(Received for publication, July 7, 1995; and in revised form, November 9, 1995)
From the
Nucleotide interactions with nitrogenase are a central part of
the mechanism of nitrogen reduction. Previous studies have suggested
that MgATP or MgADP binding to the nitrogenase iron protein (Fe
protein) induce protein conformational changes that control component
protein docking, interprotein electron transfer, and substrate
reduction. In the present study, we have investigated the effects of
MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S]
cluster using circular dichroism (CD) and x-ray absorption
spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe
protein suggested that binding of either MgATP or MgADP to the Fe
protein resulted in identical changes in the CD spectrum arising from
transitions of the [4Fe-4S] cluster. We
present evidence that MgADP or MgATP binding to the oxidized
nitrogenase Fe protein results in distinctly different CD spectra,
suggesting distinct changes in the environment of the
[4Fe-4S] cluster. The present results are consistent with
previous studies such as chelation assays, electron paramagnetic
resonance, and NMR, which suggested that MgADP or MgATP binding to the
nitrogenase Fe protein induced different conformational changes. The CD
spectrum of a [2Fe-2S]
form of the
nitrogenase Fe protein was also investigated to address the possibility
that the MgATP- or MgADP-induced changes in the CD spectrum of the
native enzyme were the result of a partial conversion from a
[4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence
was found for a contribution of a [2Fe-2S]
cluster to the CD spectrum of oxidized Fe protein in the absence
or presence of nucleotides. A novel two-electron reduction of the
[2Fe-2S]
cluster in Fe protein was apparent
from absorption, CD, and electron paramagnetic resonance data. Fe
K-edge x-ray absorption spectra of the oxidized Fe protein revealed no
changes in the structure of the [4Fe-4S] cluster upon MgATP
binding to the Fe protein. The present results reveal that MgATP or
MgADP binding to the oxidized state of the Fe protein result in
different conformational changes in the environment around the
[4Fe-4S] cluster.
Nitrogenase catalyzes the biological reduction of nitrogen according to the overall reaction.
A detailed description of the function of MgATP hydrolysis in
the nitrogenase substrate reduction mechanism has yet to
emerge(1, 2, 3, 4, 5) . The
current model suggests that MgATP hydrolysis is coupled to electron
transfer between the nitrogenase component proteins, the iron protein
(Fe protein), ()and the molybdenum-iron protein (MoFe
protein). It is known that the Fe protein can bind two MgATP molecules
and that this binding results in protein conformational changes
essential to Fe protein docking to the MoFe
protein(4, 5, 6, 7) . The series of
events following the docking of the Fe protein to the MoFe protein are
not well understood, but it has been proposed that the hydrolysis of
two MgATP molecules on the Fe protein precedes the transfer of an
electron from the Fe protein [4Fe-4S] cluster to the MoFe
protein for substrate reduction(8, 9) . Under optimal
conditions, the dissociation of the oxidized Fe protein, with two bound
MgADP molecules, is the rate-limiting step of the
reaction(10, 11) . Current models suggest that the
above sequence of events are repeated until sufficient electrons have
been transferred to the MoFe protein to carry out the reduction of the
bound substrate.
An understanding of any protein conformational
changes induced in the nitrogenase Fe protein upon binding either MgATP
or MgADP is essential to the elucidation of a detailed mechanism of
nucleotide-bound conformational changes and the mechanism of coupling
of MgATP hydrolysis to electron transfer and substrate reduction by
nitrogenase. A significant consequence of nucleotide binding to the Fe
protein are changes in the properties of its [4Fe-4S]
cluster(4, 5) . The [4Fe-4S] cluster is
bridged between the identical subunits of the Fe protein some 19
Å away from the two nucleotide binding sites(12) . Thus,
nucleotide binding-induced changes in the [4Fe-4S] cluster
are most likely communicated through protein conformational changes.
