(Received for publication, September 9, 1996, and in revised form, October 13, 1996)
From the Department of Chemistry and Biochemistry,
Utah State University, Logan, Utah 84322 and § Department
of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic
Institute and State University, Blacksburg, Virginia 24061
Nitrogenase-catalyzed substrate reduction
reactions require the association of the iron (Fe) protein and the
molybdenum-iron (MoFe) protein, electron transfer from the Fe protein
to the MoFe protein coupled to the hydrolysis of MgATP, followed by
protein-protein complex dissociation. This work examines the role of
MgATP hydrolysis and electron transfer in the dissociation of the Fe
protein-MoFe protein complex. Alteration of aspartate 39 to asparagine
(D39N) in the nucleotide binding site of Azotobacter
vinelandii Fe protein by site-directed mutagenesis resulted in an
Fe protein-MoFe protein complex that did not dissociate after electron
transfer. While the D39N Fe protein-MoFe protein complex was inactive
in all substrate reduction reactions, the complex catalyzed both
reductant-dependent and reductant-independent MgATP
hydrolysis. Once docked to the MoFe protein, the D39N Fe protein was
found to transfer one electron to the MoFe protein requiring MgATP
hydrolysis, with an apparent first order rate constant of 0.02 s1 compared with 140 s
1 for the wild-type
Fe protein. Only following electron transfer to the MoFe protein did
the D39N Fe protein form a tight complex with the MoFe protein, with no
detectable dissociation rate. This was in contrast with the
dissociation rate constant of the wild-type Fe protein from the MoFe
protein following electron transfer of 5 s
1. Chemically
oxidized D39N Fe protein with MgADP-bound did not form a tight complex
with the MoFe protein, showing a dissociation rate similar to
chemically oxidized wild-type Fe protein (3 s
1 for D39N
Fe protein and 6 s
1 for wild-type Fe protein). These
results suggest that electron transfer from the Fe protein to the MoFe
protein within the protein-protein complex normally induces
conformational changes which increase the affinity of the Fe protein
for the MoFe protein. A model is presented in which Asp-39 participates
in a nucleotide signal transduction pathway involved in component
protein-protein dissociation.
Nitrogenase-catalyzed substrate reduction involves a complex
series of reactions including the association of the two nitrogenase metalloproteins, MgATP hydrolysis coupled to interprotein electron transfer, followed by the dissociation of the two metalloproteins (1-3). The two metalloproteins that constitute nitrogenase are the
iron (Fe)1 protein, which contains two
nucleotide binding sites and a [4Fe-4S] cluster bridging the two
identical subunits (4) and the molybdenum-iron (MoFe) protein, an
2
2 tetramer with two unique
[8Fe-(7-8)S] clusters (P-clusters) and two
molybdenum-iron-sulfur-homocitrate clusters (FeMoco) (5, 6). The
generally accepted mechanism for nitrogenase-catalyzed reduction
reactions is that two MgATP molecules bind to the Fe protein, priming
this protein for docking to the MoFe protein. After complex formation,
a single electron is transferred from the Fe protein [4Fe-4S] cluster
to a MoFe protein FeMoco, with a P-cluster as a probable intermediate.
Two molecules of MgATP bound to the Fe protein are hydrolyzed to two MgADP molecules and two Pi molecules in a reaction that
appears to be coupled to electron transfer (7-9). Following electron transfer, the oxidized Fe protein, with two bound MgADP molecules, dissociates from the one-electron reduced MoFe protein in what is
considered to be the reaction rate-limiting step (10, 11). The oxidized
Fe protein is reduced by low potential electron transfer proteins
(e.g. flavodoxin or ferredoxin), and the two MgADP molecules are replaced with two MgATP molecules, preparing the Fe protein for a
second round of docking to the MoFe protein and electron transfer. This
cycle is repeated until sufficient electrons have accumulated to reduce
the substrate bound to FeMoco (12, 13).
MgATP appears to serve at least three important functions in the
proposed reaction mechanism, namely (i) MgATP binding induces protein
conformational changes in the Fe protein necessary for docking to the
MoFe protein (14); (ii) MgATP hydrolysis is coupled to intercomponent
electron transfer; and (iii) MgATP hydrolysis is coupled to the
dissociation of the Fe protein from the MoFe protein (15). One MgATP
molecule binds to each subunit of the Fe protein, about 19 Å away from
the bridging [4Fe-4S] cluster and MoFe protein docking interface (4).
MgATP binding to the Fe protein is known to result in changes in the
properties of the [4Fe-4S] cluster, suggesting long range
communication through protein conformational changes.
