(Received for publication, February 9, 1995; and in revised form, July 28, 1995)
From the
Nitrogenase is the catalytic component of biological nitrogen
fixation, and it is comprised of two component proteins called the Fe
protein and MoFe protein. The Fe protein contains a single
FeS
cluster, and the MoFe protein contains two
metallocluster types called the P cluster (Fe
S
)
and FeMo-cofactor (Fe
S
Mo-homocitrate). During
turnover, electrons are delivered one at a time from the Fe protein to
the MoFe protein in a reaction coupled to component-protein
association-dissociation and MgATP hydrolysis. Under conditions of
optimum activity, the rate of component-protein dissociation is
rate-limiting. The Fe protein's Fe
S
cluster is the redox entity responsible for intermolecular
electron delivery to the MoFe protein, and FeMo-cofactor provides the
substrate reduction site. In contrast, the role of the P cluster in
catalysis is not well understood although it is believed to be involved
in accumulating electrons delivered from the Fe protein and brokering
their intramolecular delivery to the substrate reduction site. A
nitrogenase component-protein docking model, which is based on the
crystallographic structures of the component proteins and which pairs
the 2-fold symmetric surface of the Fe protein with the exposed surface
of the MoFe protein's pseudosymmetric
interface, is
now available. During component-protein interaction, this model places
the P cluster between the Fe protein's Fe
S
cluster and FeMo-cofactor, which implies that the P cluster is
involved in mediating intramolecular electron transfer between the
clusters. In the present study, evidence supporting this idea was
obtained by demonstrating that it is possible to alter the rate of
substrate reduction by perturbing the polypeptide environment between
the P cluster and FeMo-cofactor without necessarily disrupting the
metallocluster polypeptide environments or altering component-protein
interaction.
The MgATP-dependent reduction of nitrogen gas to yield ammonia has a minimal stoichiometry usually indicated as follows.
The reaction is catalyzed by nitrogenase, which is comprised of
two component proteins called the Fe protein, a homodimer, and the MoFe
protein, an heterotetramer (reviewed
by Dean et al.(1993), Howard and Rees(1994), and Kim and
Rees(1994); see Fig. 1for structural models). Turnover requires
the sequential delivery of single electrons from the Fe protein to the
MoFe protein and involves the association and dissociation of the
protein partners in a process where MgATP hydrolysis is coupled to
electron transfer. Three different metal clusters are believed to be
involved in electron transfer and substrate reduction. These include an
Fe
S
cluster, which is bridged between the
identical subunits of the Fe protein, and two pairs of unusual metal
clusters, called the P cluster and FeMo-cofactor, both of which are
contained within the MoFe protein. There is one P cluster and one
FeMo-cofactor within each independently operating MoFe protein
-unit. Each P cluster is constructed from two
Fe
S
subcluster fragments linked by a
corner-to-corner disulfide bond and is bridged between the
- and
-subunits at an interface exhibiting pseudo-2-fold symmetry. The P
cluster is coordinated to the protein by residues
Cys
, Cys
, Cys
,
Cys
, Cys
, Cys
,
and Ser
(numbers refer to the primary sequences of
the component proteins from Azotobacter vinelandii) with
Cys
and Cys
bridging the
Fe
S
subcluster fragments. FeMo-cofactor
contains a metal-sulfide core (Fe
S
Mo) and one
molecule of (R)-homocitrate. The metal-sulfide core is
constructed from MoFe
S
and
Fe
S
subcluster fragments, joined by a ring of
three sulfide bridges connecting pairs of iron atoms. FeMo-cofactor is
contained entirely within the
-subunit and is covalently attached
to the protein through a thiolate ligand provided by Cys
to an iron atom at one end of the prosthetic group and by the
imidazole
-nitrogen atom of His
to the
molybdenum atom at the opposite end.
Figure 1:
Rees-Howard
component-protein docking model. The model shows the
-carbon trace for the Fe protein homodimer (top) and an
unit of the MoFe protein (bottom) poised at the
proposed site of interaction. The Fe
S
cluster
contained within the Fe protein, and the P cluster and FeMo-cofactor
contained within the MoFe protein, are included as space-filling
models. In the docking model, the P cluster is located between the Fe
protein's Fe
S
cluster and
FeMo-cofactor.
