe into FeMo-co of
dinitrogenase (Fig. 1A, lane 5). (iii)
Activation of apodinitrogenase with unlabeled FeMo-co prior to
synthesizing FeMo-co using the
Fe-labeled NifB-co resulted
in almost no association of
Fe with dinitrogenase (Fig. 1A, lane 3). (iv) No radiolabel was
detected at the dinitrogenase position in the lane containing only free
Fe-NifB-co (Fig. 1A, lane 4). (v)
Labeled dinitrogenase was not observed when the complete reaction
mixture was oxidized following the FeMo-co synthesis reaction, but
prior to being applied to the gel (data not shown). Note that the
C
H
reduction activities of the various assays (Fig. 1A) are consistent with the conclusion that
holodinitrogenase was only formed in the complete system (by FeMo-co
synthesis using
Fe-NifB-co; Fig. 1A, lanes 7 and 8) and in the FeMo-co activated sample
(by activation of apodinitrogenase with unlabeled FeMo-co; Fig. 1A, lane 3). Similar results were
obtained when
S-labeled NifB-co was used in the various in vitro FeMo-co synthesis reaction mixtures (Fig. 1B, compare lanes 1, 2, and 4 (control experiments) with lane 3 (complete
system)). The differences in the band intensities in reactions that
used
S-labeled NifB-co compared to those that used
Fe-labeled NifB-co (Fig. 1, compare A and B) are most likely because the cpm/ml of the
S-labeled NifB-co was only 14% that of the
Fe-labeled NifB-co. Together these data show that iron and
sulfur from NifB-co were only incorporated into dinitrogenase under
conditions where FeMo-co was synthesized and demonstrate that iron and
sulfur from NifB-co were specifically incorporated into the FeMo-co of
dinitrogenase. These data provide direct biochemical identification of
an iron and sulfur precursor of FeMo-co.
Figure 1:
Phosphorimage of a native, anoxic gel
of various in vitro FeMo-co synthesis reactions that included Fe-labeled-NifB-co (panel A) and
S-labeled-NifB-co (panel B). Arrows indicate the position of dinitrogenase and NIFNE as determined by
immunoblot analysis. Synthesis reactions used UW45 (nifB)
extract except where indicated. A, lane 1, purified
NIFNE protein (no cell-free extract); lane 2, minus MgATP
reaction; lane 3, apodinitrogenase initially activated with
purified, unlabeled FeMo-co; lane 4,
Fe-labeled-NifB-co only; lane 5,
Fe-NifB-co air-inactivated prior to addition to complete in vitro synthesis reaction mixture; lane 6, minus
MoO
reaction; lane
7, complete in vitro synthesis reaction mixture (50
µl applied); lane 8, complete in vitro synthesis
reaction mixture (100 µl applied); lane 9, minus
homocitrate reaction. B, lane 1,
S-labeled-NifB-co; lane 2, minus homocitrate
reaction; lane 3, complete in vitro synthesis
reaction mixture; lane 4, minus
MoO
and homocitrate
reaction; lane 5, minus apodinitrogenase reaction (CA11.1
(
nifHDK
vnfDGK1::spc)). Where appropriate,
the C
H
reduction activities of the assays (nmol
of C
H
formed/[min
assay]) are
reported. Table below the figures indicate components present (+)
in the reaction mixture.
The lack of Fe
associated with dinitrogenase in the sample in which apodinitrogenase
was initially activated with purified, unlabeled FeMo-co suggested
that, once bound to the protein, there was not a significant amount of
turnover of FeMo-co in this system. The absence of a labeled
dinitrogenase band indicated that the synthesized
Fe-FeMo-co did not displace the FeMo-co that originally
activated the apodinitrogenase.
The failure of NifB-co to form a complex with apodinitrogenase was also tested by investigating its possible ability to inhibit the insertion of FeMo-co into apodinitrogenase. As shown in Table 1, preincubation of apodinitrogenase with an excess of NifB-co did not result in any detectable inhibition of FeMo-co insertion, consistent with the labeling results in Fig. 1.
Accurate quantitation of the number of iron and sulfur atoms donated by NifB-co for FeMo-co synthesis remains to be accomplished. However, based on previous activity and iron analysis studies, NifB-co has been predicted to donate all of the iron for FeMo-co biosynthesis(16) .
