Nitrogenase is a two-component metalloprotein that catalyzes the
conversion of dinitrogen to ammonium(1) . Dinitrogenase
reductase is an 
dimer that contains a single
Fe
S
center. During substrate reduction,
dinitrogenase reductase (also called NIFH, Fe protein, or component II)
catalyzes the Mg
-ATP-dependent transfer of electrons
to dinitrogenase(2) . Dinitrogenase (also called MoFe protein
or component I) is an 


tetramer that
contains two pairs of unique metal clusters, known as the
iron-molybdenum cofactor (FeMo-co), (
)and the
P-cluster(3, 4, 5) . The electrons
transferred to dinitrogenase are ultimately channeled to FeMo-co, the
site of substrate reduction (5, 6) .
An in
vitro system for the synthesis of FeMo-co has been
described(7) . The system includes ATP and an ATP-regenerating
system, molybdate, homocitrate, NifB-co (an iron-sulfur cluster that is
the apparent metabolic product of the nifB gene product),
NIFNE (an 


tetramer of the nifN and nifE gene products), and dinitrogenase reductase (8, 9, 10, 11) . In addition, a
source of reductant (sodium dithionite) is routinely added to the in vitro FeMo-co synthesis assay.
Dithionite has served two
roles in the in vitro investigation of nitrogenase. Dithionite
was shown by Bulen et al.(12) to replace
ferredoxin/flavodoxin as an electron donor to the enzyme. In addition,
dithionite is an effective oxygen scavenger, thus protecting the
oxygen-labile nitrogenase components. Because NIFNE, NifB-co,
dinitrogenase reductase, and FeMo-co are all known to be extremely
oxygen labile, dithionite has been included in the in vitro FeMo-co synthesis system. Here we test the requirement for
dithionite to determine if it plays an active role in FeMo-co
synthesis.
EXPERIMENTAL PROCEDURES
Materials
AG1-X8 and Sephadex G-25 were
Pharmacia Biotech Inc. products. Sodium dithionite
(Na
S
O
, DTH) was purchased from
Fluka. Na
MoO
was obtained from
Nordion (Ontario, Canada). Indigo carmine and Ti(III) chloride were
from Sigma. (NH
)
MoS
was a gift from
D. Coucouvanis.
Azotobacter vinelandii Strain and Growth
Conditions
A. vinelandii strain UW45 (with a mutation
within nifB gene(13) ) was grown and derepressed, and
extracts were prepared as described(14) . The nifB gene product is required for the synthesis of cofactors for all
three N
fixation systems(13) . Where indicated, DTH
was excluded from the 4 M glycerol and the 25 mM Tris
(pH 7.4) used in the osmotic shock cell lysis procedure. Strain UW45
was grown and derepressed in molybdenum-free medium containing 1 mM Na
WO
(7) for preparation of
extracts used for
Mo experiments.
In Vitro FeMo-co Synthesis Assay
9-ml serum vials
were flushed with purified argon and rinsed with anoxic 25 mM Tris-HCl (pH 7.4) that contained 1.7 mM DTH. This buffer
will hereafter be referred to as Tris-HCl/DTH. The anoxic buffer was
removed, and the vials were then rinsed with DTH-free 25 mM Tris-HCl that had been sparged with nitrogen and degassed on a
gassing manifold to remove any traces of DTH from the vials. A complete
FeMo-co synthesis reaction mixture was prepared by combining the
following: 100 µl of 25 mM Tris-HCl/DTH or Tris-HCl (pH
7.4), 10 µl of 1 mM Na
MoO
, 20
µl of 5 mM homocitrate that had been treated with base to
cleave the lactone, pH 8.0, and 200 µl of an ATP-regenerating
mixture (containing 3.6 mM ATP, 6.3 mM MgCl
, 51 mM phosphocreatine, 20 units/ml
creatine phosphokinase, and 6.3 mM DTH). Assays that contained
Tris-HCl contained an ATP-regenerating system from which the DTH was
excluded. 200 µl of the appropriate A. vinelandii cell-free extract and 5 µl of purified NifB-co (9) were added to the reaction mixtures. 5 µl of the
purified NifB-co contained approximately 8.5 nmol of DTH, which is not
sufficient to support significant FeMo-co synthesis in assays that
contained indigo carmine-oxidized cell-free extract (described below,
see Table 3). The total volume of the reaction mixture was 535
µl. The vials were incubated in a rotary water bath shaker at 30
°C for 30 min. This phase of the assay is referred to as the
preincubation phase. During this phase, FeMo-co is synthesized and
inserted into apodinitrogenase (apodinitrogenase refers to the
dinitrogenase protein that lacks FeMo-co; this form of the protein
contains the P-clusters). After the 30 min of incubation, the indicated
amount of (NH
)
MoS
was added to the
assays to prevent further insertion of FeMo-co into
apodinitrogenase(15) . The
(NH
)
MoS
stock solution was prepared
in N-methylformamide that had been degassed on a gassing
manifold. The vials were incubated at room temperature for 5 min, and
the activity of the newly formed dinitrogenase was then measured using
the acetylene reduction assay as described previously (7) .