There is compelling evidence
that the Fe protein's FeS
cluster is the
obligate electron donor to the MoFe protein and that it cycles between
the 1
and 2
redox state during the
sequential single-electron deliveries (Smith and Lang, 1974; Stephens,
1985; Lindahl et al., 1985). It is also known that
FeMo-cofactor provides the substrate reduction site (Shah and Brill,
1977; Hawkes et al., 1984; Scott et al., 1990; Scott et al., 1992; Kim et al., 1995). In contrast, the
specific role of the P cluster in catalysis is much less certain, but
it is believed to be involved in accumulating electrons delivered from
the Fe protein and brokering their intramolecular delivery to the
substrate reduction site. Thus, questions concerning the role of the P
cluster in catalysis include: (i) whether or not the P cluster is
involved in mediating the delivery of electrons from the Fe
protein's Fe
S
cluster to the
FeMo-cofactor; (ii) if so, how many electrons can be accumulated by the
P cluster; and (iii) what the path is for intramolecular electron
delivery between the P cluster and FeMo-cofactor.
Kim and Rees(1992)
have previously recognized four helices, which are oriented in parallel
and located between the P cluster and FeMo-cofactor, that could
participate in electron transfer between the two clusters (Fig. 2). The Tyr residue is located on one of
these helices and is situated in a direct line between the two
clusters. This residue is also one of a group of hydrophobic residues
that provide the polypeptide environment of the P cluster without being
in contact with it. Furthermore, Tyr
approaches the
terminal carboxyl of the homocitrate moiety of FeMo-cofactor and may
indirectly interact with it by hydrogen bonding through water. (
)The homologous residue within the
-subunit,
-Tyr
, is also found on a helix located between the
clusters but its side chain is directed away rather than toward the
FeMo-cofactor (Fig. 2). To assess the role of the P cluster and
the possible participation of Tyr
in intramolecular
electron transfer, this residue was substituted by Phe, Leu, and His
and the catalytic, kinetic, and spectroscopic properties of the
resulting altered MoFe proteins were examined. Thus, the experimental
rationale was to ask whether or not it is possible to perturb
intramolecular electron transfer without disrupting the polypeptide
environments of the metalloclusters or altering component protein
interaction. In a parallel set of experiments, the
-subunit
residue, Tyr
, was also substituted by Phe, Leu, and
His, and the catalytic and kinetic properties of the altered
His
MoFe protein were examined as well. These latter
experiments were intended to serve as a control, with the substitutions
at this position considered less likely to have an effect on
intramolecular electron transfer.
Figure 2:
Coil diagram of a section of the
pseudosymmetric interface between the MoFe protein - and
-subunits. The view is approximately the same as shown in Fig. 1. The spatial relationships of the Tyr
residue and the Tyr
residue with respect to the P
cluster and FeMo-cofactor are indicated in panel A. The
pseudosymmetric nature of the two helices, which, respectively, contain
the Tyr
residue and the Tyr
residue, is also recognized in the conservation of primary
sequences between the
- and
-subunits, as shown in panel
B. Conserved residues are boxed, and the tyrosine
residues targeted for substitution and the proximal P
cluster-coordinating cysteine residues are indicated by numbers
above or below the respective
sequences.
The turnover rate was also measured by stopped-flow
spectrophotometry. In these experiments, flavodoxin (product of the nifF gene), the physiological electron donor to the Fe protein
was used as reductant. Reduction of oxidized Fe protein by the
hydroquinone form of flavodoxin to yield the semiquinone form is
accompanied by an increase in absorbance at 580 nm. Thus, under
turnover conditions, the reduced hydroquinone form of flavodoxin is
oxidized as it rapidly transfers its electron to the oxidized Fe
protein, which becomes available as the MoFe protein-Fe protein complex
slowly dissociates. In this way, the consumption of reducing
equivalents, which occurs during nitrogenase turnover, can be
continuously monitored by following the increased absorbance at 580 nm.
