From the Department of Molecular Biology and Biochemistry,
University of California, Irvine, California 92697-3900
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INTRODUCTION |
The Fe protein of nitrogenase is one example of a large family of
proteins (e.g. H-ras p21, recA, myosin) that
have energy transduction mechanisms involving switching between
conformational states upon nucleotide binding or hydrolysis (1-7).
Comparisons of the sequences and structures of this family of proteins
have revealed two highly conserved regions that are involved in
nucleotide interactions. One region, which is called the Walker A motif
or the phosphate-binding loop (P-loop), contains residues 9-16 of the
Fe protein and includes residues that are involved in both phosphate
binding and stabilization of the leaving group (5, 6, 8-10). The
second region, which in the Fe protein extends from residues 125 to
132, includes a highly conserved Asp-X-X-Gly Walker B motif
and corresponds to the switch II region in the G-protein family
(5, 7-10).
Although the nucleotide binding motifs and regions involved in energy
transduction for this family of proteins are highly conserved, the Fe
protein has the unique feature of being the only dimeric member of the
family. The two identical subunits are encoded by the nifH
gene and are bridged by a single [4Fe-4S] cluster to form a ~60-kDa
holoprotein that has two binding sites for MgATP (for recent reviews,
see Refs. 6 and 11). Although the binding site for the terminal
phosphate of MgATP is approximately 20 Å from the [4Fe-4S] cluster
(9), the binding of MgATP to the Fe protein causes a large
conformational change that affects many of the properties of that
cluster (11). After this conformational change the Fe protein is able
to bind productively to its physiological redox partner, the MoFe
protein of nitrogenase. That protein is an ~240-kDa tetramer composed
of two
and two
subunits encoded by the nifD and
nifK genes, respectively (6, 12). The MoFe holoprotein has
two distinct types of metal clusters. The P-clusters are [8Fe-7S]
clusters that are believed to accept electrons from the Fe protein
(13). The other type of metal cluster in the MoFe protein is called the
iron-molybdenum cofactor (FeMo cofactor) and is a
[Mo-7Fe-9S-homocitrate] cluster that appears to accept electrons from
the P-cluster and transfer them to dinitrogen (11-16). Although the Fe
protein alone can bind MgATP, MgATP hydrolysis only occurs after the Fe
protein binds to the MoFe protein. How the energy released by MgATP
hydrolysis is utilized by the Fe protein·MoFe protein complex is
currently not understood. However, the fact that the Fe protein is the
only known reductant that can reduce the MoFe protein in such a way
that the latter can reduce dinitrogen is no doubt related to the energy
transduction step.
There are currently two possible explanations for the fact that, unlike
other proteins that bind nucleotides and undergo conformational changes, the Fe protein is a dimer. The dominant hypothesis, which is
based on kinetic studies with dithionite as an electron donor (17),
suggests that the enzyme somehow needs to couple the hydrolysis of two
molecules of MgATP to the transfer of a single electron from the Fe
protein to the MoFe protein (11). Recent work with other electron
donors has shown that the [4Fe-4S] cluster of the Fe protein can be
reduced by two electrons to an all ferrous state (18, 19). This leaves
open a second possibility that the Fe protein is a dimer because it can
bind two molecules of its physiological electron donor, flavodoxin, in
order to accept two electrons into a single [4Fe-4S] cluster.
Regardless of which scenario is correct, the binding of two molecules
of MgATP causes a large conformational change that affects a single
[4Fe-4S] cluster. Although the structure of the Fe protein with MgATP
bound has not been reported, there is substantial evidence from x-ray
scattering experiments (20), the structure of an Fe
protein·ADP·AlF4
·MoFe
protein complex (10), and mutagenesis experiments (21-25) that the
conformational change involves interactions between the two subunits of
the Fe protein.