Nucleotide-induced changes in the properties of the Fe protein
[4Fe-4S] cluster have been monitored as changes in EPR (16), circular
dichroism (CD) (17, 18) and 1H NMR (19, 20) spectra, along
with a 120 mV shift in the redox potential of the
[4Fe-4S]2+/1+ couple (21, 22). At least one signal
transduction pathway from the nucleotide binding site to the [4Fe-4S]
cluster has recently been identified as the protein chain from Asp-125
to Cys-132, designated as switch II (14). It appears that MgATP binding induces changes in the position of this switch, resulting in changes in
the protein environment around the [4Fe-4S] cluster. This has been
suggested to ready the Fe protein for interaction with the MoFe protein
(14). Upon hydrolysis of MgATP to MgADP and Pi, the Fe
protein is proposed to undergo additional protein conformational changes (2). This is deduced from the observation that the MgADP-bound
state of the Fe protein exhibits distinct spectroscopic properties for
the [4Fe-4S] cluster compared with the MgATP-bound state (18, 20,
23). Thus, it has been suggested that the conformational changes
induced within the Fe protein in going from the MgATP-bound state to
the MgADP-bound state represent a molecular switch, which could be
coupled to electron transfer and dissociation of the protein-protein
complex (2). The status of the nucleotide bound to the protein would
have to be communicated to the [4Fe-4S] cluster and the MoFe docking
interface approximately 19 Å away. Switch II participates in this
communication event, both in changes to the cluster and in defining the
affinity for association with the MoFe protein (15). Related to these
issues is the important observation that the MoFe protein docking to the Fe protein stimulates the hydrolysis of MgATP bound to the Fe
protein, suggesting some communication from the docking interface to
the nucleotide binding site, probably by relocating residues critical
for hydrolysis.
Recently, Asp-129 on the switch II pathway has been suggested to be a
possible general base that could activate a water for hydrolysis of
MgATP (24). An examination of the x-ray structure of the Fe protein
from Azotobacter vinelandii (4) reveals that Asp-39 is also
located near Asp-129, suggesting a possible role for this residue in
the hydrolysis of MgATP (Fig. 1). In addition, Asp-39 is
part of a protein chain that connects the nucleotide binding site to
the MoFe protein docking surface of the Fe protein. This protein chain
is a second possible communication pathway in the Fe protein (25) and
has features in common with switch I in other nucleotide switch
proteins (26). Residues at the end of the proposed switch I in the Fe
protein have been previously identified as functioning in docking
interactions with the MoFe protein (27).
It seemed possible that Asp-39 could be essential for two important
functions of the nitrogenase Fe protein, (i) Asp-39 could be a general
base, activating a water molecule for nucleophilic attack on the
-phosphate of MgATP, and (ii) Asp-39 could function in switch I
nucleotide signal transduction to the MoFe protein docking interface.
To probe these possible functions of Asp-39, we have changed this
residue to the amino acids asparagine (D39N) and glutamic acid (D39E)
and characterized the properties of the purified, altered Fe proteins.
The D39N altered Fe protein was still active in MgATP hydrolytic
activity when combined with the MoFe protein, ruling out an essential
role for Asp-39 as a general base in the MgATP hydrolysis mechanism.
The D39N Fe protein was able to transfer a single electron to the MoFe
protein. However, following electron transfer, the D39N Fe protein did
not dissociate from the MoFe protein, resulting in a tight
protein-protein complex. These results suggest that Asp-39 participates
in the signal for component protein dissociation following electron
transfer and that electron transfer induces protein conformational
changes within the nitrogenase protein-protein complex.
Site-directed mutagenesis of the nitrogenase Fe protein gene from A. vinelandii, nif H, was carried out as described previously (28, 29). Expression of the altered Fe proteins in A. vinelandii and their purification were performed essentially as described previously (29), except that 20% glycerol (v/v) was included in buffers used for purification of altered Fe proteins. Prior to freezing the proteins in liquid nitrogen, the glycerol concentration was decreased to 5% by dilution with 50 mM Tris buffer, pH 8.0. Protein concentrations were determined by a modified biuret method (30), with bovine serum albumin as the standard. SDS-polyacrylamide gel electrophoresis with Coomassie staining (31) was used to confirm that all proteins were homogeneous. Protein manipulations for CD spectroscopy were performed in an argon atmosphere glove box, with an oxygen concentration of less than 1 part per million. Stopped-flow spectroscopic studies were done in a nitrogen-filled glove box. Unless noted, all buffers were saturated with argon and contained 2 mM sodium dithionite (Na2S2O4).
Activity Assays, Nucleotide Binding, and MgATP HydrolysisAcetylene reduction assays were performed as described previously (32) under argon in 100 mM MOPS buffer, pH 7.0, with 10 mM sodium dithionite as the reductant. The rates of MgATP hydrolysis were determined in assay solutions without the MgATP-regenerating system as described previously (29). The stoichiometry of MgATP molecules hydrolyzed per electron transferred was determined as described previously (29). The binding of MgATP and MgADP to the wild-type, D39E, and D39N Fe proteins were performed by an equilibrium column binding technique as described earlier (33).
