(Received for publication, November 13, 1995; and in revised form, January 12, 1996)
From the
The vnf-encoded apodinitrogenase (apodinitrogenase 2)
has been purified from Azotobacter vinelandii strain CA117.30
(nifKDB), and is an
hexamer.
Apodinitrogenase 2 can be activated in vitro by the addition
of the iron-vanadium cofactor (FeV-co) to form holodinitrogenase 2,
which functions in C
H
, H
, and
N
reduction. Under certain conditions, the
hexamer dissociates
to yield the free
subunit (the VNFG protein) and a form of
apodinitrogenase 2 that exhibits no C
H
,
H
, or N
reduction activities in the in
vitro FeV-co activation assay; however, these activities can be
restored upon addition of VNFG to the FeV-co activation assay system.
No other vnf-, nif-, or non-nif-encoded
proteins were able to replace the function of VNFG in the in vitro processing of
apodinitrogenase
2 (in the presence of FeV-co) to a form capable of substrate reduction.
Apodinitrogenase 2 is also activable in vitro by the
iron-molybdenum cofactor to form a hybrid enzyme with unique
properties, most notably the inability to reduce N
and
insensitivity to CO inhibition of C
H
reduction.
The biological conversion of atmospheric nitrogen to ammonia can occur via three distinct nitrogenase enzymes in Azotobacter vinelandii: the conventional molybdenum-containing enzyme (nitrogenase 1), a vanadium-containing enzyme (nitrogenase 2), and a third enzyme (nitrogenase 3) that is believed to contain only iron (for reviews see (1) and (2) ). Nitrogenases 1, 2, and 3 are genetically distinct, being encoded by the nif, vnf, and anf operons, respectively. Although specific structural genes encode each nitrogenase enzyme, the products of certain nif genes are also required for the full functionality of the vnf- and anf-encoded nitrogenases(3, 4) . A. vinelandii harbors all three nitrogenases; the expression of the individual nitrogenase enzymes is dependent on the metal content of the growth medium. Nitrogenase 1 is synthesized in medium containing molybdenum. Expression of nitrogenase 2 requires medium depleted of molybdenum and containing vanadium; nitrogenase 3 is expressed in medium deficient in both molybdenum and vanadium(2) .
All three nitrogenase
enzymes are two-component systems consisting of dinitrogenase and
dinitrogenase reductase. Dinitrogenase 1, an
tetramer of the nifD and nifK gene products, respectively, has been extensively
characterized(5, 6) . Dinitrogenase 2 (encoded by vnfDGK) has been purified from both Azotobacter
chroococcum(7) and A. vinelandii(8) .
Two species of dinitrogenase 3 (encoded by anfDGK) and a
hybrid form of dinitrogenase 3 have also been characterized in A.
vinelandii(9, 10) . The presence of vnfG and anfG (encoding the
subunits of dinitrogenases 2
and 3, respectively) is a genetic feature of the molybdenum-independent
nitrogenases only; however, the presence of the
subunit in
purified dinitrogenases 2 and 3 appears to
vary(7, 8, 9, 10) . The role of the
subunit in the functioning of dinitrogenase 2 was recently
addressed by Waugh et al., who found that an A. vinelandii strain lacking vnfG was unable to grow diazotrophically,
suggesting that the
subunits might be required for N
reduction(11) .
Apodinitrogenase 1 (lacking the
iron-molybdenum cofactor) is an
hexamer in certain
mutant strains of A. vinelandii; the
protein (a
non-nif gene product(12) ) was shown to be involved in
the processing of apodinitrogenase 1 to the holoenzyme(13) . By
analogy to the nitrogenase 1 enzyme system, a possible role of the
subunits of nitrogenases 2 and 3 might be in the processing of
these apodinitrogenase proteins to their respective holoenzyme forms.
The subunit composition of apodinitrogenase 2 is therefore of interest,
as it might provide insight into additional roles of the
subunit
(the VNFG protein) of dinitrogenase 2. A second property that
distinguishes the molybdenum-independent nitrogenases from nitrogenase
1 is their substrate reduction characteristics. All three enzymes
reduce N
to NH
, C
H
to
C
H
, and H
to H
;
however, in addition to reducing C
H
to
C
H
, nitrogenases 2 and 3 also catalyze the
formation of C
H
(as a minor product) from
C
H
(14, 15) .