Under normal conditions, the turnover rate estimated by the
flavodoxin-oxidation rate provides an indirect measure of the rate of
component-protein dissociation because this dissociation is
rate-limiting in catalysis (Thorneley and Lowe, 1983). In the analysis
of the altered His MoFe protein, however, where the
overall turnover rate might ultimately be limited by intramolecular
electron transfer, the observed change in optical density was used to
calculate an ``apparent'' dissociation rate because here
component-protein dissociation may not be rate-limiting. For these
experiments, flavodoxin was reduced to the hydroquinone state by using
an excess of Na
S
O
at pH 8 in an
anaerobic chamber. After incubation for approximately 20 min to ensure
complete reduction, unreacted dithionite and its oxidation products
were removed on a gel filtration column (0.5
5 cm) packed with
P-6DG (Bio-Rad, Melville, NY) that had been pre-equilibrated with
anaerobic 25 mM Hepes, pH 7.4, 10 mM MgCl
.
MoFe proteins were isolated
and purified in parallel from all three strains with substitutions at
Tyr, from the His
strain, and from
wild type. The isolated His
MoFe protein exhibited
kinetic and catalytic properties nearly identical to the wild-type MoFe
protein, confirming that this substitution has no discernible effect on
electron transfer from Fe protein through to substrate. As expected
from the good growth rates of their parent strains, the altered
Phe
and Leu
MoFe proteins exhibited
catalytic activities for H
evolution, C
H
reduction, N
fixation, and concomitant MgATP
hydrolysis comparable with those of the wild type. In contrast to the
other altered MoFe proteins, the His
MoFe protein
showed a significant decrease in the maximum specific activity for
substrate reduction, while the overall rate of MgATP hydrolysis was
maintained near the wild type rate (Table 2). In other words, the
reaction catalyzed by the altered His
MoFe protein
exhibits MgATP hydrolysis that is partially uncoupled from electron
transfer. For all of the altered MoFe proteins, the effect of 10% CO
was to divert all electron flux to H
reduction, which
was insensitive to the presence of CO, as seen for wild type. No ethane
was observed from catalyzed C
H
reduction.
A
number of experiments were performed on the His MoFe
protein to determine whether or not its lower specific activity is due
to an alteration in intramolecular electron delivery. The effect of
varying electron flux was investigated to probe if its ability to
deliver electrons to the substrate had been compromised.
Component-protein ratio titrations performed in parallel for both
wild-type and His
MoFe proteins revealed that their
specific activities maximized at different Fe protein:MoFe protein
ratios (Fig. 3). For the wild-type MoFe protein, a maximum
specific activity of 2250 nmol of H
formed/min/mg of MoFe
protein was achieved at an Fe protein:MoFe protein molar ratio of
greater than 10:1 under conditions of proton reduction. Under the same
conditions, a maximum specific activity of 1100 nmol of H
formed/min/mg of MoFe protein was observed for the His
MoFe protein, and this value was achieved at the lower Fe
protein:MoFe protein molar ratio of approximately 5:1 (Fig. 3).
This effect was also apparent in analogous titrations performed under
conditions of acetylene reduction, where the activity of the
His
MoFe protein maximizes at only about 600 nmol of
C
H
produced/min/mg of MoFe protein at the same
5:1 molar ratio, while the wild type reaches the expected maximum
activity of 2100 nmol C
H
formed/min/mg of MoFe
protein at a molar ratio of greater than 20:1. The K
values for C
H
reduction were also
determined for the wild-type and His
MoFe proteins
and found to be comparable at 0.0055 and 0.0040 atm, respectively.
These values are in the range previously reported for wild type
(Dilworth, 1966; Schollhorn and Burris, 1967; Kim et al.,
1995).
Figure 3:
Titration of wild-type and His MoFe protein with wild-type Fe protein. The component protein
ratios were varied while the total protein concentration (0.5 mg) was
kept constant. Assays were performed under an argon atmosphere, and
activities are expressed as nmol of H
produced per min per
mg of MoFe protein.