Binding of the Asp-39 Altered Fe Proteins to the MoFe ProteinThe ability of the D39N or D39E Fe proteins to bind to the MoFe protein was tested by a competition assay described earlier (24). Increasing quantities of the altered Fe protein (from 0 to 230 µg) were added to fixed concentrations of wild-type Fe protein (115 µg or 1.8 nmol) and MoFe protein (216 µg or 0.9 nmol). If the altered Fe protein binds to the MoFe protein, then a decrease in the rate of acetylene reduction should be observed. The percent of acetylene reduction activity remaining was plotted versus the ratio of altered Fe protein to wild-type Fe protein, and the data were fitted to the Hill equation.
UV-visible Absorption Spectra of Wild-type Fe Protein and Altered Fe ProteinsAbsorption spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer in 2.0-ml quartz cuvettes fitted with serum stoppers. Anaerobicity was obtained by purging the cuvettes with O2-free argon for 6 min. The buffer used for these experiments was 50 mM MOPS, pH 7.0, unless otherwise noted. A MgATP-regenerating system was included during nitrogenase catalysis to prevent the formation of MgADP, which is a known inhibitor of MgATP binding to the Fe protein (34).
Stopped-flow SpectrophotometryAbsorbance changes were monitored with a Hi-Tech SF61 stopped-flow spectrophotometer equipped with a data acquisition and curve fitting system (Salisbury, Wilts, UK) as described previously (15). Electron transfer rates, and the rates of reduction of the Fe protein by sodium dithionite, were determined as described previously (15). The rate of dissociation of the chemically oxidized Fe protein from the MoFe protein (15) and the dissociation rate of the turnover-oxidized Fe protein from the MoFe protein (11) were also determined. All protein concentrations are reported in the appropriate figure legend.
Circular Dichroism Spectra of Wild-type and Asp-39 Altered Fe ProteinsFe protein samples (20 mg or 312 nmol) containing 2 mM dithionite were desalted by passage through a Sephadex G-25 column (0.5 × 10 cm) equilibrated with 100 mM Tris buffer, pH 8.0, without dithionite. The Fe protein was then oxidized by the addition of 25 µl of a 20 mM indigo disulfonate (IDS) solution. The IDS was separated from the Fe protein on a Dowex 1 column (0.5 × 5 cm) equilibrated with 100 mM Tris buffer, pH 8.0, without dithionite. The oxidized Fe protein, free of small molecules, was then split into two equal aliquots and placed into two 4-ml sealed quartz cuvettes. The total volume of each cuvette was then brought to 2 ml by the addition of 100 mM Tris buffer, pH 8.0. Full absorption spectra were recorded to confirm the oxidation state of the Fe protein and to determine the protein concentration. CD spectra were then recorded on an Aviv model 62DS spectropolarimeter and were base-line subtracted (18).
Proton NMR Spectra of Isotropically Shifted Protons for Fe ProteinsProton NMR spectra of reduced Fe proteins were recorded as described previously (20). All spectra were recorded at 305 K in anaerobic glass NMR tubes fitted with degassed Teflon stoppers (Wilmad, Buena, NJ). Fe protein samples were exchanged into Chelex 100-treated Tris-buffered D2O solution by passage through a Sephadex G-25 column (20). The reduced Fe protein samples were concentrated to between 0.85 and 1 mM using a Centricon-30 concentrator (Amicon, Beverly, MA).
Electron Transfer Monitored by EPR SpectroscopyElectron transfer from the Fe protein to the MoFe protein was monitored by following the disappearance of the EPR signal of the Fe protein in going from the [4Fe-4S]1+ to the [4Fe-4S]2+ oxidation states. Samples were prepared essentially as described previously (15). Wild-type or D39N Fe protein (60 µM) was mixed with MoFe protein (112 µM), MgATP (1 mM), and a MgATP-regenerating system and allowed to react for 1 h. Samples were removed before the addition of MgATP and following the 1-h incubation and frozen in EPR tubes. All EPR spectra were recorded on a Bruker ESP300E spectrometer equipped with a dual mode cavity and an Oxford liquid helium cryostat. A total of 10 scans were added for each spectrum, and all other parameters were as described (15).
Changing Asp-39 by site-directed mutagenesis to the amino acids glutamic acid (D39E) and asparagine (D39N) provided a useful way to probe the function of this amino acid. Changing aspartic acid to glutamic acid represents a conservative amino acid change while maintaining the carboxylate side chain. Changing Asp to Asn would remove the negatively charged carboxylate side chain and is predicted to be a conservative structural change (35). The altered Fe proteins (D39E and D39N) were expressed in A. vinelandii cells in place of the wild-type Fe protein. This was accomplished by replacing the chromosomal wild-type Fe protein gene, nif H, with the mutated gene by a homologous recombination procedure (28). The A. vinelandii cells expressing the altered Fe protein were tested for their ability to grow under nitrogen-fixing conditions in order to analyze the functional consequences of the amino acid changes. A. vinelandii cells expressing either the D39E or D39N altered Fe proteins were unable to grow under nitrogen fixing conditions, revealing that changing Asp-39 to Glu or Asn renders the Fe protein inactive in nitrogen fixation. It was of interest to investigate the properties of the purified, altered Fe proteins to further define the functional consequences of the amino acid change and thus the function of Asp-39. The D39E and D39N Fe proteins were overexpressed in A. vinelandii cells using a derepression protocol previously described (29). The D39N Fe protein could be purified with yields similar to the wild-type Fe protein, whereas the D39E Fe protein was purified with slightly lower yields.