The substrate
reduction characteristics of the nitrogenases appear to be dictated
both by the cofactor at the active site and their polypeptide
environments. Certain altered forms of the iron-molybdenum cofactor
(FeMo-co) resulted in a loss of N and/or
C
H
reduction activities of nitrogenase 1,
indicating the involvement of the cofactor in substrate specificity (16, 17) . The role of the polypeptide in the
substrate specificity of nitrogenase 1 was demonstrated by the
conversion of glutamine 191 (of the nifD-encoded subunit) to
lysine, which caused a loss of N
reduction and the atypical
reduction of C
H
to both C
H
and C
H
(18) . The requirement for
specific polypeptide-cofactor interactions was also observed with
dinitrogenase 1 containing the iron-vanadium cofactor (FeV-co); (
)the hybrid enzyme was unable to reduce N
, and
C
H
was reduced to both C
H
and C
H
, the latter being a property of
nitrogenase 2(19) .
In this report, we describe the
purification and characterization of apodinitrogenase 2 from A.
vinelandii strain CA117.30 (nifKDB). Purified
apodinitrogenase 2 has an
subunit composition and is activable in vitro by FeV-co.
Under certain conditions, the
subunit (VNFG) dissociates from the
hexamer to yield
free VNFG and a form of apodinitrogenase 2 that is inactive in the in vitro FeV-co-activation assay. Apodinitrogenase 2 activity
can be restored upon addition of VNFG to the FeV-co-activation assay
system. In addition to being activable by FeV-co, apodinitrogenase 2 is
also activable in vitro by FeMo-co. The activation of
apodinitrogenase 2 by FeFe-co (the putative iron-only cofactor of
dinitrogenase 3) was also attempted; the substrate reduction
characteristics of the resulting hybrid enzymes were investigated.
A.
vinelandii strains CA12 (nifHDK), CA11.6
(W-tolerant,
nifHDK), CA11.1
(
nifHDKvnfDGK1::spc), and UW45 (nifB
) have been
described(9, 11, 21, 22) . All
strains were grown in Burk's medium prepared in deionized water;
all vessels used in preparing media and for cell culture were rinsed
thoroughly in 4 N HCl and then in deionized water. Strains
CA117.30, CA12, and CA11.1 were grown on Burk's medium that
lacked sodium molybdate and contained 10 µM sodium
metavanadate. Cultures of strain CA117.30 (15 liters) were grown in
20-liter polycarbonate carboys with vigorous aeration at 30 °C in
Burk's medium containing 40 µg of nitrogen/ml as ammonium
acetate; the cultures were monitored for the depletion of ammonium,
following which they were derepressed for 4 h. The cells were
concentrated using a Pellicon cassette system containing a filtration
membrane (0.45 µM, Millipore Corp., Bedford, MA) and were
centrifuged; the cell pellets were frozen in liquid N
and
stored at -80 °C. Strain CA12 was grown in 15-liter cultures
(described above) on nitrogen-free Burk's medium; the cultures
were harvested (as described above) when cell density reached
1.6-1.8 absorbance units at 600 nM. Strain CA11.6 was
grown on Burk's medium containing only iron (with no added sodium
molybdate or sodium metavanadate) as previously reported(9) .
Strain CA11.1 was grown and derepressed on Burk's medium
containing only iron by the method described for CA11.6. Cell-free
extracts of all A. vinelandii strains were prepared by the
osmotic shock method (23) .
Purified FeMo-co was prepared by the method described by Shah and Brill (23) ; when it was necessary to avoid the denaturing effects of N-methylformamide, FeMo-co was prepared by the citric acid treatment of purified dinitrogenase 1 as described by Allen et al.(24) .
FeFe-co (the iron-only cofactor) was prepared
from dinitrogenase 3 that was partially purified from A. vinelandii strains CA11.6 (W-tolerant, nifHDK) and CA11.1
(
nifHDK
vnfDGK1::spc). The dinitrogenase 3
enzymes from CA11.6 and CA11.1 exhibited specific activities of 13.8
and 1.7 nmol of C
H
formed/min/mg of protein,
respectively. Preparation of a source of FeFe-co was performed by the
method described above for the preparation of FeV-co with one
exception: 0.24 ml of 0.4 M citric acid was added to 2 ml
(approximately 31 mg of protein) of partially pure dinitrogenase 3.
