(Received for publication, October 20, 1994)
From the
The products of the nifN and nifE genes of Azotobacter vinelandii function as a 200-kDa
tetramer (NIFNE) in the synthesis of
the iron-molybdenum cofactor (FeMo-co) of nitrogenase, the enzyme
system required for biological nitrogen fixation. NIFNE was purified
using a modification of the published protocol. Immunoblot analysis of
anoxic native gels indicated that distinct forms of NIFNE accumulate in
strains deficient in either NIFB (
nifB::kan
nifDK) or NIFH (
nifHDK). During the
purification of NIFNE from the
nifHDK mutant, its
mobility in these gels changed, becoming similar to that of NIFNE from
the
nifB::kan
nifDK mutant. While NIFB
activity initially co-purified with the NIFNE activity from the
nifHDK mutant, further purification of NIFNE activity
resulted in the loss of the co-purifying NIFB activity; this loss
correlated with the change in NIFNE mobility on native gels. These
results suggest that the form of NIFNE accumulated in the
nifHDK mutant is associated with NIFB activity in crude
extract but loses this association during NIFNE purification. Addition
of the purified metabolic product of NIFB, termed NifB-co, to either
NIFNE purified from the
nifHDK strain or to the NIFNE in
crude extract of the
nifB::kan
nifDK strain
caused a change in the mobility of NIFNE on anoxic native gels to that
of the form accumulated in a
nifHDK mutant. These results
support a model where both NifB-co and dinitrogenase reductase
participate in FeMo-co synthesis through NIFNE, which serves as a
scaffold for this process.
Biological nitrogen fixation is the process by which atmospheric
nitrogen is reduced to ammonium. This process is carried out by the
nitrogenase enzyme system, which is composed of two separate protein
complexes(1, 2) . Dinitrogenase reductase (component
II, iron protein, NIFH), a 65-kDa dimer of the nifH gene product, serves as a specific electron donor to the
other protein complex, dinitrogenase. Dinitrogenase (component I,
molybdenum-iron protein, NIFKD) is a 240-kDa
tetramer of the nifK and nifD gene products. For catalytic activity, dinitrogenase
requires a unique cofactor, FeMo-co, (
)which contains iron,
molybdenum, acid-labile sulfur, and homocitrate. FeMo-co is thought to
provide the site of substrate
reduction(3, 4, 5) . FeMo-co has been
isolated and can be used to activate dinitrogenase lacking FeMo-co
(apodinitrogenase)(6) . Surprisingly, functional FeMo-co can be
synthesized in the absence of dinitrogenase in vivo and in
vitro(7) , indicating that it is synthesized elsewhere and
inserted into the apo form of dinitrogenase.
To date, the products
of six nif genes have been strongly implicated in the in
vivo synthesis of FeMo-co: nifN, nifE, nifB, nifH, nifQ, and nifV. The
purification and characterization of a number of molecules involved in
FeMo-co synthesis has been made possible by the development of an in vitro FeMo-co synthesis system, which depends upon the
above mentioned gene products as well as MgATP and
MoO(8) . The products of nifN and nifE function as a 200-kDa
tetramer (NIFNE) that has been
purified, partially characterized, and found to contain what appears to
be a single 4Fe-4S center(9) .
The role of NIFB in FeMo-co synthesis is undefined, but recently a small molecule containing iron and sulfur, thought to be a FeMo-co precursor produced by the activity of NIFB, termed NifB-co, has been isolated(10) .
Dinitrogenase reductase (NIFH), in addition to its roles in the maturation of apodinitrogenase (11, 12, 13) and donation of electrons to dinitrogenase(2, 14) , also plays an unknown role in FeMo-co synthesis (15, 16, 17, 18) .
NIFQ apparently plays a role in molybdenum processing, possibly in the formation of molybdenum-sulfur compounds required during FeMo-co synthesis(19, 20) . NIFV is probably a homocitrate synthase(21) , and homocitrate is an organic constituent of FeMo-co(21, 22) . Recently, the absence of other gene products, namely NIFW, NIFZ, and NIFM, has been shown to have some effect on FeMo-co synthesis(23, 24) .