Our earlier mutagenesis experiments have identified a highly conserved
region of the Fe protein around Ala-157 which is located at the subunit
interface and appears to be critical for the MgATP-induced conformational change (26). This region is not present in other nucleotide-binding proteins or in any other protein whose sequence is
in the data base. X-ray crystallographic analysis of the Fe protein
alone (9) and in the Fe
protein·ADP·AlF4
·MoFe protein
complex (10) reveals that this region is part of helix
5 located at
the subunit interface which extends from residue 151 to 176. This helix
moves substantially when the free Fe protein structure is compared with
the structure of the Fe protein in the complex. Our previous studies
have shown that mutation of Ala-157 to Ser results in a protein that
can still bind MgATP normally but is unable to undergo the
MgATP-induced conformational change. To understand how the mutation of
this residue can prevent the MgATP-induced conformational change, we
have begun to mutate this and neighboring conserved residues. The
mutation of Ala-157 to residues larger than Ala leads to completely
inactive protein. Here we report the construction and characterization
of an A157G Fe protein that is active.
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EXPERIMENTAL PROCEDURES |
Unless otherwise noted, all chemicals and reagents were obtained
from Fisher Scientific, Baxter Scientific, or Sigma.
Construction and Expression of the Variant Azotobacter vinelandii
Strain--
A fragment of the A. vinelandii chromosome
containing the entire nifH and nifD genes as well
as part of the nifK gene was cloned into the bacteriophage
M13mp18. Site-directed mutagenesis was performed using the Mutagene
mutagenesis kit, version 2, from Bio-Rad. The oligonucleotide used for
mutagenesis was purchased from Integrated DNA Technologies, Coralville,
IA. The oligonucleotide was 29 bases long and was complementary to the
region surrounding the Ala-157 codon. The oligonucleotide was
degenerate at the Ala-157 codon, allowing the production of several Fe
protein mutants at this position. After mutagenesis, bacteriophage
containing mutated nifH genes were selected through DNA
sequencing using the Sequenase II sequencing kit (U. S. Biochemical
Corp.). Double-stranded DNA was isolated from these phage using a
Qiagen kit (Qiagen, Chatsworth, CA). This DNA was then transformed back
into two A. vinelandii strains, the wild-type Trans strain
and the
nifH strain DJ54 using a published method (27).
Here, the chromosomal copy of the gene is replaced with the mutated
gene through homologous recombination, allowing production of variant
Fe protein in its native background, under the control of its native
promoter. The recombination of the mutated nifH gene
containing the Ala to Gly mutation into the bacterial chromosome of the
DJ54 strain resulted in a strain that was able to grow under
nitrogen-fixing conditions. All subsequent work was done on this
DJ54-derived strain.
Cell Growth and Protein Purification--
The A157G A. vinelandii strain was grown in 180-liter batches in a 250-liter
New Brunswick fermenter in Burke's minimal medium under ammonium
acetate-limiting derepressing conditions. Ammonium acetate was added to
the medium to a final concentration of 2 mM, such that the
ammonia was exhausted during mid-log phase. The decrease in ammonia
concentration was monitored both by observing the growth rate of the
culture as measured by cell density and through the use of Sigma
ammonia color reagent. After the consumption of the ammonia, the
culture was grown for an additional 3 h. The cells were then
harvested using a flow-through centrifugal harvester (Cepa, Germany).