To monitor the incorporation of
Mo into FeMo-co, in
vitro FeMo-co synthesis reactions were carried out as described
above with the following exceptions. The concentration of
nonradioactive molybdate was lowered 100-fold (10 µl of 10
µM Na
MoO
were added to each
assay), and Na
MoO
(0.5
µCi/assay, carrier free) was added to each reaction. In addition,
extracts of strain UW45 that was grown and derepressed on
tungsten-containing medium were used in these assays. At the end of the
preincubation phase of the assay, 100 µl of glycerol (that had been
degassed on a gassing manifold) were added to the reactions, and 100
µl of this mixture were then applied to anoxic, native
polyacrylamide gels as described previously(16) . Species with
associated
Mo were visualized using a PhosphorImager as
described previously with the exception that the dried gels were
exposed to the phosphor screen for 1-2 h(16) .
Treatment of Extract with Indigo Carmine
5 g of
strain UW45 (nifB) cells were broken by osmotic shock as
described previously (14) with the exception that DTH was
excluded from all solutions. 15-20 ml of the extract were
chemically oxidized by incubation with 3 mM oxidized indigo
carmine (E
= -125 mV) for 15 min. The indigo
carmine was removed from the extract using a column consisting of a 1
10-cm AG1-X8 layer on top of a 1
7-cm Sephadex G-25
layer. The column was equilibrated with 50 mM Tris (pH 7.4)
and was operated in a Vacuum Atmospheres Company glove box containing
less than 3 ppm O
.
Preparation of Reductant-free FeMo-co
A source of
reductant-free FeMo-co was needed to investigate the requirement of
reductant for in vitro activation of apodinitrogenase by
FeMo-co. Acid-treated dinitrogenase, which is a source of FeMo-co, can
be readily prepared in a form that is free of reductant. All operations
were carried out under an argon atmosphere at 0-4 °C in the
absence of dithionite. 15 mg of crystallized dinitrogenase from A.
vinelandii were added to 2 ml of anoxic distilled H
O
containing 3-4 glass beads (3-mm diameter) in a screw-capped
centrifuge tube modified (9) so that the contents could be
degassed on a gassing manifold. The contents were acidified with 125
µl of 0.4 M citric acid. After 2-3 min on ice, 250
µl of 0.4 M Na
HPO
were added, and
the contents were further incubated for 15 min before centrifugation at
500
g for 2 min. The supernatant was discarded, and
the pellet was resuspended in 2 ml of 25 mM Tris-HCl (pH 7.4)
that had been sparged with N
and degassed on a gassing
manifold. This suspension, which is free of added reductant, was used
as a source of FeMo-co.
Activation of Apodinitrogenase by FeMo-co (FeMo-co
Insertion Assay)
The FeMo-co insertion assays were performed as
described previously (5) except that the extract of A.
vinelandii strain UW45 was free of DTH and had been oxidized with
indigo carmine. In addition, reductant-free FeMo-co (described above)
was added to the reactions as the source of FeMo-co.
Preparation of DTH, Ti(III) Citrate, NADH, and
Dithiothreitol
Ti(III) citrate was prepared as described
previously(17) . When chelated by citrate, Ti(III) is a
relatively strong reductant with a midpoint potential (E
)
of -500 mV(18) . DTH was prepared in 0.1 M Tris-HCl (pH 8.0) that was sparged with N
and degassed
on a gassing manifold. DTH has a midpoint potential (E
) of
-470 mV or less(19) . NADH and dithiothreitol were
prepared in 0.025 M Tris-HCl (pH 7.4) that had been sparged
with N
and degassed on a gassing manifold. NADH and
dithiothreitol have midpoint potentials (E
) of -320
and -330 mV, respectively(20) .