The lowered maximum CH
reduction
activity for the His
MoFe protein, when compared with
its maximum H
reduction activity, is not compensated
for by increased H
reduction under 10%
C
H
to maintain a constant electron flux (Table 2). Thus, the His
MoFe protein appears
to suffer greater inhibition of electron flux under a 10%
C
H
atmosphere than under either 100% N
or 100% argon atmospheres. The inhibition of electron flux during
C
H
reduction catalyzed by the His
MoFe protein was partially relieved by carbon monoxide, bringing
it to approximately the same level observed under 100% N
or
100% argon atmospheres .
Figure 4:
Stopped-flow spectrophotometric traces of
the oxidation of the hydroquinone form of flavodoxin as a function of
time. The rate of oxidation of the hydroquinone form of A.
vinelandii flavodoxin II provides an indication of the rate of
complex dissociation. Syringe A contained dithionite free wild-type or
His MoFe protein (0.5 µM) and Fe protein
(2.5 µM), and syringe B contained flavodoxin (30
µM) and MgATP (10 mM). Panels A and B represent the wild-type MoFe protein- and His
MoFe protein-dependent reactions, respectively. The calculated
turnover rates from a linear fit are 6.3 s
for the
wild type and the first phase of the His
MoFe
protein-dependent reactions and 2.5 s
for the second
phase of the His
MoFe protein-dependent
reaction.
In the second set of stopped-flow experiments, the rates of
both primary and secondary intermolecular electron transfers to both
the wild-type and His MoFe proteins were measured, as
were the absorbance changes that occur at longer times after the
initial electron transfers (after 150 ms). The primary and secondary
electron transfer rates to both the wild-type and His
MoFe proteins were found to be identical at 158 s
(Fig. 5; data for determination of the secondary electron
transfer rate are not shown) and within the range previously reported
(Thorneley, 1975; Fisher et al., 1991). In contrast to the
wild-type MoFe protein, the reaction involving the His
MoFe protein exhibits a gradual decrease in optical absorbance
after about 150 ms in the pre-steady state experiment (Fig. 5).
Further, this decrease in absorbance occurs exponentially with a rate
constant of 2.7 s
, which is reminiscent of the
protein-protein complex dissociation rate of 2.5 s
that is measured for the latter part of this biphasic process.
Thus, the absorbance decrease may reflect reduction of the oxidized Fe
protein as it dissociates from the complex.
Figure 5:
Electron transfer from the Fe protein to
the wild type and His MoFe protein and subsequent
absorbance changes occurring after primary electron transfer. The top panel is a comparison of stopped-flow spectrophotometry
traces of His
MoFe protein- (a) and
wild-type MoFe protein-dependent Fe protein oxidation (b). The traces are an enlargement of the first 0.03 s shown in the lower trace. The lower panel is an expanded trace (0.8 s) and shows the absorbance changes that occur after primary
electron transfer. Trace c was obtained with wild-type MoFe
protein and is typical of that reported previously (Lowe et
al., 1993), whereas trace d with the His
MoFe protein-dependent reaction shows a single exponential
absorbance decrease (k
= 2.5
s
) after primary electron
transfer.
Evidence that the P cluster is the primary acceptor of
electrons from the Fe protein and that it subsequently brokers the
intramolecular delivery of electrons to the FeMo-cofactor can be
considered in the context of the proposed structural models for the
nitrogenase component proteins from A. vinelandii (Georgiadis et al., 1992; Kim and Rees, 1992). A docking model that is
based on the structures of the individual component proteins (Kim and
Rees, 1992; Howard, 1993) and that takes into account amino acid
substitution studies (Wolle et al., 1992) and chemical
cross-linking experiments has been proposed (Willing et al.,
1989; Willing and Howard, 1990). This model (Fig. 1) pairs the
2-fold symmetric surface of the Fe-protein homodimer with the exposed
surface of a MoFe-protein pseudosymmetric -unit interface. In
this arrangement, the Fe protein's Fe
S
cluster is positioned in the closest possible proximity to the
MoFe protein's P cluster, which then lies between the Fe
protein's Fe
S
cluster and FeMo-cofactor.