The cell paste was washed with 50 mM Tris-HCl, pH 8.0, and
was kept on dry ice until needed. After the initial preparation of
crude extract, all subsequent procedures, with the exception of the
measurement of the circular dichroism spectra, were performed in the
presence of 2 mM sodium dithionite. The variant Fe protein
was purified as described previously (28) with some slight
modifications. Between 0.5 and 1.0 kg of cell paste was thawed in 1.4 volumes of 0.05 M Tris-HCl, pH 8.0, and degassed
thoroughly. Sodium dithionite was added to give a final concentration
of 2 mM, and the cells were then broken by passing them
through a Gaulin cell homogenizer two or three times at 6,000 p.s.i. to
make a crude extract. The crude extract was then made 10 µg/ml with
both DNase and RNase, degassed for an additional 2 h,
ultracentrifuged for 80 min at 31,000 rpm in a Beckman Ti 45 fixed-angle rotor, and then loaded onto a 5 × 25-cm
DEAE-cellulose (Whatman) ion-exchange column. Throughout the
purification procedure the protein was monitored by absorbance at 405 nm. The Fe protein was eluted with a linear 0.1-0.5 M NaCl
gradient. The Fe-containing fraction was loaded onto a 2.5 × 100-cm Ultrogel AcA34 (ICF, France) gel filtration column and eluted
with 0.05 M Tris-HCl, pH 8.0, 0.1 M NaCl. The
Fe protein-containing fraction was then loaded onto a 1.5 × 10-cm
Q-Sepharose (Amersham Pharmacia Biotech) column and eluted with a
linear 0.1-0.5 M NaCl gradient. The eluate was then loaded
onto a 1.5 × 10-cm DEAE-cellulose column and concentrated by
elution in the reverse direction using 0.5 M NaCl. The
purified Fe protein was analyzed by polyacrylamide gel electrophoresis, and its concentration was determined using the Biuret method (29).
Spectroscopy--
For spectroscopic experiments, all samples
were made in a Vacuum Atmospheres dry box under argon. For EPR, the
samples were 2 mM in sodium dithionite. The spectra were
recorded on a Bruker ESP 300 EZ spectrophotometer equipped with an
Oxford Instruments ESR-9002 liquid helium continuous flow cryostat. For
CD experiments, the [4Fe-4S] cluster was oxidized by one of two
methods. In one case, a degassed solution of 20 mM indigo
disulfonate (MC/B, Norwood, OH) was added to the protein until a blue
color remained. The protein was incubated in the dye longer than 15 min
and was then separated from the dye by passage over a 1 × 10-cm
Sephadex G-25 (Amersham Pharmacia Biotech) column. The other method
consists of oxidizing the protein by passage over a specially prepared column as described previously (30). The column (1 × 10 cm) consists of, from top to bottom, 5 cm of indigo disulfonate dye bound
to AG 1-X8 (Bio-Rad) and 5 cm of P6DG (Bio-Rad). The protein was loaded
onto the top of the column and was allowed to incubate longer than 15 min. It was then eluted with 0.05 M Tris-HCl, pH 8.0. The
CD spectra were obtained using a Jasco J720 spectropolarimeter. Ultraviolet/visible spectra were recorded on a Hewlett Packard 8452/A
diode array spectrophotometer.
Activity Assays--
Enzyme activity was determined by measuring
both hydrogen evolution under an argon atmosphere and ethylene
production under an atmosphere of 10% acetylene and 90% argon. Assays
were performed by incubating the Fe and MoFe proteins together at
30 °C in the presence of a reaction mixture and 20 mM
sodium dithionite for 3-10 min in calibrated stoppered vials. Reaction
mixture consists of 250 mM
TES,1 pH 7.4, 100 mM ATP, 250 mM MgCl2, 300 mM creatine phosphate, and 500 units/ml creatine
phosphokinase. 0.6 ml of the reaction mix and 0.3 ml of 250 mM TES, pH 7.4, were degassed in calibrated vials. 0.1 ml
of a 200 mM sodium dithionite solution was then added. The
ratio of Fe protein to MoFe protein used was 0.5:1, and the total
protein concentration was 0.5 mg. The reaction was initiated by the
addition of Fe protein. After incubation, the reaction was killed by
the addition of 100 µl of 30% trichloroacetic acid. The amount of
product evolved was determined on a Varian 3700 gas chromatograph using
either a flame ionization detector (ethylene production) or a thermal
conductivity detector (hydrogen evolution). The crude extract ethylene
production assay was carried out as above except that the reaction was
initiated by the addition of 0.1 ml of either wild-type or A157G crude
extract to a calibrated vial containing degassed reaction mix, water,
and 20 mM sodium dithionite. The total protein
concentration used in the crude extract activity assay was determined
using the Biuret method (29) and was found to be 45.2 and 36.6 mg/ml
for the wild-type and A157G crude extracts, respectively. The
ATP/2e
ratio was determined by performing a standard
hydrogen evolution assay in the absence of creatine phosphate and
creatine phosphokinase and then determining the amount of ADP generated
over the course of the assay. The assays were incubated for increasing
amounts of time. Times were selected such that the rate of hydrogen
evolution was linear with respect to time so as to avoid complications
resulting from inhibition of the assay by the formation of ADP. ADP
concentrations were determined using a high performance liquid
chromatography method (24, 31). An aliquot of the liquid from the
killed activity assay was centrifuged to remove precipitated protein. The nucleotide concentration of a portion of this was measured by
loading it onto a Supelco LC-18-T C18 reversed phase column and eluting
with 100 mM potassium phosphate buffer, pH 6.0. Nucleotides were detected using a Waters 486 tunable absorbance detector at 259 nm.