The in vitro activation of apodinitrogenase 2 by FeMo-co or
FeFe-co was carried out as outlined above with some exceptions.
Purified FeMo-co (10 µl, in N-methylformamide) or
acid-denatured dinitrogenase 1 (50 µl) was used as a source of
FeMo-co (in place of FeV-co); acid-denatured dinitrogenase 3 (200
µl, described above) was used as a source of FeFe-co. Where
indicated, 200 µl of CO gas was added to the vials prior to the
addition of CH
.
Conditions under
which apodinitrogenase 2 was detected (by the FeV-co insertion assay)
are summarized in Table 1. Apodinitrogenase 2 was detected in
cell-free extracts of CA117.30 (nifKDB) only under
derepressing (nitrogen-fixing) conditions in media lacking molybdenum
and containing 10 µM NaVO
(compare lines
a-c, Table 1). Varying the length of derepression of strain
CA117.30 showed that apodinitrogenase 2 levels were highest at
approximately 4 h from the start of derepression (data not shown).
Extracts of CA117.30 derepressed on NaVO
exhibited no
cross-reactive material to antibodies prepared against dinitrogenase 1,
indicating that no contaminating apodinitrogenase 1 was present (data
not shown), and confirming a previous report that dinitrogenases 1 and
2 are immunologically distinct(8) . As predicted, extracts of
an A. vinelandii strain deleted in the structural genes for
dinitrogenase 2 (CA11.1(
nifHDKvnfDGK1::spc)) exhibited no
activity in the FeV-co insertion assay (Table 1, line d). In
cell-free extracts, apodinitrogenase 2 was inactivated by oxygen (upon
exposure to air) and was also denatured by heat treatment at 60 °C (Table 1, lines e and f), the latter appears to be characteristic
of the apoprotein, since holodinitrogenase 2 is stable under similar
conditions(8) . (
)
Figure 1:
SDS-PAGE of highly purified
apodinitrogenase 2 (lanes 2 and 3). Lanes 1 and 2 were from a 10.5% gel, and lanes 3 and 4 were from a 15% gel, which allowed visualization of VNFG
(the subunit). Approximately 14 µg of protein was loaded in lanes 2 and 3. Molecular weight standards (lanes
1 and 4) are indicated.
The 14-kDa protein (Fig. 1, lane 3) that
copurified with the and
polypeptides (encoded by vnfD and vnfK, respectively) was identified as VNFG (the
subunit encoded by vnfG) by a comparison of its N-terminal
amino acid sequence (the 2nd to 12th amino acids were identified as
SQSHLDDLFAY) with the predicted amino acid sequence for the protein
product of vnfG(30) . Scanning densitometry of the
highly pure apodinitrogenase 2 fraction (Fig. 1, lanes 2 and 3) showed that the VNFK, VNFD, and VNFG polypeptides
are present in equimolar ratios. The molecular mass of apodinitrogenase
2, estimated by Superose 12 gel filtration chromatography, is
consistent with a protein of approximately 200 kDa. These data indicate
that apodinitrogenase 2 has an
subunit
composition.
In order to determine whether VNFG (the subunit)
formed a complex with the
and/or
subunits, a native/SDS
two-dimensional gel analysis was performed. Highly pure
apodinitrogenase 2 was first subjected to anaerobic native PAGE as
described previously(29) ; a lane from the native gel that
contained apodinitrogenase 2 was cut lengthwise, and the proteins were
resolved (in the second dimension) by SDS-PAGE. The native/SDS
two-dimensional gel (visualized both by Coomassie Blue R-250 and silver
stain) revealed that VNFG (the
subunit) was not in a complex with
the
and
polypeptides (data not shown). These observations,
together with the fact that highly pure apodinitrogenase 2 is an
species, suggests
that some weak interactions (such as ionic and/or hydrogen bonding)
exist between
and the
and/or
subunits and that these
interactions are not stable under the native PAGE conditions. In this
aspect, the physical properties of the apodinitrogenase 2 complex
differ from those of the
complex of apodinitrogenase 1 in which the
subunits were
dissociable from the
and
subunits only upon treatment with
6 M urea (29) . Purified dinitrogenase 2 from A.
vinelandii and A. chroococcum exhibit different subunit
compositions. The A. chroococcum enzyme was purified as an
hexamer(7) , while the A. vinelandii enzyme has
an
subunit composition(8) .