Changes in the [4Fe-4S] cluster upon the binding of
nucleotides to the Fe protein have been monitored by several
techniques. For example, the [4Fe-4S] cluster becomes
sensitive to a time-dependent chelation by iron-specific chelators upon
Fe protein binding MgATP(13, 14) . MgADP binding to
the Fe protein does not result in an increased rate of chelation.
Likewise, MgATP binding to the reduced Fe protein has been shown to
result in changes in the lineshape of the EPR spectrum of the
[4Fe-4S] cluster, which converts from rhombic to axial
forms(15, 16) . MgADP binding to the Fe protein,
however, does not result in changes in the EPR spectrum. These studies
suggested that MgATP binding to the Fe protein results in significant
changes in the environment of the [4Fe-4S] cluster, but
provided little information about any MgADP-induced changes. It has
been found that MgATP binding to the Fe protein lowers the redox
potential of the 2/1
couple of the
[4Fe-4S] cluster from -310 to -430 mV (versus the standard hydrogen electrode) (15, 17) . MgADP
binding to the Fe protein results in the same lowering of the reduction
potential, clearly suggesting that MgADP binding does affect the
properties of the [4Fe-4S] cluster. While these results
demonstrated an effect on the [4Fe-4S] cluster by both MgADP
and MgATP binding, little information exists to distinguish between
MgATP- and MgADP-induced changes in the [4Fe-4S] cluster.
Proton NMR spectra of the reduced Fe proteins from Clostridium
pasteurianum(18) and Azotobacter vinelandii(19) revealed a series of isotropically shifted proton
resonances arising from cysteinyl protons, which are ligands to the
paramagnetic [4Fe-4S]
cluster. The chemical
shifts of these protons were sensitive to MgATP or MgADP binding to the
Fe protein, providing evidence for differences in the cluster
environment upon binding of either nucleotide.
Circular dichroism spectroscopy in the visible wavelength region is a useful way to monitor the type and environment of [Fe-S] clusters in proteins(20, 21, 22, 23, 24) . The circular dichroism spectrum of proteins containing [Fe-S] clusters is more structured and much more sensitive to changes in environment than the corresponding visible absorption spectra(20) . Thus, CD spectroscopy should provide a useful method to monitor changes in the environment of the [4Fe-4S] cluster upon nucleotide binding to the Fe protein. Previous CD studies of nitrogenase proteins from a variety of organisms have been carried out (20, 21, 22, 25, 26, 27) . These studies showed that the CD of the Fe protein is measurable but weak in both oxidation states of the [4Fe-4S] cluster. Spectra from C. pasteurianum, Klebsiella pneumonia, and A. vinelandii were essentially identical, leading to the conclusion that the [4Fe-4S] cluster environment of these proteins is highly conserved. These studies further demonstrated that the oxidized Fe protein CD spectrum changed upon binding MgATP or MgADP. Both MgADP and MgATP binding were found to result in essentially the same CD spectrum(22, 26, 28) .