An extinction coefficient of 15,400 M
1
cm
1 was used to determine the concentration of
nucleotide.
Chelation Assay--
The chelation assay followed published
methods (21, 32). Chelation was performed by degassing a solution of 50 mM Tris-HCl, pH 8.0, and 6.25 mM
,
'-dipyridyl in a stoppered quartz cuvette. Fe protein was added
to a concentration of 0.8 mg/ml and the spectrophotometer blanked. The
chelation reaction was started by the addition of MgATP to a final
concentration of 5 mM ATP and 10 mM
MgCl2. The progress of the reaction was followed by
monitoring the absorbance of the solution at 520 nm. The ability of the
Fe protein to interact with the wild-type MoFe protein was assessed by
performing the chelation assay in the presence of MoFe protein (1.29 mg/ml) (25, 32). In this case, a regenerating system was used to
prevent the accumulation of ADP. The regenerating system used consisted of 6 mM creatine phosphate and 0.125 mg/ml creatine
phosphokinase.
 |
RESULTS AND DISCUSSION |
Growth of an A. vinelandii Strain Expressing A157G Fe
Protein--
When A. vinelandii is grown under
nitrogen-fixing conditions its growth rate is limited by the
availability of fixed nitrogen and is therefore controlled by the
activity of the enzyme nitrogenase. In a previous study we established
that an A157S Fe protein variant could bind MgATP but could not undergo
the MgATP-induced conformational change (26). Because that Fe protein
was inactive, expression of the protein in its native background in
A. vinelandii led to a strain that was unable to grow under
nitrogen-fixing conditions. This Nif
phenotype was also
observed when other residues larger than Ala were substituted for
Ala-157.2. However, a strain
expressing an A157G variant could grow under nitrogen-fixing conditions
at 55% of the wild-type rate. The A157G variant strain has a much
longer doubling time, 5.6 h, compared with 3.1 h for the
wild-type strain. Based on purification profiles, SDS-polyacrylamide
gel electrophoresis of crude extracts, and purification yields, the
A157G Fe protein is present in approximately wild-type levels, thus we
do not believe that the longer doubling time is caused by lower amounts
of A157G Fe protein in vivo.
Purification and Activity Measurements--
The Fe protein of
nitrogenase has at least three functions: electron transfer to the MoFe
protein (11); the initial biosynthesis of FeMo cofactor (33-35); and
the insertion of preformed FeMo cofactor into an inactive, FeMo
cofactor-deficient MoFe protein (36). Because the FeMo cofactor center
of the MoFe protein has a characteristic EPR signal (37), the ability
of an Fe protein to function in FeMo cofactor biosynthesis and
insertion can be readily tested by EPR. Analysis of extracts from the
strains expressing the A157G Fe protein (Fig.