Substrate Reduction and MgATP HydrolysisGiven the
observation that the D39N and D39E Fe proteins were inactive in
nitrogen fixation in whole cells, it was important to ascertain any
activities of the purified Fe proteins when combined with purified MoFe
protein. Three activities were monitored, namely the two-electron
reduction of acetylene to ethylene, the two-electron reduction of
protons to H2, and the hydrolysis of MgATP. The last is
particularly important to determine because the wild-type Fe protein
will hydrolyze MgATP to MgADP at a considerable rate when combined with
the MoFe protein in the absence of reductant, in a reaction termed
reductant-independent MgATP hydrolysis (36-38). Neither the D39N nor
the D39E Fe proteins were found to support acetylene or proton
reduction activities when combined with MoFe protein. Interestingly,
the D39N Fe protein did show a significant MgATP hydrolysis activity
(53 nmol of MgADP·min1·mg of Fe
protein
1) when combined with MoFe protein in the presence
of reductant. The rate was much lower than that catalyzed by the
wild-type nitrogenase complex (8772 nmol of
MgADP·min
1·mg Fe protein
1) but
significantly above background rates determined for each protein alone
of 1.0 nmol of MgADP·min
1·mg of Fe
protein
1. The D39N Fe protein also catalyzed
reductant-independent MgATP hydrolysis (5 nmol of MgADP formed
·min
1·mg of Fe protein
1) when combined
with the MoFe protein, which compared with the wild-type rate of 817 nmol of MgADP formed·min
1·mg of Fe
protein
1. For both the wild-type and D39N Fe proteins,
the reductantindependent rates of MgATP hydrolysis were about 10%
the reductant-dependent rates, consistent with previously
published results for wild-type nitrogenase (36-38). The MgATP
hydrolysis catalyzed by the D39N Fe protein-MoFe protein complex
clearly indicates that Asp-39 is not absolutely required in the
mechanism of MgATP hydrolysis. In addition, the requirement for MoFe
protein suggests that both the oxidized and reduced states of the D39N
Fe protein can dock with the MoFe protein, forming a complex capable of
MgATP hydrolysis, even in the absence of substrate reduction.
Given the location of Asp-39 in the Fe protein near the probable binding site of the phosphate portion of a bound nucleotide, it was important to determine how changing Asp-39 affected the affinity of the Fe protein for binding nucleotides. Both the stoichiometry of binding and the affinity for binding MgATP or MgADP to the Fe protein can be determined by an equilibrium column binding procedure (33). The reduced states of the D39N and D39E Fe proteins were found to bind a maximum of 2 nucleotides, with apparent dissociation constants (Kd) similar to those found for the wild-type Fe protein (Table I). The D39E Fe protein bound both MgATP and MgADP with a slightly lower affinity compared with wild-type Fe protein. In contrast, the D39N Fe protein bound MgATP with a slightly greater affinity and MgADP with a slightly lower affinity when compared with wild-type Fe protein. These results clearly show that both the D39N and D39E Fe proteins bind 2 nucleotides with similar affinity when compared with the wild-type Fe protein and thus that Asp-39 is not essential to nucleotide binding to the Fe protein. In addition, both the D39N and D39E Fe proteins demonstrated strong positive cooperativity in binding both MgATP and MgADP, consistent with the results for the wild-type Fe protein, indicating that Asp-39 does not participate in this cooperative mechanism.
|
The MoFe
protein-dependent MgATP hydrolysis activity of the D39N Fe
protein suggested that this altered protein could bind to the MoFe
protein. To probe this possible interaction, the Asp-39 altered Fe
proteins were used in a competitive binding activity assay (24). In
this assay, the inactive altered Fe protein is titrated against
wild-type Fe and MoFe proteins to determine competition for binding of
Fe proteins to the MoFe protein. The formation of a D39N Fe
protein-MoFe protein complex would be inactive and thus would reduce
the total substrate reduction activity. Fig. 2 shows
that while the D39E Fe protein did not inhibit the wild-type nitrogenase activity, the D39N Fe protein did show strong inhibition. A
fit of the D39N Fe protein inhibition data to the Hill equation revealed that 50% inhibition of activity was achieved at a ratio of
two D39N Fe proteins to one MoFe protein. A similar pattern of
inhibition has recently been observed for another altered Fe protein
(L127), which was shown to form a tight, irreversible complex with
the MoFe protein (15). The D39N Fe protein clearly binds to the MoFe
protein, possibly forming a tight protein-protein complex.