In
order to define the function of MgATP binding and hydrolysis in the
nitrogenase mechanism in conjunction with studies using
site-specifically altered Fe
proteins(6, 7, 8, 29, 30) ,
we have re-examined the CD spectra of nitrogenase Fe protein in the
absence or presence of MgATP or MgADP. In accord with the earlier
studies, we find a distinctive CD spectrum for the oxidized Fe protein
and changes in this spectrum upon binding of nucleotides. However,
unlike the earlier CD studies, we have found that MgATP or MgADP
binding to the Fe protein result in very different CD spectra. These
results clearly show that MgATP or MgADP binding to the Fe protein are
communicated as different changes in the environment of the
[4Fe-4S] cluster and demonstrate that CD is a sensitive way
to monitor these changes. Resonance Raman studies of the
[4Fe-4S] cluster of the Fe protein (31) have suggested that MgATP binding to the Fe protein
results in different resonance bands that could be modeled by the
partial conversion of the Fe protein [4Fe-4S] cluster to a
[2Fe-2S] cluster. Since proteins containing
[2Fe-2S] clusters (with four Cys ligands) generally have more
intense CD spectra relative to proteins containing [4Fe-4S]
clusters, it was suggested that the change in the CD spectrum of the Fe
protein observed upon binding MgATP was the result of the partial
conversion to a [2Fe-2S] cluster(31) . To test this
possibility, we have prepared a [2Fe-2S] cluster form of the
Fe protein using a published procedure (32) and examined it by
absorption, CD, and EPR spectroscopy. CD spectra of plant/algal type
[2Fe-2S] cluster containing proteins, unlike those exhibited
by other types of protein bound [Fe-S] clusters, are
relatively insensitive to protein environment(20) . The
[2Fe-2S] cluster containing Fe protein exhibited CD spectra
very similar to the classical plant/algal ferredoxin [2Fe-2S]
type, which does not resemble the nucleotide-bound spectra of the
[4Fe-4S] form of the Fe protein. Therefore, we can preclude
any contribution of a [2Fe-2S]
cluster to
the observed CD spectrum of the [4Fe-4S] cluster form of the
Fe protein in the absence or presence of MgADP or MgATP. Fe K-edge XAS
studies of the [4Fe-4S]
cluster of the
oxidized Fe protein were also performed and suggested no major
structural changes in the cluster upon Fe protein binding MgATP.
Finally, a novel two-electron reduction of the
[2Fe-2S]
cluster in the Fe protein by
dithionite was inferred from dithionite titration experiments using
absorption, CD, and EPR spectroscopies.
Reduction of the
[2Fe-2S] Fe protein was accomplished by the
addition of aliquots of a calibrated dithionite
(Na
S
O
) solution in 50 mM Tris buffer, pH 8.0. The dithionite concentration of the stock
solution was quantified by titration with redox dyes(34) . An
aliquot of the dithionite solution was added to an anaerobic solution
of methylene blue or potassium ferricyanide in 50 mM Tris
buffer, pH 8.0. From the change in the absorption spectra of the dye
and the known absorption coefficients (32.8
mM
cm
at 600 nm for
methylene blue and 1.0
mM
cm
at 420 nm for
potassium ferricyanide), the dithionite concentration was determined to
be 2.3 mM(34) .
Data analysis using
amplitude and phase functions derived from FEFF 5.05 (or 6)
calculations (35, 36) was as described previously (37) except that the least-squares residual reported
() and the method for estimating
parameter uncertainties now follow recommendations of the International
Workshops on Standards and Criteria in XAFS(38) . New amplitude
and phase functions were derived for Fe-S and Fe-Fe interactions using
the crystallographic coordinates of
[(CH
)
N]
[Fe
S
(SPh)
] (39) . Fitting EXAFS of this model complex, iron metal, and low
and room temperature samples of Fe(S
CNEt
)
(40) revealed that the FEFF-generated functions for Fe-S
and Fe-Fe overestimate the amplitude of the EXAFS, but good fits (with n within ± 20% of the true value and r within
0.01 Å of crystallography) can be obtained by using empirical
amplitude reduction factors of 0.75 and 0.55, respectively, and E
= 7122 eV. For plots and weighting, k is calculated using 7125 eV(37) . Disorder factors,
, are relative to the disorder calculated by
FEFF using Debye temperature of 100 K.
We have measured a CD spectrum for indigo disulfonate-oxidized Fe
protein from A. vinelandii (Fig. 1, trace 1), which
is essentially identical to that previously reported for thionine
oxidized Fe protein(21, 22) . The addition of MgADP to
a final concentration of 1 mM resulted in a significant change
in the CD spectrum (Fig. 1, trace 3), consistent with
previously observed CD spectra(21, 22) . Titration of
the oxidized Fe protein with MgADP revealed a conversion from the
oxidized Fe protein CD to the MgADP-bound form CD over a MgADP
concentration range from 0 to 150 µM (Fig. 2A).