1) clearly show the EPR signal that
arises from protein-bound FeMo cofactor, leading to the conclusion that
like the previously characterized A157S Fe protein (26), the A157G Fe
protein does function normally in FeMo cofactor biosynthesis and
insertion. The slower than wild-type growth rate of the A157G strain
therefore results from some defect in the electron transfer function of
the protein. To identify the defect, the A157G Fe protein was purified
to homogeneity using a modification of the original anaerobic
purification procedure (28) described under "Experimental
Procedures." The A157G Fe protein behaved normally throughout the
purification process. As shown in Table
I, the protein was then assayed for
catalytic activity as described under "Experimental Procedures."
The activity of the purified protein was approximately 20% of the
wild-type level for both hydrogen evolution and ethylene production. We
note that this is lower than expected based on our observed growth
rate.

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Fig. 1.
EPR spectra of reduced (A)
wild-type and (B) A157G crude extracts. The spectra
were measured at a microwave power and frequency of 50 mW and 9.43 GHz,
respectively. The receiver gain was 5 × 103,
modulation frequency was 100 kHz, modulation amplitude was 5.131 G, and
the temperature was 10 K. The total protein concentration of the
wild-type crude extract was 68.7 mg/ml, and that of the A157G crude
extract was 57.2 mg/ml. Both extracts contained 2 mM sodium
dithionite.
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Nucleotide-induced Conformational Change--
A critically
important step in the overall nitrogenase mechanism is the change in
the conformation of the Fe protein which occurs upon MgATP binding
(11). As shown in Fig. 2 one of the most
striking effects of MgATP binding by the Fe protein is the change in
the reactivity of the [4Fe-4S] cluster with iron chelators. In the
absence of MgATP the [4Fe-4S]+ cluster is not accessible
to attack by
,
'-dipyridyl (32). When MgATP binds, the chelator is
able to remove all of the Fe from the Fe protein rapidly (38). Fig. 2
shows that the same chelation reaction occurs for the A157G Fe protein
but that the initial rate of Fe chelation is much slower. As expected
(32), neither wild-type nor A157G Fe protein was chelated in the
presence of MgADP. Although chelation of iron from the Fe protein by
,
'-dipyridyl has been shown to be biphasic (38), a
pseudofirst-order rate can be fitted to the initial portion of the
data. The wild-type observed rate of Fe chelation was calculated to be
8.5 × 10
3 s
1, whereas that of the
A157G Fe protein was found to be 1.5 × 10
3
s
1. This chelation rate of only 17% the wild-type rate
corresponds well with the unexpectedly low activity we see in our
in vitro activity assays. Thus, either the mutant undergoes
the conformational change more slowly than the wild-type protein, or
the final conformation is different. To distinguish these possibilities
spectroscopic methods were employed.

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Fig. 2.
MgATP-dependent Fe chelation of
purified wild-type and A157G Fe proteins. Each chelation reaction
was carried out as described under "Experimental Procedures." The
observed Fe chelation rates were found to be 8.5 × 10 3 s 1 and 1.5 × 10 3
s 1 for the wild-type and A157G Fe proteins, respectively.
Although the chelation reaction looks like it reaches a plateau, after
exposure to oxygen the absorbance of the chelated A157G Fe protein
solution reaches the same value as that of the wild-type protein,
indicating that both proteins contain the same amount of iron. We do
not believe that the chelation reaction is stopping after the formation
of a [2Fe-2S] cluster because CD spectroscopy of chelated A157G Fe
protein does not show the expected signal characteristic of a
[2Fe-2S] cluster.
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As shown in Fig. 3, the EPR spectrum of
the reduced [4Fe-4S]+ cluster in the Fe protein exhibits
an S = 1/2 signal with a rhombic line shape
(37). The addition of MgATP results in a change in the shape of the
S = 1/2 signal from rhombic to axial (39). The S = 1/2 EPR signal exhibited by the A157G Fe
protein is qualitatively and quantitatively indistinguishable from the
signal exhibited by the wild-type Fe protein both in the presence and
absence of MgATP. Thus, by this criterion, the A157G Fe protein appears
to adopt the same final conformation as the wild-type protein.

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Fig. 3.