Electron Transfer
Given the fact that the D39N Fe protein
bound to the MoFe protein and still hydrolyzed MgATP, but did not
reduced substrates, it was of interest to determine if a single
electron could be transferred to the MoFe protein. A simple way to
monitor electron transfer from the Fe protein to the MoFe protein is to
follow the increase in absorbance in the 430 nm region as a result of the oxidation of the Fe protein [4Fe-4S] cluster from the 1+ to the
2+ oxidation state (15). When the wild-type Fe protein is combined with
the MoFe protein, MgATP and reductant, a rapid increase in absorbance
in the 430 nm region of the UV-visible spectrum is observed,
corresponding to the oxidation of the [4Fe-4S] cluster upon electron
transfer (Fig. 3, panel A). Under these
conditions, protons are reduced to H2. When mixed with MoFe
protein, MgATP, and reductant, the D39N Fe protein [4Fe-4S] cluster
was also found to oxidize to the 2+ state (panel B). By
following this oxidation at 430 nm using a stopped-flow
spectrophotometer, apparent first order rate constants for primary
electron transfer from the Fe protein to the MoFe protein were
determined (15). A rate constant of 140 s1 was determined
for the wild-type Fe protein and 0.02 s
1 for the D39N Fe
protein. Clearly, the D39N Fe protein transfers an electron to the MoFe
protein, although at a much slower rate when compared with the
wild-type Fe protein. No electron transfer was observed for the D39E Fe
protein (data not shown).
It is noteworthy to compare the 350 nm region of the spectra in Fig. 3. This region of the spectrum is dominated by the absorbance of the reductant dithionite. In the case of the wild-type nitrogenase mixture, the absorbance at 350 nm decreased rapidly, corresponding to the nitrogenase-catalyzed oxidation of dithionite coupled to the reduction of protons. In the case of the D39N Fe protein, no decrease in the absorbance in the 350 nm region was observed even after the D39N Fe protein was fully oxidized. This observation suggests two important points. First, the D39N Fe protein does not undergo multiple rounds of electron transfer to the MoFe protein, but rather a single electron transfer. Second, the fact that the D39N Fe protein becomes fully oxidized in the presence of excess dithionite suggests that the D39N Fe protein is protected from reduction. This could be explained by formation of a tight complex between the D39N Fe protein and the MoFe protein. Previous studies have established that when the oxidized Fe protein is bound to the MoFe protein, it is protected from reduction by dithionite (11).
Additional support for the observation of a single electron transfer
from the D39N Fe protein to the MoFe protein comes from an examination
of the MoFe protein concentration dependence of D39N oxidation
(Fig. 4). As can be seen, the maximum extent of oxidation of the D39N Fe protein occurred at a molar ratio of 0.52 MoFe
proteins to one D39N Fe protein. This is consistent with the MoFe
protein having two Fe protein binding sites (39). In addition, these
data clearly show that the oxidation of the D39N Fe protein requires
stoichiometric amounts of MoFe protein, supporting a single electron
transfer between the proteins.
It seemed possible that the oxidation of the D39N Fe protein by the MoFe protein could be catalyzed by traces of active, wild-type Fe protein that might act as a mediator for electron transfer between the D39N Fe protein and the MoFe protein. To test this possibility, traces of wild-type Fe protein were added to the D39N Fe protein-MoFe protein reaction mixture. No changes in the rate or extent of electron transfer from the D39N Fe protein to the MoFe protein was observed, ruling out wild-type Fe protein mediation of electron transfer.
Finally, the transfer of an electron from the D39N Fe protein to the MoFe protein was confirmed by monitoring with EPR spectroscopy (data not shown). In these studies, the reduced D39N or wild-type Fe proteins was mixed with MgATP and the MoFe protein and allowed to react for 1 h before being frozen in EPR tubes. Both the D39N and wild-type Fe proteins showed a MgATP-dependent disappearance of the S = 1/2 EPR signal consistent with the oxidation of the Fe protein [4Fe-4S] cluster from the 1+ to the 2+ oxidation state. There was also a small decrease in the intensity of the S = 3/2 region EPR signal, consistent with the reduction of FeMoco.
Affinity of Fe Protein Binding to the MoFe ProteinTo
quantify the affinity of binding of oxidized D39N Fe protein to the
MoFe protein, stopped-flow spectrophotometry was used to define the
dissociation rate constants for Fe protein-MoFe protein complexes. In
this experiment, Fe protein is oxidized with a mediator
(e.g. indigo disulfonate) prior to the addition of reduced,
but dithionite-free MoFe protein and MgADP. This solution is rapidly
mixed in the stopped-flow apparatus against a solution containing
reduced Fe protein ([4Fe-4S]1+), dithionite, and MgADP.