In the
complete in vitro FeMo-co synthesis reaction mixture (Fig. 1A, lanes 7 and 8), the iron
from NifB-co was obviously associated with more than one protein. The
slowest migrating species is currently unidentified (Fig. 1A, lane 8). However, the species was
also present in extracts of wild type cells grown on
NH (data not shown) and was likely binding
iron from denatured NifB-co because it was also observed in samples
containing oxidized NifB-co (Fig. 1A, lane 5)
and, in fact, became more prominent as the labeled NifB-co samples lost
activity, most likely due to oxygen inactivation. The slowest migrating
species was not detected when
S-labeled NifB-co was used (Fig. 1B).
The labeled species that migrated slightly
faster than dinitrogenase (Fig. 1A, lanes 7 and 8) was identified as NIFNE by immunoblot analysis
(data not shown) and by use of purified NIFNE protein (Fig. 1A, lane 1). Purified NIFNE with
associated Fe from NifB-co (Fig. 1A, lane 1) comigrated with the labeled species observed in a
number of the in vitro FeMo-co synthesis reaction mixtures
including: (i) reaction mixtures from which MgATP (Fig. 1A, lane 2), molybdenum (Fig. 1A, lane 6), homocitrate (Fig. 1A, lane 9), or dinitrogenase reductase
(data not shown) were excluded, (ii) complete in vitro FeMo-co
synthesis reaction mixtures in which all of the apodinitrogenase was
activated with unlabeled FeMo-co prior to the addition of the
Fe-NifB-co (Fig. 1A, lane 3), and
(iii) the complete reaction mixture (Fig. 1A, lanes
7 and 8). Active NifB-co was required for this
association, as determined by the absence of this species when
air-inactivated
Fe-NifB-co was used in the FeMo-co
synthesis reactions (Fig. 1A, lane 5). These
data suggest that in the absence of molybdenum, homocitrate, MgATP, or
dinitrogenase reductase (each of which is a requirement for FeMo-co
synthesis) NifB-co is associated with NIFNE. These results are
consistent with a model where these compounds are all necessary for
later steps of FeMo-co synthesis on NIFNE and suggest that FeMo-co
might be completed on NIFNE. The
Fe-NifB-co data presented
here definitively demonstrate that the previously observed shift in the
mobility of NIFNE on native gels in the presence of NifB-co was due to
the association of iron and sulfur from NifB-co with NIFNE.
It is
currently unclear why less Fe from NifB-co was
reproducibly associated with NIFNE when homocitrate was excluded from
the reaction mix compared to when MgATP or molybdenum were excluded (Fig. 1A, compare lane 9 with lanes 2 and 6). At least two hypotheses might explain this
observation. Although the activity assay showed no C
H
reduction activity (which suggests that no FeMo-co was
synthesized), an organic acid other than homocitrate might have been
present in the reaction mixture, and thus a low amount of an aberrant
form of FeMo-co (containing an organic acid other than homocitrate)
might have been synthesized and proceeded along the biosynthetic
pathway (i.e. been passed to another protein). The ability of
other organic acids to substitute for homocitrate in vitro has
been demonstrated(27) . Alternatively, it is possible that in
this reaction system (that lacked homocitrate) the iron-sulfur
precursor was converted to a form (possibly with added molybdenum, but
not the finished FeMo-co) that had a lesser affinity for NIFNE than did
the iron-sulfur precursor.