The purification of apodinitrogenase 2 from A. vinelandii provides a framework for understanding the function(s) of VNFG
(the
subunit) and the processing of the apoprotein to the
holoenzyme.
Apodinitrogenase 2 in cell-free extracts of CA117.30
(nifKDB) separated into two distinct fractions upon
DEAE-cellulose chromatography, the first species eluted with 0.2 M NaCl (and was purified to near homogeneity) and the second with
0.3 M NaCl (Table 2). The difference between the two
apodinitrogenase 2 species is most likely not a result of the presence
or absence of VNFG (the
subunit) because both DEAE-cellulose
fractions retain their activity, and loss of VNFG was seen to result in
a loss in activity in the FeV-co insertion assay (discussed below). We
are currently examining chromatography methods that might be used to
purify and characterize the 0.3 M NaCl apodinitrogenase 2
species.
Figure 2:
SDS-PAGE of Sephacryl S-100 fractions
2-8 (lanes 2-8) and VNFG (
subunit)-containing HPLC fraction 5 (lane 9). The
and
subunits in fractions 2 and 3 (lanes 2 and 3)
are indicated by a and, VNFG (
subunit) in fractions
7-9 (lanes 7-9) is indicated by b. The 15% SDS
gel does not allow optimal separation of the
and
subunits.
Molecular weight standards (lane 1) are
indicated.
In order to establish that the stimulating factor
was VNFG, a side fraction (from the final HPLC step in the purification
of apodinitrogenase 2) containing VNFG (Fig. 2, lane 9)
as the only detectable low molecular weight protein, was added to
fractions 2 and 3. Addition of the VNFG-containing fraction (designated
fraction 5 (HPLC) in Table 3) resulted in a 14-20-fold
stimulation of activity of fractions 2 and 3 (Table 3, lines i
and j), indicating that the low molecular weight protein involved in
reconstitution of apodinitrogenase 2 activity (in the presence of
FeV-co) was indeed VNFG (the subunit). The low levels of activity
in fractions 2 and 3 (Table 3, lines a and b) can be attributed
to the presence of some VNFG that was visible upon silver staining a
SDS gel of the Sephacryl S-100 fractions (data not shown).
Apodinitrogenase 2 activity of fractions 2 and 3 was dependent on the
level of the VNFG-containing fraction added; the addition of increasing
levels of fractions 7 and 8 (Fig. 2, lanes 7 and 8) to fractions 2 and 3, respectively, resulted in a linear
increase in apodinitrogenase 2 activity of the latter fractions,
indicating that VNFG was most likely the limiting component in the
assay (Fig. 3). The ability of VNFG to stimulate
apodinitrogenase 2 activity (in the presence of FeV-co) was observed to
be both heat- and O
-labile (Table 3, lines k and l).
The difference in the level of stimulation obtained by the addition of
the Sephacryl S-100 fractions (fractions 7 and 8) and the HPLC fraction
(fraction 5) to fractions 2 and 3 might result from a lower VNFG
concentration in the HPLC fraction (the protein concentration of the
HPLC fraction was approximately half that of the Sephacryl S-100
fractions).
Figure 3:
Titration of Sephacryl S-100 fractions 2
and 3 with VNFG ( subunit)-containing fractions 7 and 8,
respectively. The dotted line indicates the titration of
fraction 2 with fraction 7, and the solid line indicates the
titration of fraction 3 with fraction 8.