As the oxidized Fe protein dissociates from the MoFe protein, it is
reduced by the dithionite, resulting in a decrease in absorbance at 430 nm. Thus, the decrease in absorbance at 430 nm can be directly
correlated with the dissociation of the Fe protein from the MoFe
protein (11). The results of such an experiment with IDS-oxidized
wild-type or D39N Fe proteins are shown in Fig. 5
(panel A). As can be seen, both the wild-type and D39N Fe
proteins showed similar rates of dissociation from the MoFe protein,
with apparent first order rate constants of 6 s1 for the
wild-type Fe protein and 3 s
1 for the D39N Fe protein.
These experiments were also performed over a range of Fe protein
concentrations from 50 to 250 µM as described previously
(11).
These results suggest that the IDS-oxidized D39N Fe protein with bound
MgADP does not bind to the MoFe protein with a significantly higher
affinity than does the wild-type Fe protein. This is in contrast to the
suggestion of a tight complex between the oxidized D39N Fe protein and
the MoFe protein from the earlier steady-state spectroscopic data. A
significant difference between the steady-state data and the
stopped-flow spectroscopic data is the method of oxidation of the D39N
Fe protein. In the stopped-flow experiment, the Fe proteins were
oxidized first to the 2+ state with a dye (i.e. IDS) and
then incubated with MgADP and the MoFe protein. In the steady-state
experiments, the Fe proteins were oxidized by electron transfer
directly to the MoFe protein under turnover conditions. It seemed
possible that the electron transfer from the Fe protein to the MoFe
protein as part of a complex might result in a tighter protein-protein
complex, explaining the different results between the two experiments.
To test this possibility, the stopped-flow dissociation experiment was
repeated, except that the Fe proteins were oxidized under turnover
conditions by incubation with MgATP and the MoFe protein for 30 min
prior to rapidly mixing with dithionite and the reduced Fe protein. As can be seen in Fig. 5 (panel B), the wild-type Fe protein
showed an apparent first order dissociation rate constant from the MoFe protein of 5 s1. This is essentially identical to the
rate constant observed in the experiment where the Fe protein was
IDS-oxidized. In contrast, the D39N Fe protein showed essentially no
dissociation from the MoFe protein when turnover oxidized. This
contrasts with the rapid dissociation of the D39N Fe protein from the
MoFe protein when the D39N Fe protein was oxidized by IDS. It is
important to note that in both cases, the Fe protein is oxidized by one
electron to the 2+ state and that MgATP hydrolysis continues. In a
control reaction, incubation of the IDS-oxidized D39N Fe protein with MgATP and the MoFe protein did not result in a tight Fe protein-MoFe protein complex, indicating that MgATP hydrolysis was not sufficient to
allow D39N Fe protein-MoFe protein tight complex formation. These
results provide compelling evidence that the D39N Fe protein forms a
tight, essentially irreversible complex with the MoFe protein only
following electron transfer to the MoFe protein.
To understand why the D39N Fe protein formed a tight complex with the MoFe protein only under conditions of electron transfer and MgATP hydrolysis, it was important to understand how nucleotide-induced conformational changes were affected in the D39N altered Fe protein. A number of spectroscopic techniques have been used to monitor the MgATP- or MgADP-induced protein conformational changes in the environment of the [4Fe-4S] cluster. The visible region CD spectra of the oxidized Fe protein provides the only spectroscopic technique that gives distinct spectra for the three states of the oxidized Fe protein, namely the nucleotide-free, the MgATP-bound, and the MgADP-bound states (17, 18).
Fig. 6 shows the CD spectra for the wild-type, D39N, and
D39E Fe proteins in the absence of nucleotides (panel A), in
the presence of MgATP (panel B), and in the presence of
MgADP (panel C). As can be seen, all three proteins showed
very similar CD spectra in the absence of nucleotides, indicating that
changing Asp-39 to Asn or Glu did not have a significant impact on the [4Fe-4S] cluster environment. Likewise, the addition of MgATP to all
three proteins resulted in similar CD spectra (panel B), consistent with previously reported spectra for the MgATP-bound state
of wild-type Fe protein (18). This revealed that all three proteins
underwent similar MgATP-induced protein conformational changes around
the [4Fe-4S] cluster, suggesting that Asp-39 is not critical for
communicating the MgATP-induced conformational change to the [4Fe-4S]
cluster. In marked contrast, the wild-type and Asp-39 altered Fe
proteins showed very different CD spectra when MgADP was added.
Notably, the CD spectrum of the D39N Fe protein was most similar to the
spectra observed for the Fe protein with MgATP bound. These results
indicate that changing Asp-39 to Asn has had a significant impact on
the MgADP-induced conformational changes communicated to the [4Fe-4S]
cluster.