RESULTS AND DISCUSSION
Requirement of Reductant for in Vitro FeMo-co
Synthesis
The initial approach employed to address the
requirement of reductant for FeMo-co biosynthesis involved preparing
two cell-free extracts of A. vinelandii strain UW45 (nifB) in parallel, one that contained 1.7 mM DTH and
the other that lacked DTH. When added to the FeMo-co synthesis assays
from which DTH was excluded during the preincubation phase (during
which FeMo-co is synthesized and inserted into apodinitrogenase), both
extracts exhibited similar FeMo-co synthesis activities (Table 1,
compare lines b and d). These results suggest that either reductant is
not required for in vitro FeMo-co synthesis or that there is a
form of physiological reductant present in the A. vinelandii cell-free extract that functions in the in vitro reaction. To address the possibility that a physiological
reductant in the A. vinelandii extract might be functioning in vitro, extract of strain UW45 (prepared in the absence of
DTH) was chemically oxidized using indigo carmine.
The results
presented in Table 1(lines e and f) demonstrate that when
chemically oxidized extract of strain UW45 is used in the in vitro reaction, the formation of holodinitrogenase is dependent on the
addition of reductant (DTH) to the assay. These results suggest that
reductant is required for in vitro FeMo-co synthesis. However,
the in vitro FeMo-co synthesis reaction monitors the activity
of the newly formed holodinitrogenase and thus actually is a measure of
both the amount of FeMo-co synthesized and the amount of FeMo-co
inserted into apodinitrogenase. Therefore, another possible explanation
of the results presented in Table 1(lines e and f) is that
FeMo-co is synthesized in the absence of reductant but is unable to be
inserted into the oxidized apodinitrogenase present in the A.
vinelandii extracts that had been oxidized with indigo carmine.
Requirement of Reductant for in Vitro FeMo-co Synthesis
versus FeMo-co Insertion into Apodinitrogenase
Two independent
experiments were performed to determine if FeMo-co might be synthesized
in the absence of reductant though not inserted into the form(s) of
apodinitrogenase present in the chemically oxidized cell-free extract.
First, the ability of preformed FeMo-co (reductant-free) to activate
the apodinitrogenase in the oxidized extract was investigated. The
reductant-free FeMo-co was added to the in vitro FeMo-co
synthesis reactions following the preincubation phase of the assay to
demonstrate the presence of apodinitrogenase that could be activated in
the absence of reductant. Following the addition of FeMo-co,
(NH
)
MoS
was added to prevent
subsequent insertion of FeMo-co into apodinitrogenase prior to the
addition of DTH and ATP-regenerating mixture (Table 2, line b).
FeMo-co DTH and ATP-regenerating mixture (prepared as described under
``Experimental Procedures'') were added to the reaction
presented in Table 2(line a) following the preincubation phase
of the assay. Comparison of the activities of these assays shows that a
significant amount of apodinitrogenase (>25%) present in the
oxidized extract could be activated in the absence of reductant. Note
that when (NH
)
MoS
, which inhibits
the insertion of FeMo-co into apodinitrogenase(15) , was added
prior to addition of the reductant-free FeMo-co, an insignificant
amount of FeMo-co was inserted into apodinitrogenase (and thus very
little holodinitrogenase activity was detected; Table 2, line c).
This result confirms that the activity observed in the assay reported
in Table 2(line b) is due to a population of apodinitrogenase
that is activable by FeMo-co in the absence of reductant (and is not
due to FeMo-co activation of apodinitrogenase during the acetylene
reduction phase of the assay). Comparison of the activities of the
assays reported in Table 2(line a), to which DTH was added as a
component of the ATP-regenerating mixture after the initial 30-min
preincubation phase, and Table 2(line b), to which
(NH
)
MoS
was added to prevent
subsequent insertion of FeMo-co into apodinitrogenase prior to the
addition of DTH to the assay, suggests that in the presence of
reductant, apodinitrogenase is more amenable to activation by FeMo-co.