The stimulation of apodinitrogenase 2 activity (in the
presence of FeV-co) was further characterized by the addition of
various extracts/fractions to Sephacryl S-100 fractions 2 and 3; data
obtained with only fraction 2 are shown, as similar results were
obtained with fraction 3 (Table 3, lines m-p). That the
addition of CA11.1 (nifHDK
vnfDGK1::spc,
derepressed on NaVO
) extract did not stimulate
apodinitrogenase 2 activity (Table 3, line n) demonstrates that
apodinitrogenase 2 specifically
requires VNFG as the stimulatory component in the in vitro assay system (as CA11.1 contains a deletion in vnfG) and
that other vnf proteins do not function to restore
apodinitrogenase 2 activity in vitro. Extracts of CA11.1
derepressed on W-containing medium (growth conditions allowing the
expression of nif proteins) and
NH
-grown UW (wild-type A.
vinelandii) also failed to stimulate activity (Table 3,
lines n and o), indicating that nif proteins and non-nif proteins (specifically the
protein (a non-nif protein) which is required for the activation of apodinitrogenase
1 with FeMo-co (13) ) do not replace VNFG in vitro.
The addition of partially purified
(73% in purity,
0.04-0.31 mg total protein added) to fraction 2 had no effect on
the activity of the fraction (Table 3, line p), indicating that
although the partially pure
protein (and
present in
extracts of CA11.1 and UW) was competent in binding
FeMo-co(12) , it was unable to function in place of VNFG in
vitro.
Separation of VNFG (the subunit) from the
form of
apodinitrogenase 2 also resulted in a loss of both H
and N
reduction activities of Sephacryl S-100
fractions 2 and 3 (as detected by the FeV-co insertion assay). Both
H
and N
reduction activities were restored
upon addition of VNFG-containing fractions to the FeV-co insertion
assays (Table 4). These data indicate a requirement for VNFG (in
addition to FeV-co) in the in vitro assay system.
In
summary, the results discussed above indicate that VNFG (the
subunit) can be dissociated (to a large extent) from the
form of
apodinitrogenase 2 by gel filtration chromatography. The separation of
VNFG results in an inability to convert the
form of apodinitrogenase 2 (in the
presence of FeV-co) to a form capable of C
H
,
H
, and N
reduction; substrate reduction
activities are restored upon addition of VNFG alone to the in vitro FeV-co insertion assays. The ability of VNFG to stimulate
apodinitrogenase 2 activity was observed to be heat- and
O
-sensitive. Various vnf, nif, and
non-nif proteins (in cell-free extracts) failed to replace the
function of VNFG in vitro. These results indicate a specific
requirement for VNFG for the in vitro activation of
apodinitrogenase 2 by FeV-co, and/or for substrate reduction by
holodinitrogenase 2.
The dissociation of VNFG under the
chromatography conditions of the Sephacryl S-100 column could be a
result of the prolonged length of time that the apoprotein is exposed
to the matrix in the absence of glycerol. It is interesting that in the
purification of dinitrogenase 2 from A. vinelandii, Hales et al. (8) observed an irreversible loss in
dinitrogenase 2 activity when Sephacryl S-200 chromatography was
performed using a long column; whether this was due to the slow
dissociation of VNFG under the experimental conditions remains unknown.
It should be noted that in the final HPLC step in the purification of
apodinitrogenase 2, fractions immediately preceding the highly pure
apodinitrogenase 2-containing fractions contained significant levels of
VNFG but did not exhibit activity when monitored using the FeV-co
insertion assay (data not shown). This suggests that VNFG might be
dissociating from a population of the
species in the
presence of 1.0 M (NH
)
SO
(see ``Experimental Procedures'') or that a population
of free VNFG might be copurifying with the
species up to the
last HPLC step, during which they separate. Addition of a
VNFG-containing side fraction (from the final HPLC step) to the highly
pure
-containing
fraction did not result in a stimulation in activity of the latter
fraction, implying that VNFG was not limiting the activity of the
highly pure
fraction. We have recently demonstrated that VNFG cannot be
dissociated from holodinitrogenase 2 under the Sephacryl S-100
chromatography conditions used to separate VNFG from apodinitrogenase
2, indicating that apodinitrogenase 2 is distinct from
holodinitrogenase 2 with respect to the ability of VNFG to dissociate
from the former.