The change in the CD spectrum was saturated by 150 µM MgADP and did not change further when the MgADP concentration was
increased to 1 mM. Two isosbestic points (at 380 and 450 nm)
were observed, suggesting that only two states contribute to the CD.
The concentration dependence of the CD change is consistent with
dissociation constants (K) for MgADP binding to Fe
protein (143 µM), previously measured by direct binding
methods(30) .
Figure 1:
Circular dichroism spectra of oxidized A. vinelandii Fe protein in the absence or presence of MgATP
or MgADP. CD spectra of IDS oxidized nitrogenase Fe protein (Fe
protein) in 100 mM Tris buffer, pH 8.0, were
recorded as described under ``Materials and Methods.'' Trace 1, Fe protein
; trace 2, Fe
protein
with 1 mM MgATP; trace 3, Fe
protein
with 1 mM MgADP. The molar absorption
coefficient (
) was plotted against the wavelength. All
spectra were base line subtracted.
Figure 2:
Circular dichroism spectra of oxidized
nitrogenase Fe protein titrated with MgATP or MgADP. Circular dichroism
spectra of IDS oxidized nitrogenase Fe protein in 100 mM Tris,
pH 8.0, were recorded as described under ``Materials and
Methods.'' Panel A, CD spectra of Fe protein in the absence (trace 1) or presence of 75 µM (trace 2), 150 µM (trace 3), or 1
mM (trace 4) MgADP. Panel B, CD spectra of
Fe protein
in the absence (trace 1) or presence
of 75 µM (trace 2), 150 µM (trace 3), 300 µM (trace 4), or 1
mM (trace 5) MgATP.
The addition of MgATP to the oxidized Fe
protein resulted in a significant change in the CD spectrum (Fig. 1, trace 2), which was dramatically different
from that observed in the absence of nucleotides or in the presence of
MgADP. This spectrum was clearly different from the CD previously
reported for oxidized Fe protein in the presence of
MgATP(21, 22) . It is concluded from the present data
that the MgADP and MgATP bound forms of the oxidized Fe protein undergo
significant but different conformational changes in the environment of
the [4Fe-4S] cluster. CD titrations of the Fe protein with
MgATP were carried out (Fig. 2B). Conversion of the
oxidized Fe protein CD to the MgATP-bound Fe protein CD spectrum
occurred over a range of MgATP concentrations from 0 to 150
µM. The CD spectrum did not undergo further change when
MgATP was added up to a concentration of 1 mM. An isosbestic
point was observed in the titration at approximately 380 nm, suggesting
that only two states contribute to the CD. It should be noted that
previously reported CD titrations of Fe protein with MgATP did not
reveal isosbestic points (26) , suggesting the possibility that
multiple species contributed to the CD spectrum. The previous CD
studies of Fe protein were done at pH 7.4 with dithiothreitol and
excess Mg(21, 22) , while the
present studies have been done at pH 8.0 without dithiothreitol or
excess Mg
. To determine if these different conditions
could account for the dramatically different CD spectrum observed for
the MgATP bound state, we recorded CD spectra for Fe protein prepared
under conditions used in the previous studies. Fig. 3reveals
that the CD observed for the three states of the Fe protein (oxidized,
+MgATP or +MgADP) are essentially identical to the spectra
recorded at pH 8.0 (Fig. 2). Clearly, the differences in the CD
spectra of the MgATP-bound Fe protein presented here compared with
previous works (21, 22, 25) cannot be
accounted for by differences in pH or dithiothreitol and Mg
addition. In our studies, the MgATP-bound state of the Fe protein
could be converted to the MgADP-bound state by addition of excess MgADP
to a sample containing 150 µM MgATP. Finally, samples were
checked for activity following the acquisition of CD and in all cases
were found to retain at least 85% of the original activity.