The order and time of component addition to these assays clearly
reveals that this difference in activity is not due to any instability
of the apodinitrogenase or the FeMo-co in the absence of reductant. One
explanation for this observation is that there is a mixed population of
apodinitrogenase in the oxidized extract (most likely apodinitrogenases
at various oxidation states) and that addition of reductant during the
activation assay significantly increases the percentage of the
apodinitrogenase population that is activable by FeMo-co. The DTH
presumably reduces the P-clusters of apodinitrogenase and the reduced
form(s) of apodinitrogenase appears to be more amenable to FeMo-co
activation. A thorough analysis of the redox properties of the
P-clusters of A. vinelandii dinitrogenase previously revealed
that mixing dinitrogenase with chemical oxidants (as was done in this
study) does not result in the P-clusters being in a single well defined
redox state(21) . The results presented here, however,
demonstrate that the indigo carmine-oxidized extract contains a
sufficient amount of apodinitrogenase that is activable in the absence
of reductant and thus suggest that the lack of dinitrogenase activity
when reductant is excluded from the preincubation phase of the in
vitro FeMo-co synthesis reaction (that utilizes oxidized extract)
is due to the lack of FeMo-co synthesis (Table 1) as opposed to
the lack of insertion of FeMo-co into apodinitrogenase.
The second
experiment that investigated whether FeMo-co is synthesized in the
absence of reductant but is not inserted into the apodinitrogenase
present in the oxidized extract employed in vitro FeMo-co
synthesis reactions that contained
Na
MoO
. Following the preincubation
phase of the in vitro FeMo-co synthesis reaction, the proteins
were separated by electrophoresis on anoxic, native gels, and
Mo-labeled FeMo-co associated with proteins in the
reaction mixture was detected. Previous studies have demonstrated that
in the absence of apodinitrogenase, FeMo-co synthesized in vitro is associated with the gamma protein (16) . The gamma
protein becomes associated with the 


form of apodinitrogenase to generate the



form, which can be activated by
FeMo-co(22, 23) . If FeMo-co insertion into
apodinitrogenase but not FeMo-co synthesis requires that reductant be
added to the oxidized extract,
Mo-labeled FeMo-co might be
expected to be associated with gamma in a reaction system that uses
oxidized extract and lacks DTH. The results presented in Fig. 1clearly show that when the oxidized extract is used in the in vitro FeMo-co synthesis assay, no
Mo-labeled
FeMo-co is associated with gamma (or any other protein other than the
molybdenum storage protein, a non-nif protein that has been
observed to accumulate
Mo both in vivo and in
vitro ( (24) and (25) ; Fig. 1, lane
2)). When DTH is added to an identical assay, however,
Mo-labeled FeMo-co is observed to be associated with both
dinitrogenase and gamma (in addition to the molybdenum storage protein (Fig. 1, lane 1)). The lack of
[
Mo]FeMo-co associated with gamma protein in the
assay employing the oxidized extract provides further evidence that
reductant is required for in vitro FeMo-co biosynthesis.
Figure 1:
Phosphor image of a native, anoxic gel
of various in vitro FeMo-co synthesis reactions that included
Na
MoO
. Arrows indicate
the position of dinitrogenase and the position of gamma protein as
determined by immunoblot analysis. The position of the molybdenum
storage protein is also indicated. Synthesis reactions used indigo
carmine-oxidized extract of tungsten-grown A. vinelandii strain UW45 (except lane 3). Lane 1, DTH
included during preincubation phase of the in vitro FeMo-co
synthesis assay; lane 2, DTH excluded during preincubation
phase of the in vitro FeMo-co synthesis assay; lane
3, UW45 extract prepared in the absence of DTH (but not oxidized)
used in an in vitro FeMo-co synthesis reaction from which DTH
was excluded during the preincubation
phase.
In
agreement with the results presented in Table 1,
Mo-labeled FeMo-co is associated with dinitrogenase in the
assay that lacked DTH during the preincubation phase and contained
extract of strain UW45 prepared in the absence of DTH (but not
oxidized; Fig. 1, lane 3). These data provide further
evidence that the A. vinelandii extract contains a
physiological reductant(s) that functions in the in vitro synthesis of FeMo-co. From the results presented here, however, it
is not clear if a stoichiometric amount of reductant is required for
each molecule of FeMo-co synthesized or if the reductant is merely
required to reduce the proteins involved in FeMo-co biosynthesis (e.g. NIFNE and dinitrogenase reductase) to the redox state in
which they are active in the biosynthesis of FeMo-co.