Evidence supporting the docking model has come from biochemical and
kinetic analyses of altered component proteins having one or more amino
acid substitutions located within the respective docking sites (Wolle et al., 1992; Kim et al., 1993; Thorneley et al., 1993; Peters et al., 1994; Seefeldt, 1994).
In the
present work, the possibility that substitutions for Tyr might alter intramolecular electron transfer was investigated
because this residue is located on a helix, which spans the P cluster
and FeMo-cofactor, and yet does not directly contact either the P
cluster or FeMo-cofactor (Fig. 2). Thus, the primary objective
was to determine whether or not it is possible to alter intramolecular
electron transfer between these prosthetic groups without disrupting
either of their respective polypeptide environments. Studies of the
effects of substitutions at Tyr
were carried out in
parallel with identical amino acid substitutions at the corresponding
residue in the
-subunit, Tyr
, which served as an
internal control because the side chain of this residue is directed
away from rather than across the direct line from the P cluster to the
FeMo-cofactor. Substitutions at Tyr
were, therefore,
considered much less likely to affect intramolecular electron transfer.
Of the six mutant strains resulting from the substitution of either
Tyr
or Tyr
by Phe, Leu, or His,
only the His
-substituted strain showed a significant
increase in diazotrophic growth-doubling time, which correlated with
the His
MoFe protein being the only one to exhibit
significantly reduced maximal specific activity for N
fixation, H
evolution, and C
H
reduction. These results were consistent with our hypothesis that
the Tyr
residue would have no role in intramolecular
electron transfer.
The decreased steady-state maximum activity
observed for the His MoFe protein is best explained
as arising from an alteration in electron transfer capability that
occurs after intermolecular electron transfer between the Fe protein
and the MoFe protein because the primary and secondary rates of
intermolecular electron transfer were found to be identical for both
the altered His
MoFe protein and the wild-type MoFe
protein (Fig. 5). Moreover, the component-protein titration
experiments can be explained by a model where, under high flux
conditions (i.e. high Fe protein:MoFe protein ratios),
substrate reduction catalyzed by the His
MoFe protein
becomes limited by intramolecular electron transfer rather than complex
dissociation. In other words, the amount of Fe protein required to
achieve maximum His
specific activity is lowered in
the titration experiments shown in Fig. 3because the maximum
flux through the system is limited as a consequence of a defect in
intramolecular electron transfer per se.
This conclusion is
supported by MCD spectrophotometric analysis of the thionine-oxidized
state of the altered His MoFe protein, which was
unchanged when compared with the wild type, indicating no apparent
changes in the P cluster structure or its electronic environment. (
)Similarly, no perturbation of FeMo-cofactor's S
= 3/2 EPR spectra was observed for any of the altered
dithionite-reduced MoFe proteins when compared to the wild-type
spectrum. Also, none of the altered MoFe proteins having substitutions
at the Tyr
position exhibited any of the
characteristic substrate reduction changes associated with perturbation
of the FeMo-cofactor's polypeptide environment (Scott et
al., 1990, 1992; Kim et al., 1995; Table 2). These
results, together with comparable K
values for
acetylene reduction for both the wild-type and His
MoFe protein, indicate that the lowered maximum specific activity
for the His
MoFe protein under conditions of high
flux is unlikely to occur as a result of an alteration in the substrate
reduction site. However, the unusually low electron flux, plus the
increased uncoupling of MgATP hydrolysis (see below) observed only
under 10% C
H
with the His
MoFe protein, both of which are relieved by CO, suggests that
different substrates may be served by different or multiple
electron-transfer pathways.