EPR spectra of purified wild-type Fe protein
(A), A157G Fe proteins without nucleotide (B),
and wild-type (C) and A157G Fe proteins (D) in
the presence of 5 mM MgATP. All protein concentrations
were 15 mg/ml, and the solutions contained 2 mM sodium
dithionite. The spectra were measured at a microwave power of 1 mW and
at a microwave frequency of 9.43 GHz. Receiver gain was 5 × 104, modulation frequency was 100 kHz, modulation amplitude
was 2.886 G, and the temperature was 13 K.
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A second method that has been used to study the MgATP-induced
conformational change is visible region CD spectroscopy which, in
general, is a useful way to monitor the environment of [Fe-S] clusters in proteins. Studies of Fe proteins from three organisms have
shown that the CD is measurable in the oxidized
[4Fe-4S]2+ oxidation state and is very sensitive to the
addition of nucleotides (11, 40-42). Fig.
4 shows that, as was the case with the
EPR, the shapes of the CD spectra for the wild-type and A157G Fe
proteins are the same before and after the addition of MgATP. The
binding of MgADP also causes a change in the conformation of the Fe
protein which is quite distinct from that induced by MgATP. The
addition of MgADP does not allow chelation of the Fe atoms of the
[4Fe-4S]+ cluster and does not cause substantial change
in the EPR spectra (11). However, the addition of MgADP has been
observed to cause a large change in the CD spectra of the oxidized
protein (41, 42). As shown in Fig. 4, the changes in shape observed in
the spectra of the wild-type and the A157G Fe proteins upon addition of
MgADP are indistinguishable from each other.

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Fig. 4.
CD spectra of wild-type and A157G Fe
proteins. A final concentration of 5 mM ATP or ADP and
10 mM MgCl2 was used in the measurements
containing nucleotide. The protein concentrations ranged between 4.2 and 11.7 mg/ml.
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Binding to the MoFe Protein--
Taken together, the above data
provide strong evidence that the A157G and wild-type proteins begin in
the same conformation, that upon addition of MgATP it takes much longer
for the A157G protein to adopt the correct conformation, but that the
final conformation obtained for the two proteins in the presence of a
large excess of MgATP appears to be the same. The next step in a normal
nitrogenase reaction is for the reduced Fe protein with MgATP bound to
bind to the MoFe protein. One method that has been used to examine the
binding of the Fe protein to the MoFe protein involves the addition of
MoFe protein to a chelation experiment of the type shown in Fig. 2.
When the MoFe protein is complexed with the Fe protein the [4Fe-4S]
cluster is covered up (10), protecting it from exogenous chelator (32,
43). Fig. 5 shows that, unlike the
situation for the wild-type Fe protein, the MoFe protein is not able to
protect the A157G Fe protein [4Fe-4S]+ cluster from
chelation.

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Fig. 5.
MgATP-dependent iron chelation of
the [4Fe-4S] cluster of the Fe protein in the presence of wild-type
MoFe protein. The experiment was performed as in Fig. 2 with the
addition of 1.29 mg/ml MoFe protein and a regenerating system as
described under "Experimental Procedures."
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In a previous study, we showed that the A157S protein that failed to
undergo the MgATP-induced conformational change also could not compete
with the wild-type Fe protein in an activity assay. That study (26)
combined with other data (21, 25, 41) show that the MgATP-induced
conformational change is a prerequisite for productive binding of the
Fe protein to the MoFe protein. The A157G protein must be able to bind
productively to the MoFe protein at least some of the time because it
has catalytic activity (Table I), which again supports the conclusion
that the final conformation of the mutant protein is the same as that
of the wild-type protein. However, if the A157G protein takes a
relatively long time to adopt that conformation, then at any point in
time only a relatively small percentage of the molecules might be in the required conformation to bind productively to the MoFe protein, a
phenomenon that could explain the lack of protection from chelation (Fig. 5) and the low activity (Table I).