Stability of the Components Involved in the in Vitro
Synthesis of FeMo-co
To more definitively demonstrate that the
lack of FeMo-co synthesis observed in the absence of DTH (when using
the oxidized extract) is due to the requirement of reductant for
FeMo-co synthesis and is not the result of the instability of the
components required for FeMo-co biosynthesis in the absence of DTH, the
following assays were performed. The components required for FeMo-co
synthesis (molybdate, homocitrate, MgATP, oxidized extract of strain
UW45, and NifB-co) were incubated in the absence of DTH for the
standard 30-min incubation time. At this time, DTH was added to the
assays and the reactions were incubated for an additional 30 min to
allow FeMo-co synthesis and insertion into apodinitrogenase to occur.
Comparison of the activities of the assays presented in Table 2,
line d, to which DTH was added after the 30 min preincubation period,
and line e, which contained DTH from the start of the preincubation
phase, shows that almost identical in vitro FeMo-co synthesis
activities were detected whether DTH was added after the preincubation
phase or was present from the start of the reaction. These results
demonstrate that the components required for FeMo-co synthesis are
stable during the incubation in the absence of DTH. Independent assays
to monitor the NIFNE and dinitrogenase reductase activities of the
oxidized UW45 extract following preincubation in the absence of
reductant confirmed that both of these components are stable (data not
shown). The stability of the components required for FeMo-co synthesis
in the absence of DTH further suggests that the lack of dinitrogenase
activity observed when DTH is excluded from the preincubation phase of
the assay (that contains oxidized extract) is due to the lack of
FeMo-co synthesis in the absence of reductant.
Amount of DTH Required to Support in Vitro FeMo-co
Synthesis
The amount of DTH required to support in vitro FeMo-co synthesis was investigated. The typical in vitro FeMo-co synthesis assay contains 1,300 nmol of DTH during the
preincubation phase. The indigo carmine-oxidized extract was used to
test the requirement for DTH and the results presented in Table 3demonstrate that 700 nmol of DTH supported maximum in
vitro FeMo-co synthesis activity. However, when the amount of DTH
was reduced to 500 nmol, a substantial reduction in activity was
observed. It is likely that a significant amount of the DTH added to
the assay was consumed by non-nif components present in the
oxidized extract. It will be interesting to further investigate the
requirement of reductant when a purified in vitro FeMo-co
biosynthesis system is available.
Other Reductants in in Vitro FeMo-co
Synthesis
Ti(III) citrate has been reported to serve as a source
of reductant for a number of enzyme-catalyzed reduction reactions,
including nitrogenase-catalyzed reduction
reactions(17, 18) , and was therefore tested in the in vitro FeMo-co synthesis system. Ti(III) citrate supported
the synthesis of FeMo-co. Approximately 80 nmol Ti(III) citrate added
to the preincubation phase of the assay supported an activity of
approximately 5 nmol C
H
formed per min/assay.
The addition of increasing amounts of Ti(III) citrate to the assay
resulted in an inhibition of activity. This inhibition might be due to
the increasing amounts of citrate added as the amount of reductant is
increased. Citrate can be utilized by the FeMo-co biosynthetic
machinery to produce an aberrant form of FeMo-co that is less efficient
in the reduction of C
H
to
C
H
(25) .The abilities of NADH and
dithiothreitol to fulfill the requirement for reductant in the in
vitro synthesis of FeMo-co were also investigated. NADH supported
the synthesis of FeMo-co in assays that included indigo
carmine-oxidized cell-free extracts. Assays that contained 50 nmol of
NADH and 500 nmol of DTH exhibited almost identical activities. NADH
has been used as a physiological electron donor for acetylene reduction
with crude extracts(26) . Dithiothreitol was ineffective in the
FeMo-co synthesis system. Ineffectiveness of dithiothreitol to recover
activity of the oxidized extracts suggests that the loss of activity
upon oxidation is not the result of oxidation of a free sulfhydryl
group on apodinitrogenase (27) but rather the loss of a source
of reducing equivalents.