Although the
exact role of NIFNE in FeMo-co synthesis is unknown, it is thought to
serve as a scaffold on which FeMo-co is built for the following
reasons: first, the genes nifN and nifE exhibit
striking similarity to nifK and nifD,
respectively(25, 26) , and the cysteine residue which
serves as the FeMo-co ligand in NIFKD is also conserved in
NIFNE(25) . Second, both proteins function as
tetramers; and functional FeMo-co
can be synthesized in the absence of dinitrogenase, suggesting another
protein might serve as a scaffold(7) .
The purpose of this work is to further our understanding of the process of FeMo-co synthesis by examination of electrophoretic mobility in anoxic native gels of NIFNE accumulated in mutants blocked at different steps in FeMo-co synthesis. The rationale of this approach is that if NIFNE does function as a scaffold for FeMo-co synthesis, different FeMo-co precursors might accumulate on NIFNE in mutants blocked in different steps in FeMo-co biosynthesis.
A. vinelandii strains CA12 and DJ
were grown by inoculating 220 ml of modified Burk's medium (33) + 5 mM NHCl with 1 ml of a cell
stock frozen at -80 °C in 20% glycerol. This culture was
grown overnight at 30 °C in a baffled flask with vigorous shaking.
It was then divided between two 20-liter carboys containing 20 liters
of modified Burk's medium + 2.8 mM NH
Cl. These carboys were placed in a 32 °C water
bath and sparged vigorously with filter-sterilized air until 5.5 h
after exhaustion of ammonium, as determined by Nessler's reagent.
Cells were harvested by concentration using a Pellicon 0.45-µm
tangential flow cell concentrator (Millipore) followed by
centrifugation. Cell pellets were frozen in liquid nitrogen and stored
at -80 °C until use. Cells were broken anoxically by osmotic
shock as described(34) . All crude extracts were made 10% in
glycerol and stored as 5-ml aliquots in anoxic vials at -80
°C.
A. vinelandii strain DJ677 was grown as above
except that the carboys had 30 ml of 15.3% NHCl added to
assure excess ammonium. Three hundred eighty liters of modified
Burk's medium in a 400-liter fermentor was inoculated with 1
liter of 22% ammonium acetate (Fisher) and the 20-liter overnight
culture. The cells were harvested at 5.5 h after exhaustion of ammonium
with a Sharples continuous flow centrifuge.
In the previous report(9) , the activity of NIFNE was given as a specific activity in nmoles of ethylene formed per min per mg of protein in NIFNE-containing fractions. We feel this nomenclature should be revised for the following reasons. First, the formation of ethylene in the assay reflects dinitrogenase activity and is therefore only an indirect measure of NIFNE activity. Secondly, the assay that is used to measure NIFNE activity involves a 30 min incubation prior to the acetylene reduction assay. During this time, it is believed that FeMo-co synthesis has gone to completion (8) and the FeMo-co synthesis activity of NIFNE is exhausted. Because FeMo-co synthesis has gone to completion, NIFNE activity should not be expressed as a function of time. This aspect of NIFNE activity is currently being investigated in detail.
In this study, NIFNE activity is expressed as the nanomoles of ethylene produced per min by dinitrogenase formed per mg protein in the NIFNE-containing fraction. This nomenclature is distinctly different than the previous nomenclature in that it avoids both the implication of NIFNE directly producing ethylene, and the implication of NIFNE activity being measured as a rate. Until we determine the exact nature of NIFNE function and the in vitro FeMo-co synthesis assay and also determine an efficient way in which to separately analyze FeMo-co synthesis and insertion, a direct analysis of NIFNE's specific activity cannot be performed.
NIFB activity was determined by the previously described method(10) .
Figure 1: Purification of NIFNE. Coomassie Blue-stained SDS-polyacrylamide (12%) gel separation of proteins in NIFNE-containing fractions during the purification of NIFNE from DJ677 and purified NIFNE from CA12 and wild type. Lane 1, DJ677 crude extract; lane 2, DJ677 NIFNE-containing fraction from a DE52 cellulose column; lane 3, DJ677 NIFNE-containing fraction from a Reactive Red column; lane 4, DJ677 NIFNE-containing fraction from a TSK phenyl column; lane 5, CA12 NIFNE-containing fraction from a TSK phenyl column; lane 6, wild-type NIFNE-containing fraction from a TSK phenyl column. Molecular mass markers are (in kilodaltons): rabbit muscle phosphorylase B (97.4), bovine serum albumin (66.2), hen egg white ovalbumin (45.0), bovine carbonic anhydrase (31.0), and soybean trypsin inhibitor (21.5).