Figure 3:
Circular dichroism spectra of oxidized
nitrogenase Fe protein at pH 7.4 in the absence or presence of
nucleotides. Circular dichroism spectra of IDS oxidized nitrogenase Fe
protein in 25 mM HEPES buffer, pH 7.4, with 0.1 mg/ml
dithiothreitol and 2 mM MgCl were recorded as
described under ``Materials and Methods.'' Trace 1,
Fe protein
; trace 2, Fe protein
with
1 mM MgADP; trace 3, Fe protein
with 1
mM MgATP. All spectra were base line
subtracted.
We have observed that when an oxidized Fe protein sample with MgATP at pH 7.0 was allowed to sit for several hours at 25 °C, a slow conversion from the MgATP-bound CD spectrum to the MgADP-like CD spectrum was observed. It seemed reasonable to conclude that over long time periods, MgATP could be hydrolyzed to MgADP. We have confirmed that partially pure Fe protein samples hydrolyze a significant amount of MgATP to MgADP. Additional purification of Fe protein to a more homogeneous state was found to significantly reduce the hydrolysis of MgATP while increasing the specific activity of the Fe protein. Thus, it seems reasonable that in previous CD studies (21, 22, 25, 26) a portion of the added MgATP was hydrolyzed to MgADP during the course of the experiment, accounting for the MgADP-like CD spectra upon addition of MgATP or MgADP and the lack of isosbestic points in titrations with MgATP.
The results of the present study, therefore, reveal that MgATP or MgADP binding to the Fe protein result in distinct conformational changes in the [4Fe-4S] cluster, which can be monitored by CD spectroscopy.
To determine if the formation of a
[2Fe-2S] cluster could account for the CD spectrum recorded
for oxidized Fe protein or the nucleotide bound forms of oxidized Fe
protein, we prepared the [2Fe-2S] form of A. vinelandii Fe protein and measured the CD spectrum. Fig. 4shows the
CD spectrum for the oxidized, [2Fe-2S] form of the Fe protein (trace 4) compared with CD spectra recorded for the
[4Fe-4S] form of Fe protein in the absence
or presence of MgATP or MgADP. It is clear from these results that the
CD spectra observed for the [4Fe-4S]
states
of the Fe protein, either with or without nucleotides, are not the
result of a conversion to a [2Fe-2S]
cluster but would rather be consistent with changes in the
environment of the [4Fe-4S] cluster upon Fe protein binding
nucleotides. The CD spectra observed for the [2Fe-2S] form is
very similar to the CD spectra exhibited by plant/algal type
[2Fe-2S] cluster containing
ferredoxins(20, 24) , confirming that the
[2Fe-2S] cluster in this form of the Fe protein is of this
type.
Figure 4:
Comparison of the circular dichroism
spectra of oxidized Fe protein with the [2Fe-2S] form of Fe
protein. Circular dichroism spectra of IDS oxidized
nitrogenase Fe protein and preparation of the oxidized
[2Fe-2S] form of the Fe protein were as described under
``Materials and Methods.'' Oxidized Fe protein in the absence (trace 1) or presence of 1 mM MgADP (trace
2) or 1 mM MgATP (trace 3) or the oxidized
[2Fe-2S] form of Fe protein in the absence of nucleotides (trace 4).