Binding of Fe and
S from NifB-co to NIFNE was also observed in reactions
that utilized cell extracts of strains with mutations in nifDK (apodinitrogenase mutants; CA11.1 (
nifHDK
vnfDGK1::spc), which produces both NIFNE and NifB-co (Fig. 2, lane 2); DJ677 (
nifB::kan
nifKD), which produces NIFNE (Fig. 1B, lane 5)). Interestingly, the
observed amount of
Fe from NifB-co associated with NIFNE
in extracts of strain CA11.1 in the presence and absence of added
dinitrogenase reductase suggests that the NIFNE-NifB-co complex is a
physiologically relevant precursor along the FeMo-co biosynthetic
pathway. Significantly more
Fe (from NifB-co) was
associated with NIFNE from CA11.1 (
nifHDK
vnfDGK1::spc) extracts when dinitrogenase reductase
was added in addition to all of the other requirements for in vitro FeMo-co synthesis (Fig. 2, compare lanes 2 and 3). Extracts of strain CA11.1 derepressed for the Nif proteins
exhibited high levels of NIFB and NIFNE activities as monitored by the
FeMo-co synthesis assay. Immunoblot analysis of a native gel containing
crude extract from this strain showed that the NIFNE present migrated
at the position for NIFNE with bound NifB-co (data not shown; (15) ). We hypothesize that the majority of NIFNE in lane 3 (Fig. 2) had bound NifB-co (unlabeled, of in vivo origin), and therefore little association of the added
Fe-NifB-co with NIFNE was observed in the absence of a
complete in vitro FeMo-co synthesis reaction system. When
dinitrogenase reductase was added to complete the reaction mixture
containing CA11.1 extract, molybdenum, homocitrate, and MgATP, all of
the components known to be required for FeMo-co biosynthesis were
present and therefore FeMo-co was synthesized. Thus, the unlabeled
NifB-co (of in vivo origin) was apparently incorporated into
FeMo-co and proceeded along the biosynthetic pathway (discussed below).
The NIFNE protein was then available to interact with the
Fe-NifB-co that had been added to the system, and
therefore an increase in
Fe associated with NIFNE was
observed upon addition of dinitrogenase reductase to the reaction
mixture (Fig. 2, lane 2). These results demonstrate the
relevance of the interaction of NifB-co with NIFNE. The apparent
ability to chase NifB-co from NIFNE in CA11.1 extracts by addition of
dinitrogenase reductase to the reaction mixture suggests that NIFNE is
not only capable of binding NifB-co, but, in fact, the NIFNE-NifB-co
complex appears to be a precursor of FeMo-co.
Figure 2:
Phosphorimage of a native, anoxic gel of
various in vitro FeMo-co synthesis reactions that included Fe-labeled-NifB-co. Arrows indicate the position
of dinitrogenase and NIFNE as determined by immunoblot analysis.
Extracts of strain CA11.1 (
nifHDK
vnfDGK1::spc) were used in the reactions applied to lanes 1 and 2. Lane 1, minus
apodinitrogenase reaction; lane 2, minus apodinitrogenase and
dinitrogenase reductase reaction; lane 3, complete in
vitro FeMo-co synthesis reaction (UW45 (nifB) extract).
Where appropriate, the C
H
reduction activities
of the assays (nmol of C
H
formed/[min
assay]) are reported. Table below the figure
indicates components present (+) in the reaction
mixture.
It is currently
unclear if FeMo-co synthesis is completed on NIFNE, however there is
evidence that NIFNE can bind to FeMo-co in crude extracts. When
purified FeMo-co was added to extracts of strain DJ677
(nifB::kan
nifKD), which lacks
apodinitrogenase and is unable to synthesize NifB-co, and the mixture
was applied to a Sephacryl S-200 sizing column, the NIFNE-containing
fraction contained FeMo-co as determined by the ability to activate
apodinitrogenase in extracts of strain UW45 (nifB; data not
shown). When a similar experiment was done by adding purified FeMo-co
to extracts of strain DJ678 (
nifDK
YENX::kan), which lacks both apodinitrogenase and
NIFNE, no FeMo-co was associated with the Sephacryl S-200 column
fraction that corresponded to the NIFNE-containing fraction in the
DJ677 experiment (data not shown). These data suggest that NIFNE is
capable of associating with the completed FeMo-co molecule and are
consistent with the hypothesis that FeMo-co is completed on NIFNE. The
observation that
Fe from NifB-co was associated with NIFNE
in extracts of strain UW45 that had been activated with purifed FeMo-co (Fig. 1A, lane 3) might suggest that NIFNE has
a greater affinity for NifB-co than for FeMo-co.