There are at least two possible explanations for
the requirement of VNFG for in vitro apodinitrogenase 2
activity. Analogous to the role of the protein in the activation
of apodinitrogenase 1, VNFG might be required for the activation
process whereby apodinitrogenase 2 is converted to holodinitrogenase 2
in the presence of FeV-co. Alternatively, VNFG might play a role in
substrate reduction by the newly formed holodinitrogenase 2. Recently,
Waugh et al. (11) noted that a vnfG
strain was unable to grow
diazotrophically but that cell-free extracts of the strain exhibited
some C
H
reduction activity; our results
indicate a loss in C
H
, H
, and
N
reduction activity in fractions lacking VNFG, implying
that VNFG might be involved in the activation of apodinitrogenase 2 by
FeV-co in vitro. A direct comparison, however, cannot be made
between our results and those of Waugh et al., as our studies
were performed on an in vitro system. It is interesting that
while
nifK and/or nifD strains synthesize and
accumulate FeMo-co in vivo on a 65-kDa
protein(31, 32) , strain CA11.1
(
nifHDK
vnfDGK1::spc) did not accumulate
FeV-co in vivo when derepressed on NaVO
(data not
shown). It is possible that the in vivo accumulation of FeV-co
requires the presence of VNFG; however, the possibility that FeV-co
synthesis does not occur in the absence of the structural polypeptides
for dinitrogenase 2 cannot be excluded. Future studies are aimed at
examining the interactions of VNFG (the
protein) with FeV-co and
the
form of apodinitrogenase 2 in
order to investigate the role of VNFG in the processing of
apodinitrogenase 2 to the holoenzyme.
The activation of apodinitrogenase 2 by FeMo-co
resulted in a hybrid enzyme with properties distinct from both
dinitrogenase 1 and dinitrogenase 2. FeMo-co-activated apodinitrogenase
2 was unable to reduce N, and C
H
reduction by the hybrid enzyme was resistant to inhibition by CO
under conditions where C
H
reduction by
FeMo-co-activated apodinitrogenase 1 was inhibited by 98%. The
C
H
and H
reduction activities
of FeMo-co-activated apodinitrogenase 2 were approximately 6- and
1.5-fold lower, respectively, than that of the FeV-co-activated
protein. The lower activities of FeMo-co-activated apodinitrogenase 2
might result from the inefficient activation of the apoprotein by
FeMo-co. The low activities are probably not due to FeMo-co being a
limiting component of the assay, as apodinitrogenase 1 (in cell-free
extracts of UW45) was activated to 20.3 nmol C
H
formed per min per assay by the level of FeMo-co added. Our
results are in disagreement with those of Moore et al. (33) who reported that FeMo-co-activated apodinitrogenase 2
reduced N
as effectively as dinitrogenase 1 and that the
hybrid enzyme retained CO sensitivity of C
H
reduction. C
H
reduction by
FeMo-co-activated apodinitrogenase 2 was characterized by the formation
of C
H
as a minor product; the
C
H
:C
H
ratio of the
hybrid enzyme was increased (0.21) compared with the FeV-co-activated
protein (0.08). Pau et al. (10) noted
C
H
reduction to both C
H
and C
H
in a hybrid form of dinitrogenase
3 that contained FeMo-co at its active site; Gollan et al. (34) independently observed an increase in the
C
H
:C
H
ratio of
dinitrogenase 3 from Rhodobacter capsulatus upon the in
vivo incorporation of FeMo-co into the anf1 polypeptides.
In both cases it was unclear whether the formation of
C
H
arose from FeMo-co-containing dinitrogenase
3 or from a mixed population of dinitrogenase 3 containing both FeMo-co
and FeFe-co.
The role of the cofactor in specifying substrate
reduction properties and inhibitor susceptibilities was observed in
dinitrogenase 1 when homocitrate (an integral component of
FeMo-co(16) ) was substituted with its analogs. Incorporation
of threo-fluorohomocitrate resulted in a complete loss of
N reduction while C
H
and
H
reduction activities were retained(17) ,
substitution of homoisocitrate and 2-oxoglutarate resulted in a loss of
both C
H
and N
reduction(16) . Altering the structure of FeMo-co by the
incorporation of homocitrate analogs did not result in CO-resistance of
C
H
reduction(17) , indicating that the
CO-resistance of C
H
reduction exhibited by
FeMo-co-activated apodinitrogenase 2 is a result of altered
interactions between residues at the cofactor binding site of
apodinitrogenase 2 and FeMo-co. That the polypeptide also dictates
substrate specificity of nitrogenase 1 was demonstrated by the
substitution of glutamine 191 in the
polypeptide of dinitrogenase
1 by lysine(18) ; the mutant strain was unable to grow
diazotrophically, and cell-free extracts reduced C
H
to both C
H
and
C
H
, the latter being a property of the vnf- and anf-encoded nitrogenases ((14) ).