Effective nitrogenase catalysis requires
the coupled hydrolysis of about four MgATP for each pair of electrons
transferred to substrate. However, MgATP hydrolysis can become
partially uncoupled from electron transfer under certain conditions,
such as extremely low flux (Ljones and Burris, 1972; Hageman and
Burris, 1978), high or low pH (Jeng et al., 1970; Imam and
Eady, 1980), and high or low temperature (Watt et al., 1975;
Watt and Burns, 1977). Certain amino acid substitutions that alter
either component-protein interaction (Wolle et al., 1992;
Seefeldt, 1994) or the substrate reduction site (Kim et al.,
1995) have also been shown to uncouple MgATP hydrolysis from electron
transfer. Such uncoupling of MgATP hydrolysis from substrate reduction
can be explained either by the back donation of an electron from the
MoFe protein to an oxidized Fe protein, called futile cycling
(Orme-Johnson and Davis, 1977) or by the occurrence of MgATP hydrolysis
upon component-protein interaction without electron transfer (Thorneley et al., 1991). A reasonable explanation for the uncoupled
MgATP hydrolysis in reactions catalyzed by the His MoFe protein (Table 2) is that, as a consequence of
disturbing the intramolecular electron transfer pathway, the capacity
for the P cluster to accept electrons becomes saturated (after one or
more rounds of component-protein interaction and intermolecular
electron transfer), and any further component-protein interactions
result in MgATP hydrolysis but not necessarily a net electron transfer
from the Fe protein to the MoFe protein.
This explanation is
consistent with the biphasic nature of the apparent component-protein
dissociation rate. Here electron transfer to the P cluster of the
His MoFe protein occurs at an initial rate comparable
with the wild type only until its capacity for storing electrons is
reached, at which time the apparent component-protein dissociation rate
becomes substantially lowered due to a defect in intramolecular
electron transfer. Moreover, the gradual absorbance decrease observed
after 150 ms for the His
MoFe protein contrasts
dramatically with the absorbance increase observed with the wild-type
MoFe protein. Thus, according to the model of Lowe et
al.(1993), it appears that P cluster oxidation within the
His
MoFe protein occurs only at a relatively slow
rate and is masked by the reduction of the oxidized Fe protein as it
dissociates from the complex, consistent with the hypothesis that the
altered MoFe protein is unable to achieve normal intramolecular
electron transfer. Further, the apparent ability of the P cluster to
accumulate at least two electrons is consistent with the proposed role
of the P cluster as an electron storage unit and the proposed
corner-to-corner disulfide link between the P cluster subfragments
(Rees et al., 1993). Although the present results provide no
insight as to whether the P cluster donates single electrons or
electron pairs (or both) during substrate reduction, they do provide
some credence to the possibility that a two-electron transfer from the
P cluster to the substrate reduction site could occur during turnover.
Finally, the helix containing Tyr has been
suggested as one possible electron transfer pathway from the P cluster
to FeMo-cofactor (Kim and Rees, 1992), in particular the portion from
the P cluster-ligating Cys
to Tyr
and then through a hydrogen bond to homocitrate, which ligates
the molybdenum atom of FeMo-cofactor. However, both the Phe
and Leu
MoFe proteins, each of which would be
incapable of hydrogen bonding to homocitrate, exhibit rates of
substrate reduction similar to wild type. Thus, this pathway could be
viewed as unattractive and the simple interpretation invoked that
neither the hydroxyl group nor the aromatic feature of the
Tyr
residue is critical for productive intramolecular
electron transfer. In this context, however, it should be noted that
intramolecular electron transfer rates measured for certain other
proteins (reviewed by Farid et al. (1993)) are orders of
magnitude faster than the rate reported for nitrogenase
component-protein dissociation (Thorneley and Lowe, 1983), the
rate-limiting step in nitrogenase catalysis. Consequently, a dramatic
decrease in the rate of intramolecular electron transfer might be
necessary to become manifested as a lower rate of enzyme turnover.
Thus, because the rate of intramolecular electron transfer within the
MoFe protein cannot be directly measured, it remains premature to
conclude that neither the hydroxyl group nor the aromatic nature of the
Tyr
residue is involved in intramolecular electron
transfer. Moreover, the significant changes in the rates of MgATP
hydrolysis and catalyzed substrate reduction exhibited by the
His
MoFe protein, likely resulting from an introduced
structural perturbation, suggest that this helix could provide a
significant electron transfer pathway.
Similar studies to those described above on the residues constituting the other prosthetic group-spanning helices should provide insight into how electrons are both accommodated within and intramolecularly transferred among the redox-active moieties of the MoFe protein. Such information will be necessary to describe the complete mechanism of biological nitrogen fixation and could impact generally on our understanding of electron transfer processes in complex biological systems.