Possible Roles for Reductant in the in Vitro Synthesis of
FeMo-co
The results presented here suggest that reductant is
required for in vitro FeMo-co biosynthesis when indigo
carmine-oxidized extract is the source of the proteins required for
FeMo-co synthesis. The role of reductant in the in vitro synthesis of FeMo-co is currently unclear. The reductant might
simply reduce a protein(s) required in the reaction to the state in
which it is active in the FeMo-co synthesis assay. The initial
reduction of such a protein might be the only requirement for reductant
in the in vitro synthesis of FeMo-co. Alternatively, there
might be a stoichiometric requirement for reductant for each molecule
of FeMo-co synthesized. One possible function of the reductant is to
reduce the molybdenum added to the assay to the form of molybdenum that
is incorporated into the FeMo-co. The molybdenum added to the assay (as
Na
MoO
) is in the +6 oxidation state.
Spectroscopic analysis of the molybdenum atom in FeMo-co suggests that
the molybdenum atom is in the more reduced +4 oxidation
state(28) . Assays that contain an extract of molybdenum-grown A. vinelandii require reductant for in vitro FeMo-co
synthesis, although they are not dependent on added
Na
MoO
(data not shown). From these observations
we deduce that the reductant is not solely required in an initial step
of processing or reducing the molybdenum atom to a form that can be
used in FeMo-co synthesis because this processing step should have
occurred in vivo in molybdenum-grown A. vinelandii cells. The ability of A. vinelandii to sequester
significant amounts of molybdenum is attributed to the molybdenum
storage protein(24) . Although the oxidation state of
molybdenum that is associated with the molybdenum-storage protein is
unknown, the results presented in Fig. 1suggest that
incorporation of molybdenum (added as Na
MoO
)
into the molybdenum storage protein does not require reductant. It is
possible, however, that the reductant is required to reduce molybdenum
associated with the molybdenum-storage protein prior to its
incorporation into FeMo-co. Perhaps the reductant is required to reduce
some other component(s) involved in FeMo-co synthesis or a precursor(s)
of FeMo-co.Neither flavodoxin nor ferredoxin I is required for in vitro FeMo-co synthesis in DTH-free extracts. A.
vinelandii flavodoxin and ferredoxin I have each been implicated
as members of the electron transport chain to
nitrogenase(2, 29) . If either flavodoxin or
ferredoxin I is the physiological reductant functioning in the in
vitro synthesis of FeMo-co (in reductant-free extract that is not
oxidized), it was hypothesized that a requirement for reductant might
be observed in reactions that contained extract of flavodoxin and/or
ferredoxin mutant strains. The addition of reductant was not required
even when reductant-free extract of the strain (DJ138) containing
mutations in the genes that encode both flavodoxin and ferredoxin I was
tested in the in vitro FeMo-co synthesis assay (data not
shown). A. vinelandii is able to fix N
in the
absence of either or both flavodoxin and ferredoxin I and thus at least
one other protein can also function in vivo as an electron
donor to nitrogenase(30) .
The results presented here
suggest that FeMo-co activation of apodinitrogenase might be more
efficient when the apodinitrogenase is in a reduced state(s). Pierik et al.(21) have shown that oxidized dinitrogenase
prepared as in this study contains the P-clusters in a mixture of
oxidation states, and such a mixture of oxidation states is consistent
with the results presented in this paper. The oxidation state of the
apodinitrogenase might affect the interaction of apodinitrogenase with
the gamma protein that is known to be required for the insertion of
FeMo-co into apodinitrogenase. Alternatively, the oxidation state of
the apodinitrogenase might affect the interaction of apodinitrogenase
with FeMo-co itself. Ensign et al.(31) demonstrated
that the reduced but not the oxidized apo-carbon monoxide dehydrogenase
from Rhodospirillum rubrum was able to accept nickel into its
active site, and it is possible that a similar situation occurs for the
insertion of FeMo-co into apodinitrogenase.
Conclusion
A requirement for reductant in the in vitro FeMo-co synthesis system has been established. The
addition of reductant, however, is only required if extract of A.
vinelandii is chemically oxidized prior to addition to the assay.
This observation suggests that a physiological reductant that is able
to function in FeMo-co biosynthesis is present in extracts of A.
vinelandii or that the reductant requirement in assays that
utilize the oxidized extract is simply to reduce the protein(s)
required for FeMo-co biosynthesis to the appropriate redox state(s).
DTH, Ti(III) citrate, and NADH were found to satisfy the requirement
for reductant in the in vitro FeMo-co synthesis assay.