More recently, Kim et al. (35) have shown that substitution of
histidine 195 (of
dinitrogenase 1) with glutamine results in an altered enzyme that binds
but does not reduce N
and that exhibits hypersensitivity to
CO inhibition of C
H
reduction.
The altered
substrate reduction properties conferred by FeMo-co activation of
apodinitrogenase 2 are possibly due to the inability of FeMo-co to
properly contact the protein ligands necessary for optimal reduction of
all three substrates. An altered orientation of the cofactor in the
binding site could explain the lowered CH
and
H
reduction and also the CO-resistance of
C
H
reduction, which might arise from a
decreased accessibility of CO to its binding site. Although the two
amino acid residues implicated in covalently binding FeMo-co (cysteine
275 and histidine 442) are conserved in all three dinitrogenase
proteins(36, 30) , specific amino acid-cofactor
interactions unique to each dinitrogenase protein are probably required
for optimal substrate reduction, the ligations necessary for N
reduction being the most stringent.
Substrate reduction by
apodinitrogenase 2 activated with the putative iron-only cofactor
(FeFe-co) was dependent on the source of dinitrogenase 3 used in the
preparation of the cofactor. Partially pure dinitrogenase 3 (previously
characterized(9) ) from A. vinelandii strain CA11.6
(W-tolerant, nifHDK) was initially used in the
preparation of FeFe-co. Acid-denatured dinitrogenase 3 (from CA11.6)
preparations activated apodinitrogenase 2, forming an enzyme that
exhibited C
H
, H
, and N
reduction activities, albeit 4-5-fold lower than
FeV-co-activated apodinitrogenase 2 (data not shown). In order to
confirm these results, we used partially purified dinitrogenase 3 from A. vinelandii strain CA11.1
(
nifHDK
vnfDGK1::spc) as a source of
cofactor. It should be noted that although strains CA11.6 and CA11.1
were derepressed under identical conditions, the specific activity of
dinitrogenase 3 from CA11.1 was repeatedly observed to be
8-10-fold lower than dinitrogenase 3 from CA11.6 (see
``Experimental Procedures''). No C
H
,
H
, or N
reduction was detected upon
addition of acid-denatured dinitrogenase 3 (from CA11.1) to
apodinitrogenase 2. It is possible that the cofactor from CA11.1
dinitrogenase 3 activated apodinitrogenase 2, but the hybrid enzyme was
unable to function in C
H
, H
,
and N
reduction; however, the possibility that the cofactor
failed to activate apodinitrogenase 2 also exists. The activities
observed by activation of apodinitrogenase 2 by the cofactor from
CA11.6 dinitrogenase 3 might be attributed to low levels of a
molybdenum- or vanadium-containing cofactor. Establishing the substrate
reduction pattern of FeFe-co-activated apodinitrogenase 2 awaits a more
thorough characterization of the cofactors of dinitrogenase 3 from A. vinelandii strains CA11.6 and CA11.1.
In this study, we have shown that highly purified
apodinitrogenase 2 from A. vinelandii is an
protein and that
the
subunit (VNFG) is specifically required for the processing of
apodinitrogenase 2 (in the presence of FeV-co) to a form capable of
C
H
, H
, and N
reduction. The function(s) of VNFG might be elucidated by further
characterization of apodinitrogenase 2 and its processing to the
holoenzyme. We have also demonstrated that apodinitrogenase 2 is
activable in vitro by FeMo-co to form a hybrid enzyme with
unique substrate reduction properties, most notably a loss of N
reduction and insensitivity of C
H
reduction to inhibition by CO. The formation of hybrid
dinitrogenase enzymes (both in vivo and in vitro)
indicates a lack of specificity with respect to the activation of the
apodinitrogenase proteins by a particular cofactor. Specificity does
occur, however, at the level of cofactor biosynthesis(25) . A
study of the ability of certain nif and vnf gene
products involved in cofactor biosynthesis to function in the in
vitro synthesis of FeMo-co and FeV-co might provide insight into
the component(s) specifying the heterometal that is incorporated into
the dinitrogenase proteins.