As another, more global way to monitor the nucleotide-induced
conformational changes in the Fe protein, the proton NMR spectra for
the wild-type and Asp-39 altered Fe proteins were compared (Fig. 7). The downfield portion of the proton NMR
spectrum of the reduced Fe protein contains resonances from eight
-CH2 and four
-CH protons of the four cysteinyl
ligands to the [4Fe-4S] cluster, which appear as four isotropically
shifted peaks between 13 and 50 ppm (20). These proton resonances are
primarily shifted by through bond interactions with the paramagnetic
[4Fe-4S]1+ cluster and are thus sensitive to distance and
orientation relative to the cluster. Both MgATP and MgADP binding to
the wild-type Fe protein have been shown to result in changes in the
chemical shifts of these proton resonances, reflecting changes induced in the protein environment around the cluster (20). In the absence of
nucleotides, the downfield proton NMR spectrum of the D39N and D39E
altered Fe proteins were found to be nearly identical to that of the
wild-type Fe protein (Fig. 7, panel A). Upon the addition of
MgATP, the D39E, D39N, and wild-type Fe proteins showed nearly
identical shifts in
-CH2 proton (49 and 23 ppm)
resonances (Fig. 7, panel B). Notably, only the D39N Fe
protein demonstrated a significant shift in the
-CH protons (17-13 ppm) that was similar to the wild-type Fe protein. Upon the addition of
MgADP, both the D39N and D39E Fe proteins showed very small shifts in
the proton resonances, in contrast to the substantial shifts observed for the wild-type Fe protein. Differences between the MgADP-bound state
of the D39N and D39E Fe protein NMR spectra were evident for the
-CH
protons (Fig. 7, panel C). These results suggest different
conformational changes between the D39N and D39E Fe proteins upon the
addition of MgATP and MgADP and further confirm the lack of
MgADP-induced conformational changes in the D39N Fe protein upon
binding MgADP when compared with the wild-type Fe protein.
As mentioned earlier, an important reaction in the catalytic mechanism of nitrogenase is the dissociation of the Fe protein from the MoFe protein following MgATP hydrolysis and electron transfer. This has been suggested to be the overall reaction rate-limiting step. The results from the present study can be discussed in the context of two important questions relating to the signal for dissociation of the nitrogenase proteins. (i) What is the role of MgATP hydrolysis in signaling protein-protein dissociation, and (ii) what is the role of electron transfer from the Fe protein to the MoFe protein in signaling protein-protein dissociation?
MgATP HydrolysisIt is becoming clear that MgATP serves
several important functions in the nitrogenase reaction mechanism (2,
3). MgATP binding to the Fe protein clearly induces protein
conformational changes that are communicated from the nucleotide
binding sites at the center of the protein to the [4Fe-4S] cluster
and MoFe protein docking surface approximately 19 Å away (2, 14). These conformational changes dramatically alter both the
physicochemical properties of the [4Fe-4S] cluster and the binding
equilibrium in favor of the Fe protein-MoFe protein complex (15). In
addition to influencing the initial binding of the Fe protein to the
MoFe protein, it has been held that MgATP hydrolysis is somehow coupled to the transfer of an electron from the Fe protein [4Fe-4S] cluster to the MoFe protein (3). This model has been challenged recently by the
observation that an altered Fe protein locked in the MgATP-like state
(L127) could transfer one electron to the MoFe protein in the
absence of MgATP hydrolysis, thus suggesting that only the MgATP-bound
protein conformation of the Fe protein was required for electron
transfer, not necessarily MgATP hydrolysis (15). It is important to
note that the rate of electron transfer from the L127
Fe protein was
significantly slower than that observed from the wild-type Fe protein
with MgATP hydrolysis, suggesting that one possible role for MgATP
hydrolysis is to accelerate electron transfer. How then is MgATP
hydrolysis initiated? Since the Fe protein alone shows no MgATP
hydrolysis activity, it can be concluded that MoFe protein docking to
the Fe protein initiates MgATP hydrolysis. Several models have been
suggested to explain this MoFe protein dependence of MgATP hydrolysis,
the most likely being that residues within the Fe protein nucleotide
binding site are moved into position to catalyze the hydrolysis
reaction (2, 8). It would seem reasonable that this would occur by long
range protein conformational changes from the MoFe protein docking
surface back to the nucleotide binding sites. The results of the
present work clearly indicate that Asp-39 cannot be an essential
residue (e.g. general base) in the MgATP hydrolysis
mechanism as changing the Asp to Asn did not stop MgATP hydrolysis
activity. The significantly slower rate of MgATP hydrolysis catalyzed
by the D39N Fe protein-MoFe protein complex suggests that Asp-39 could
participate either directly or indirectly in positioning residues
critical for MgATP hydrolysis.
Taken together, these observations suggest that an important function
for MgATP hydrolysis in the nitrogenase mechanism might be to induce
protein conformational changes that would be coupled to protein-protein
dissociation. The results from the present study support the notion (2)
that the change in protein conformation in the Fe protein in going from
the MgATP-bound state to the MgADP-bound state during MgATP hydrolysis
could dramatically alter the interactions (and hence affinity) between
the Fe protein and the MoFe protein by altering the position of a
series of amino acid residues involved in protein-protein interaction.