Because of the structural similarities
between NIFNE and dinitrogenase, the latter protein can be a major
contaminant during the purification of NIFNE. Therefore, strains
deleted for nifK and nifD, the genes encoding
dinitrogenase, simplify the purification. When NIFNE accumulated in
DJ54 (nifH) extracts was directly compared with NIFNE
accumulated in CA12 (
nifHDK) extracts, there was no
detectable difference in anoxic native gel mobility nor activity (data
not shown). Similarly, when NIFNE accumulated in extracts from UW45 (nifB) and DJ677 (
nifB::kan
nifDK)
were compared, no differences were observed. Therefore the absence of
dinitrogenase does not appear to alter these properties of NIFNE in
either of these strains.
Figure 2: Immunoblot analysis of NIFNE. Western blot of a 7-10% anoxic native gel was developed with antibody to NIFNE. Lane 1, wild-type extract; lane 2, DJ677 extract; lane 3, CA12 extract; lanes 4-9, DJ677 extract preincubated with increasing amounts of NifB-co; lane 10, CA12 NIFNE-containing fraction from a DE52 cellulose column; lane 11, CA12 NIFNE-containing fraction from a Reactive Red column; lane 12, CA12 NIFNE-containing fraction from a TSK phenyl column; lanes 13 and 14, CA12 NIFNE-containing fractions from a TSK phenyl column preincubated with increasing amounts of NifB-co; lane 15, DJ677 NIFNE-containing fraction from a TSK phenyl column; lane 16, DJ677 NIFNE-containing fraction from a TSK phenyl column preincubated with NifB-co; lane 17, wild-type NIFNE-containing fraction from a TSK phenyl column; lane 18, wild-type NIFNE-containing fraction from a TSK phenyl column preincubated with NifB-co; lane 19, wild-type extract pre-incubated with NifB-co.
To test the first of these
possibilities, a partially pure source of NifB-co was added to crude
extracts of DJ677 (nifB::kan
nifDK).
Addition of increasing amounts of NifB-co to crude extracts from DJ677
changed the form of NIFNE from the slower, diffuse species (Fig. 2, band A) to the faster migrating, sharper
species (Fig. 2, band B, lanes 4-9).
Identical results were obtained with NifB-co that had been further
purified. Addition of NifB-co or Sarkosyl elute to crude extracts of
wild type (DJ) caused a similar effect (Fig. 2, lane 1
versus 19) suggesting much of the accumulated form of NIFNE in
wild-type can also interact with NifB-co. Incubation of NIFNE with the
detergent that solubilizes NifB-co leads to a loss of NIFNE
cross-reacting material from the described bands (Fig. 2, lane 2 versus 4-9). This effect is exacerbated when low
levels of protein (lane 1 versus 19) or highly purified
protein (lane 12 versus 13-14, 15 versus 16 and 17 versus 18) is assayed (see below). Oxygen treatment of
NifB-co prior to preincubation, which destroys its activity in a
FeMo-co synthesis assay, eliminated the appearance of the faster
migrating, sharp band (band B) (data not shown), suggesting
that the effect is specific to functional NifB-co. A solvent-only
control showed no effect on the NIFNE unless allowed to incubate for an
extended period of time (30 min) after which the NIFNE was no longer
detected by immunoblot analysis of the native gels (data not shown). As
determined by examination of immunoblots of native gels, NIFNE from
CA12 (NIFNE associated with NifB-co) is relatively stable to the same
treatment. No mobility shifts of NIFNE were seen with the addition of
up to 1000-fold molar excess of ferrous ammonium sulfate to DJ677
(
nifB::kan
nifDK) extract, with the
addition of isolated FeMo-co to DJ677 extract, nor with the addition of
dinitrogenase reductase to CA12 (
nifHDK) extract (data
not shown).