The [2Fe-2S] cluster form of the Fe protein was confirmed by its absorption spectra, which also exhibit features characteristic of plant/algal ferredoxin type [2Fe-2S] clusters (20, 24) and by total iron analysis(6) . The [4Fe-4S] cluster containing Fe protein was found to contain 3.5 ± 0.2 Fe/protein, while the [2Fe-2S]-form was found to contain 1.7 ± 0.2 Fe/protein. We found that addition of MgATP or MgADP to the [2Fe-2S] form of the Fe protein did not result in any noticeable changes in the CD spectrum. It is likely, however, as noted above, that the CD spectra of the [2Fe-2S] form will be relatively insensitive to the same kinds of protein conformational changes to which the [4Fe-4S] cluster form is sensitive. We also examined CD spectra for the reduced state of the [2Fe-2S] form of the nitrogenase Fe protein. The CD spectrum of dithionite reduced [2Fe-2S] cluster containing Fe protein was not of the plant/algal ferredoxin type, and was generally quite featureless. The absorption spectrum, furthermore, was bleached on addition of dithionite by approximately 80 percent at 400 nm, about twice the bleaching typical of one electron reduction of a plant/algal type [2Fe-2S] cluster(20) . Titration of this protein with dithionite (Fig. 5) revealed two isosbestic points (at 420 and 520 nm) in the CD, suggesting conversion from the oxidized state to a single reduced state. The titration was observed to be complete upon the addition of one molar equivalent of dithionite. Since dithionite is a two-electron reductant, this suggested that complete reduction of the nitrogenase Fe protein [2Fe-2S] cluster required the addition of two electrons without a significant population of a one-electron reduced intermediate. This reduction stoichiometry was independently determined by titration of the Fe protein [2Fe-2S] cluster by absorption spectroscopy (Fig. 6). The fully reduced [2Fe-2S] form of the Fe protein could be reoxidized to the original absorption spectrum, confirming the reversibility of the redox reaction.
Figure 5: Circular dichroism spectra of the oxidized [2Fe-2S] form of nitrogenase Fe protein titrated with sodium dithionite. Circular dichroism spectra and preparation of the oxidized [2Fe-2S] form of nitrogenase Fe protein were as described under ``Materials and Methods.'' The concentration of the [2Fe-2S] form of Fe protein was 56 µM. Spectra were recorded in the absence of added sodium dithionite (trace 1) and following the addition of sodium dithionite to final concentrations of 10.4 µM (trace 2), 20.8 µM (trace 3), 31.2 µM (trace 4), 41.6 µM (trace 5), and 52 µM (trace 6).
Figure 6: Absorption spectra of the oxidized [2Fe-2S] form of nitrogenase Fe protein titrated with sodium dithionite. Absorption spectra of the [2Fe-2S] form of nitrogenase Fe protein were recorded in 100 mM Tris buffer, pH 8.0, and the concentration of dithionite in the stock solution was determined as described under ``Materials and Methods.'' The concentration of the [2Fe-2S] form of the Fe protein was 44 µM. Spectra were recorded in the absence of added sodium dithionite (trace 1) and following the addition of sodium dithionite to final concentrations of 6.9 µM (trace 2), 13.7 µM (trace 3), 20.5 µM (trace 4), 27.3 µM (trace 5), 34 µM (trace 6), 40.7 µM (trace 7), 47.3 µM (trace 8), 53.9 µM (trace 9), and 60.5 µM (trace 10). Spectra were recorded until no further change was observed. Inset, the change in absorbance at 400 nm was plotted against the electron equivalents added per protein.
Recently, Im et al.(42) revealed the first reported case of a two-electron reduction of a [2Fe-2S] cluster in a ferredoxin using a novel Cr(II) macrocycle as reductant. At 400 nm, the absorption spectrum of the ferredoxin is 80% bleached relative to the oxidized state. This bleaching and the lineshape of the two-electron reduced absorption spectrum are virtually identical to those of the reduced [2Fe-2S] cluster containing Fe protein. Thus, the two equivalent reduction of the nitrogenase Fe protein [2Fe-2S] cluster to a formally diferrous state observed in this work represents only the second such reduction reported and the first to occur with the common reductant dithionite.
EPR spectra
were obtained at helium temperatures on samples of [2Fe-2S]
cluster containing Fe protein as prepared (oxidized) and reduced with
0.5 and 6 mol eq of dithionite. Oxidized [2Fe-2S] cluster Fe
protein was EPR silent near g 2, and exhibited only an
isotropic adventitious Fe(III) signal at g = 4.3 (data
not shown). This is consistent with the presence of only
[2Fe-2S]
and [4Fe-4S]
clusters. [2Fe-2S] cluster Fe protein treated with a
0.5 mol eq of dithionite was EPR silent, with the exception of a small
residual g = 4.3 signal (data not shown).