MgATP Hydrolysis Is Coupled to Electron Transfer--
In the next
step in nitrogenase turnover, the Fe protein transfers one electron to
the MoFe protein, and two molecules of MgATP are hydrolyzed to two
molecules of MgADP and inorganic phosphate (11). Because the Fe protein
does not hydrolyze MgATP by itself, complex formation must cause a
second conformational change in the Fe protein which brings the
catalytic residues in the Fe protein into the correct position to
hydrolyze MgATP (6, 10). The minimum stoichiometry for the reaction is
two molecules of MgATP hydrolyzed per electron transferred, but there
are many examples of mutations or unfavorable assay conditions that
cause the two reactions to be uncoupled such that MgATP hydrolysis
occurs in the absence of electron transfer (11). The stoichiometry of MgATP hydrolyzed to electrons transferred is only slightly larger for
the A157G mutant than it is for the wild-type protein. The values are
5.9 ± 0.2 and 3.8 ± 0.3 MgATP/two electrons for A157G and
wild-type protein, respectively. Thus, once the A157G Fe protein has
bound to the MoFe protein, it can adopt the conformation required for
MgATP hydrolysis.
The rate-limiting step in the Fe protein cycle is dissociation of the
Fe protein·MgADP complex from the MoFe protein (17). The chelation
protection assay shown in Fig. 5 shows that, unlike an Fe protein
variant with a deletion at Leu-127 (25, 44), the A157G protein does not
have a problem with dissociation. The CD data shown in Fig. 4 further
show that the conformation of the oxidized A157G Fe protein·MgADP
complex is likely to be the same as that of the wild-type complex.
A Model for the Role of Residue 157--
The A157S mutant could
not undergo the initial MgATP-induced conformational change and was
also blocked for all subsequent steps in the reaction. Using the active
A157G mutant, it is now possible to examine the role of this residue in
subsequent reactions. Taken together, the above data indicate that upon
MgATP binding, the A157G mutant is slow to adopt the conformation
required for productive binding to the MoFe protein, but once that
conformation is obtained it can undergo all subsequent reactions. Thus,
residue 157 is critical only for the initial conformational change.
Using the structure of the Fe protein in the absence of nucleotide and the structure of the Fe protein·ADP·
AlF4
·MoFe protein complex we
can address two questions: How does mutation of Ala-157 to Ser prevent
the initial conformational change? Why does putting a Gly in that
position increase the time required to get into the correct
conformation?
As stated above, Ala-157 is part of helix
5, which includes residues
151-176. Fig. 6 shows that this helix
changes orientation in the complex structure as opposed to the
noncomplex structure. A comparison of these two structures in this
region shows that the formation of the correct orientation is important
for two reasons. First, the structure of helix
5 changes near its
COOH-terminal end in the complex structure (Fig. 6). A bend occurs in
the helix near the complex interface around Gly-167, and a 2.8 Å salt
bridge is formed between the backbone oxygen of Asn-173 and the
amino group of MoFe
subunit residue Lys-171 (10). Changes in the protein which alter these observed movements of helix
5 would therefore affect the ability of the Fe protein to bind to the MoFe
protein. Because of the lack of symmetry observed in the Fe
protein·MoFe protein complex, only Asn-173 from one of the Fe protein
subunits (monomer 2) interacts with the MoFe protein. In this
discussion, monomer 2 refers to polypeptide chain E, and monomer 1 refers to polypeptide F in the complex crystal structure (10). When
necessary, the individual subunits of the noncomplexed form of the
protein are referred to as monomer A or B, which correspond, respectively, to polypeptide chains A and B in the noncomplex crystal
structure PDB file (9).

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Fig. 6.
A comparison of the structure of helix 5
as found in (A) the original crystal structure and in
(B) the Fe
protein·ADP·AlF4 ·MoFe protein
complex crystal structure. Residues 157 and 173 from the Fe
protein and 171 from the MoFe protein are labeled. All computer models
were generated using Rasmol (45).
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Second, the NH2-terminal region of helix
5 is in close
proximity to the trigger region composed of residues 9-16 (Fig.