To test the second of these predictions, NifB-co was
added to the NIFNE purified from CA12 (nifHDK). If this
species has lost the associated NifB-co, the model predicts that the
original mobility might be regained upon the addition of NifB-co. Fig. 2shows that, when NifB-co is added, the NIFNE purified from
CA12 is converted from the predominantly slowly migrating diffuse band (Fig. 2, lane 12, band A) to the form which
migrates more rapidly as a sharp band (Fig. 2, lanes 13 and 14, band B). Addition of NifB-co to NIFNE
purified from DJ677 (
nifB::kan
nifDK) and
wild type also exhibited the same effect (Fig. 2, lanes 15
versus 16 and 17 versus 18, respectively). These results
indicate that functional NifB-co associates with NIFNE, and that the
forms of NIFNE accumulated in different genetic backgrounds can be
converted from one to another by the addition or loss of this factor.
Thus, it appears that the form of NIFNE accumulated in CA12 is
associated with NifB-co, and that during purification NifB-co is
stripped from NIFNE. The results also suggest that the NIFNE
accumulated in DJ677 (
nifB::kan
nifDK) is
not associated with NifB-co but can associate with NifB-co in
vitro. The association of NIFNE and NifB-co activities is
addressed below.
As a
necessary control that the NIFB activity was associated with NIFNE and
not co-purifying coincidentally, the first step of the NIFNE
purification protocol was followed using an extract of a nifE mutant (DJ35). This strain has comparable NIFB activity
(approximately 50% of CA12), but lacks the NIFNE complex. In this
strain, where the NIFNE complex is absent, only 9% of the NIFB activity
was found to elute in the fraction that would contain NIFNE if present (Table 2). The simplest explanation for this difference in the
elution of NIFB activity is that NifB-co and NIFNE are physically
associated.
When the in vitro FeMo-co
synthesis activities of NIFNE in crude extracts of CA12
(nifHDK) and DJ677 (
nifB::kan
nifDK) are normalized to the amounts of NIFNE
protein accumulated in those extracts, the activities are again
comparable (Table 3, column 2). This suggests that the similarity
in activities is not due to the changes observed during purification.
It is not possible to quantitate the activity of NIFNE in wild-type
crude extracts due to the overwhelming amount of nitrogenase activity
caused by the fully functional nitrogenase system in these extracts.
The general role of NIFNE in FeMo-co synthesis has been thought to be that of a scaffold protein upon which FeMo-co is built (25, 40) both because of sequence identity when compared to dinitrogenase (25) and because dinitrogenase is not required for the synthesis of FeMo-co(7) . This report provides the first biochemical evidence for that function.
Our current working model is that construction of FeMo-co occurs on NIFNE in steps, shown diagrammatically in Fig. 3. It is based on the assumption that when FeMo-co synthesis is blocked by a mutation affecting a critical step in the process, the accumulated NIFNE is expected to harbor the form of immature FeMo-co that is the substrate of the mutationally absent gene product. Because a nifH mutant (lacking dinitrogenase reductase) accumulates a ``NifB-co-charged'' version of NIFNE, it is likely that NIFNE interacts with NifB-co prior to any interaction with dinitrogenase reductase. Because the form of NIFNE accumulated in a NIFB mutant lacks molybdenum(9) , and is dependent upon homocitrate in the current in vitro FeMo-co synthesis assay, it appears that molybdenum and homocitrate are incorporated subsequent to NIFNE's interaction with NifB-co. Although the model suggests that dinitrogen-ase reductase functions directly after NIFNE's association with NifB-co, it is possible that intermediate steps occur in vivo which may not cause detectable changes in anoxic native gel mobility.
Figure 3:
A model for the role of NIFNE in FeMo-co
synthesis. Large circles and squares represent the dinitrogenase
analogous and
subunits of NIFNE, respectively. Small
circles represent different forms of maturing
FeMo-co.
The number of steps needed to build the form of
NIFNE that interacts with NifB-co (NIFNE accumulated in
DJ677(nifB::kan
nifDK)) is unknown, but it
does appear that this form contains a minimum of 4Fe arranged in what
is believed to be a single 4Fe-4S center(9) . Rather than being
a FeMo-co precursor, however, this 4Fe-4S center may be in a region of
NIFNE analogous to dinitrogenase's P-cluster and could
conceivably be involved in electron transfer to a FeMo-co precursor.