[2Fe-2S] cluster Fe protein reduced by 6 equivalents of
dithionite exhibited a small amount of an axial EPR signal at g
2 (Fig. 7), with apparent g values identical
to those of the MgATP-bound state of the reduced [4Fe-4S]
cluster form of Fe protein. The intensity of this signal, compared with
a sample of reduced Fe protein with bound MgATP represents
approximately 5% of the signal intensity expected for the MgATP treated
[4Fe-4S]
cluster at this concentration of
Fe protein. Obviously, this small signal may also arise in part from
the presence of some [2Fe-2S]
cluster.
Nevertheless, the sample is essentially EPR silent with respect to the
majority species, supportive of the presence of a formally diferrous
metal center.
Figure 7: EPR spectra of the reduced [4Fe-4S] and [2Fe-2S] cluster forms of Fe protein. EPR spectra are shown for the dithionite reduced [4Fe-4S] cluster form of the Fe protein (30 mg/ml) with a 10-fold molar excess of MgATP (trace 1) and the dithionite (6-fold molar excess) reduced [2Fe-2S] cluster form of the Fe protein (32 mg/ml) (trace 2). Trace 3 is the same as trace 2 expanded by a factor of three. EPR spectra were acquired at a temperature of 12 K, a microwave frequency of 9.50 GHz, a microwave power of 0.5 milliwatts, a modulation amplitude of 5 G, a modulation frequency of 100 kHz, and equivalent gain settings.
Fe K-edge x-ray spectroscopy of the Fe protein
[4Fe-4S] cluster has also been used to monitor possible
changes in the cluster structure. Previous studies on the reduced Fe
protein [4Fe-4S] cluster have revealed that
no structural changes within the cluster could be detected upon the
addition of MgATP(44) . The oxidized Fe protein
[4Fe-4S]
cluster was not previously
examined. We have now examined the Fe K-edge x-ray absorption spectra
of the IDS oxidized Fe protein in the absence or presence of MgATP and
can detect no changes in either the edge or EXAFS spectra ( Fig. 8and Fig. 9). The EXAFS spectra can be best fit
assuming four sulfur and three iron scatterers around each iron atom
(as in [4Fe-4S] clusters), but they are fit poorly if only
one Fe-Fe scatterer is assumed (as would be the case if
[2Fe-2S] clusters were formed) (Fig. 9). Parameters
from least-squares fits are within estimated error for both forms of
the protein, with refined bond distances essentially identical to those
obtained from data for a [4Fe-4S] model compound (Table 1). These results from x-ray spectroscopy indicate that
the iron is in the [4Fe-4S] cluster form in samples of
oxidized Fe protein with and without added MgATP, and so rule out
extensive formation of a [2Fe-2S] cluster induced by addition
of MgATP.
Figure 8:
Similarities in Fe K-edge XAS of Fe
protein in the absence (thin line) or presence (thick line) of MgATP and of
[(CH
)
N]
[Fe
S
(SPh)
] (dots). Spectra have been base line corrected and normalized
to edge jump height = 1.0. Successive spectra are offset by
0.1.
Figure 9:
Fe
K-edge EXAFS spectra of Fe protein in the absence
(
) or presence (
) of MgATP. The solid line is the
fit from Table 1to the data in the presence of MgATP. The dashed line is the best fit (
= 14.8) obtainable if n
is fixed at
1 (as in [2Fe-2S] clusters).
In summary, we have demonstrated unique CD spectra arising
from the [4Fe-4S] cluster upon nitrogenase
Fe protein binding either MgATP or MgADP. These results suggest that
each nucleotide communicates different conformational changes to the
environment of the cluster. We provide direct evidence that the
observed CD changes in the Fe protein upon binding nucleotides are not
the result of a conversion of the [4Fe-4S] cluster to a
[2Fe-2S] cluster. The Fe K-edge XAS results support this
conclusion and suggest that nucleotides do not result in major changes
in the cluster structure but rather in changes in the environment
around the cluster.