Altering Asp-39 to Asn resulted in an Fe protein that could not access
the MgADP-bound protein conformation as detected by changes in the CD
and proton NMR spectra of the [4Fe-4S] cluster. The resulting tight
complex between the D39N Fe protein and the MoFe protein could be the
result of the lack of the Fe protein attaining the MgADP-bound protein
conformation, even though MgATP is hydrolyzed to MgADP as part of the
complex. Together with the observation of a tight complex between the
L127 modified Fe protein and the MoFe protein, this suggests that
the MgADP-bound state of the Fe protein has a significantly reduced affinity for binding to the MoFe protein, essentially functioning as a
trigger for protein-protein dissociation. This idea is supported by the
recent observation that if AlF4
was
included with nitrogenase under turnover conditions, a tight, protein-protein complex was formed (25, 40).
AlF4
is thought to be a phosphate
analog and to bind to the Fe protein nucleotide site as an
ADP-AlF4
complex, preventing the
formation of the MgADP-bound state required for complex dissociation.
Hence, the Fe protein in the ADP-AlF4
bound conformation cannot achieve the correct conformation for dissociation (25, 40). A reasonable model would be that one important
function of MgATP hydrolysis is as a trigger to alter the Fe
protein-MoFe protein equilibrium to favor protein-protein dissociation.
Whereas the above model for the function of MgATP hydrolysis in protein-protein dissociation is reasonable, results from the present studies suggest that it is not that simple. Rather, it appears that electron transfer is also somehow coupled to protein-protein dissociation. The results from the present work with the D39N altered Fe protein clearly point to a significant role for electron transfer in defining the affinity for Fe protein binding to the MoFe protein. This comes from the observation that only following electron transfer from the D39N Fe protein to the MoFe protein did a tight, irreversible protein-protein complex form. MgATP hydrolysis to MgADP and Pi continued in both the reduced and oxidized D39N Fe protein-MoFe protein complexes, suggesting that MgATP hydrolysis is not a sufficient signal to initiate protein-protein dissociation. Instead, it would appear that electron transfer is linked to the nucleotide-bound status of the Fe protein to trigger dissociation. This suggests a complex mechanism for protein dissociation in which electron transfer induces protein conformational changes within the Fe protein (and possibly the MoFe protein), which are linked to conformational changes induced by MgATP hydrolysis. There is growing precedent for oxidation or reduction (i.e. electron transfer) of redox active proteins resulting in significant protein conformational changes. For example, flavodoxins (41, 42), cytochrome c (43), and the [Fe-S] cluster containing adrenodoxin (44) have all been observed to undergo significant protein conformational changes upon oxidation or reduction. In addition, extended x-ray absorption fine structure data for the nitrogenase Fe protein has revealed a 0.02-Å shortening of the Fe-S bond distance in the [4Fe-4S] cluster upon oxidation from the 1+ to the 2+ state (45). This change in the [4Fe-4S] cluster could easily be translated into changes in the protein environment around the cluster, thus affecting the affinity of the Fe protein for the MoFe protein.
So, a reasonable model would suggest that electron transfer from the Fe protein to the MoFe protein results in the oxidation of the Fe protein [4Fe-4S] cluster, inducing protein conformational changes affecting the affinity for MoFe protein docking. This event might also initiate the signal for MgATP hydrolysis, consistent with recent data suggesting that MgATP hydrolysis follows electron transfer (9). Once in the [4Fe-4S] cluster oxidized state, MgATP hydrolysis then initiates the nucleotide-induced protein conformational changes as the protein goes to the MgADP-bound state, which then favor protein-protein dissociation. Asp-39, being located near the MgATP binding site, may initiate a communication pathway that then proceeds to a region of the Fe protein involved in MoFe protein interactions. This communication pathway may be a second such pathway identified in the Fe protein, going from Asp-39 to Val-67. This putative domain in the Fe protein is highly homologous with similar domains in other nucleotide binding proteins, which have been designated as switch I (26). Thus, as has been suggested for several other nucleotide switch proteins, the nucleotide status of the protein results in different movements in the protein chain switch I, which are communicated over a distance to the binding site for another protein (26). In the Fe protein, it seems reasonable that the MgADP- or MgATP-bound state could be communicated to amino acid residues in the 59 to 67 region critical for docking with the MoFe protein. The 59 to 67 region of the Fe protein has recently been suggested to participate in MoFe protein interactions (27).
In summary, the present work provides evidence that the conserved Asp at position 39 of the nitrogenase Fe protein plays an important role in defining a shift in equilibrium toward Fe protein-MoFe protein dissociation. This could be communicated through a protein domain designated as switch I, which might originate at Asp-39. In addition, evidence was presented suggesting that electron transfer from the Fe protein to the MoFe protein within the complex induces protein conformational changes that are critical in defining the protein-protein binding equilibrium.
We thank Professor L. E. Mortenson, Dr. M. E. Rasche, M. J. Ryle, and J. M. Chan for helpful discussions and Professor W. E. Newton for use of the stopped-flow spectrophotometer and advice on the rapid reaction kinetic studies.