Molecular modeling of NIFNE based on the structure of dinitrogenase and
sequence alignment is consistent with NIFNE containing a 4Fe-4S cluster
in each analogous P-site. (
)The number of steps subsequent
to NifB-co's interaction with NIFNE is also unknown. Indeed, it
is not yet clear whether or not FeMo-co synthesis is completed on NIFNE
or on another protein.
Anoxic native gel electrophoresis described here revealed that different NIFNE complexes accumulate in different genetic backgrounds of A. vinelandii. Although the causes of mobility differences in anoxic native gels are unknown, these differences are reminiscent of the differences between the active form of dinitrogenase and that lacking FeMo-co (apodinitrogenase) (13, 41) in which the acquisition of a small molecule containing molybdenum, iron, and sulfur (FeMo-co) induces a large change in mobility on native gels. These results also suggest that, in mutants blocked in FeMo-co synthesis, these mutants predominantly accumulate a single form of NIFNE, which is likely to be the normal substrate of the mutationally altered gene product.
The observation
that the original mobility of NIFNE purified from CA12
(nifHDK) could be regained upon incubation with NifB-co,
the product of NIFB, suggests that (i) NifB-co and NIFNE directly
contact each other before FeMo-co is mature and (ii) the interaction of
NifB-co with NIFNE is prior to the interaction of dinitrogenase
reductase with NIFNE during FeMo-co synthesis. Addition of NifB-co to
crude extracts of DJ677 (
nifB::kan
nifDK)
revealed the same mobility change. The change in mobility of the NIFNE
from CA12 during purification is therefore likely to be due to the loss
of the associated NifB-co from the NIFNE. This association of NifB-co
with NIFNE is not stable enough to survive purification. Disruption of
this complex does not, however, render the NIFNE inactive in the in
vitro assay. Moreover, once stripped of the NifB-co, NIFNE and
NifB-co can again associate, suggesting the association is a transient
one. The in vitro association of NifB-co with NIFNE does not
require the addition of any nucleotides. Efforts toward analyzing the
metal content of isolated NIFNE containing NifB-co are underway and
should allow some characterization of the NifB-co molecule.
The purified NIFNE has an activity of 10,000-18,000 nmol of ethylene produced by dinitrogenase formed per mg of NIFNE added to the assay. Thus, approximately 5-9 mol of apodinitrogenase are activated per mol of NIFNE added, assuming a maximum specific activity of dinitrogenase of 2000 nmol ethylene formed per min per mg of dinitrogenase. This result can be explained only if NIFNE is functioning catalytically in the in vitro assay. This also suggests that our ability to measure a reliable specific activity of NIFNE is dependent upon whatever other molecules are necessary for this turnover and may become limiting in vitro. We expect that further investigation of NIFNE function and a purified FeMo-co synthesis system will help resolve these questions.
Finally, we report that wild type accumulates significantly less NIFNE than do mutants blocked in FeMo-co synthesis. While this feature might be due to the effects of differential transcriptional or post-transcriptional regulation, there is no known mechanism whereby synthesis or translation of nif transcripts is independently regulated. If protein stability is responsible for these differences, the result suggests the surprising possibility that NIFNE accumulated in mutants unable to complete synthesis of FeMo-co is more stable that the NIFNE accumulated in wild type.
The precise function of NIFNE in FeMo-co synthesis both in vitro and in vivo remains unknown and could include one or any combination of the following. (i) NIFNE is simply the scaffold on which FeMo-co is synthesized. (ii) NIFNE associates with a small molecule, possibly containing iron and sulfur, which it then donates to the synthesis process. (iii) NIFNE functions as a catalyst during FeMo-co synthesis. While the data presented here do not address the above possibilities directly, they do support the scaffold model and provide an approach for further analysis.
In conclusion, the results of this study are that (i) distinct forms of NIFNE accumulate in different genetic backgrounds, (ii) NifB-co directly interacts with NIFNE, (iii) dinitrogenase reductase performs a necessary step in FeMo-co synthesis subsequent to the interaction between NifB-co and NIFNE, and (iv) NIFNE is apparently a scaffold on which FeMo-co is synthesized.