FeMo-co synthesis assays
were performed using extracts from strain DJ677 (nifB::kan
nifDK), which lacks apodinitrogenase and NIFB. The
absence of apodinitrogenase allows the accumulation of FeMo-co on other
proteins to be detected. To visualize dinitrogenase and gamma on the
same system (Fig. 3), samples were electrophoresed on a gel for
significantly fewer V
h than the gels shown in Fig. 1and Fig. 2. The fastest migrating species (marked
``unknown'' in Fig. 3) is unidentified
(discussed below). The smear on the gel (that obscures
Fe
associated with NIFNE) is attributed to
Fe from
oxygen-inactivated NifB-co (see Fig. 3, lane 1). The
position of gamma (with bound FeMo-co) on the gel was determined by
immunoblot analysis (data not shown).
Figure 3:
Phosphorimage of a native, anoxic gel of
various in vitro FeMo-co synthesis reactions that included Fe-labeled-NifB-co. Arrows indicate the positions
of dinitrogenase and gamma (with bound FeMo-co) as determined by
immunoblot analysis. The unknown species is also indicated by an arrow. Synthesis reactions used extracts of strain DJ677
(
nifB::kan
nifDK), except in lane
6. Lane 1, minus apodinitrogenase with
Fe-NifB-co inactivated by air prior to addition to the
assay; lane 2, minus apodinitrogenase and homocitrate; lane 3, minus apodinitrogenase and MgATP; lane 4,
minus apodinitrogenase with purified, unlabeled FeMo-co added; lane
5, minus apodinitrogenase (all other FeMo-co synthesis
requirements present); lane 6, complete in vitro FeMo-co synthesis reaction (UW45 (nifB)). Where
appropriate, the C
H
reduction activities of the
assays (nmol of C
H
formed/[min
assay]) are reported. Table below the figure
indicates components present (+) in the reaction
mixture.
The data in Fig. 3revealed that significant amounts of Fe from
Fe-NifB-co accumulated on a protein that co-migrated with
gamma in extracts of strain DJ677 only when all of the components
required for FeMo-co biosynthesis were present (Fig. 3, lane
5).
Fe was not associated with gamma when FeMo-co
synthesis could not occur due to omission of the MgATP (Fig. 3, lane 3) or homocitrate (Fig. 3, lane 2) from
the reaction mixture or when purified, unlabeled FeMo-co was added
prior to the synthesis reaction (Fig. 3, lane 4). In
addition, no label was associated with this protein when the
Fe-NifB-co was inactivated by exposure to air prior to
addition to the FeMo-co synthesis reaction (Fig. 3, lane
1). Because all components of the in vitro FeMo-co
synthesis system were required to observe
Fe-NifB-co-dependent labeling of gamma, these data support
the conclusion that the iron associated with gamma was in the form of
FeMo-co and definitively demonstrate that the iron previously observed
to be associated with gamma (in the form of FeMo-co) was from NifB-co.
A Fe-labeled species that migrated slightly faster than
gamma was observed in some reaction mixtures (Fig. 3, lanes
2, 3, and 5). The band did not correspond to
free
Fe-NifB-co and was dependent on active NifB-co
because the species was absent in samples where air-inactivated
Fe-NifB-co was utilized (Fig. 3, lane 1).
The species was most prominent in samples that contained all of the
components required for FeMo-co synthesis (Fig. 3, lane
5) and in samples that contained all of the components required
for FeMo-co synthesis except homocitrate (Fig. 3, lane
2). Immunoblot analysis indicated that this species did not
correspond to gamma or NIFNE. The possibility that this is another
relevant protein involved in FeMo-co biosynthesis is currently under
investigation. A similar species appears to have been observed when
MoO
was used to investigate
incorporation of molybdenum into dinitrogenase in the presence of
various homocitrate analogs (see Fig. 3of (27) ).
A major question that remains concerns the source
of iron and sulfur for NifB-co biosynthesis. Exciting results from the
laboratory of Dean and colleagues indicate that the nifU and nifS gene products are likely to be involved in the
mobilization of inorganic iron and sulfur for the synthesis of the
nitrogenase iron-sulfur clusters (see (7) for a recent
review). A system that utilized NIFS for the reconstitution of the
FeS
cluster of dinitrogenase reductase was
recently described(29) . It will be interesting to attempt to
generate NifB-co using a similar system.