7). Although the two regions do not
contact each other directly, examination of both crystal structures
indicates that these two regions are very close. In the complex
structure, the
methyl group of Ala-157 is 4.1 Å away from the
methylene of Lys-10 and 3.8 Å away from the
methyl group of Ile-13
(Table II). Additionally, Glu-154 comes
within 3.4 Å of the Ala-157 side chain, and Ser-152 comes within 3.3 Å. Thus, in the complex crystal structure, the amino acid packing in
this region is such that there is a pocket formed in which there is no
room for a larger residue at position 157. Another observation from the
original crystal structure is that the distances between amino acids
vary when comparing this pocket in both subunits. The distances between
the Ala-157 side chain and residues 10, 13, 152, and 154 are as much as
1.0 Å longer in monomer B than those in monomer A, making the pocket
formed by the residues around Ala-157 slightly larger in monomer B
(Table II). This variation between the two subunits is not seen in the complex crystal structure. In the complex structure, this pocket around
Ala-157 has almost identical dimensions in both subunits, suggesting
that the structure and interactions seen in the complex crystal
structure are more critical than those seen in the MgATP-free crystal
structure.

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Fig. 7.
A stereo image of the region around Ala-157
as seen in the Fe
protein·ADP·AlF4 MoFe protein complex
crystal structure. The dotted spheres indicate the
space occupied by the atoms in the indicated residues.
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It is thought that the binding of MgATP triggers a conformational
change by breaking a salt bridge between Lys-15 and Asp-125 (22, 23).
Replacement of Ala-157 with a larger residue would introduce the
problem of steric clashes between it and residues Ile-13, Lys-10,
Ser-152, and Glu-154. The availability of the complex crystal structure
has greatly assisted us in our understanding of the importance of
specific interactions in this region. Lys-10 is seen to interact across
the subunit interface with the
phosphate of the ADP molecule and
has been proposed to be involved in the stabilization of the leaving
group during ATP hydrolysis (10). To avoid steric clashes with these
important residues, one could envision two possibilities, one being the
movement or rearrangement of the trigger region and the other being the
movement or rearrangement of helix
5. Interfering with the trigger
region would very likely affect the affinity of the protein for MgATP.
However, the A157S Fe protein was shown to bind MgATP in a manner
comparable to wild-type Fe protein (26). Presumably, the proper binding
of MgATP in the A157S mutant would still break the salt bridge between
Lys-15 and Asp-125. However, in the A157S Fe protein, the subsequent signal transduction to the [4Fe-4S] cluster through the switch II
sequence, which consists of residues 125-132 (23, 25), is somehow
prohibited. Helix
5 does not interact directly with the switch II
sequence. The closest distance between these two regions in the complex
structure is a 4.1 Å separation between the side chain of Ile-164 and
the oxygen of Gly-128. Thus, this region exerts its influence apart
from the signal transduction sequence and highlights the importance of
concerted interactions throughout the protein for the formation of the
proper conformation upon MgATP binding. A second possibility is that of
the movement or rearrangement of helix
5. As stated above, in the
complex this helix has interactions at its COOH-terminal end with the MoFe protein. At its NH2-terminal end, it contains residues
that help to stabilize the binding of nucleotide through intersubunit interactions between Glu-154 and Arg-213 (10) (Fig.
8). Also, Met-156 has close interactions
both with residue Asp-43 at a distance of 3.4 Å, a residue on the
other subunit which is about 3.5 Å from the AlF group in the complex
structure and with the ADP molecule, at a distance of 300 Å from the
phosphate group. These interactions further suggest that the proper
orientation of this helix, both at its COOH terminus and especially at
its NH2 terminus, is critical to attaining the conformation
observed in the complex structure. As indicated above, in the complex
structure, replacement of Ala-157 with Ser would cause steric
interference between this residue and residues in the trigger region,
probably causing a reorganization of the structure of the
NH2-terminal portion of helix
5 which would result in a
failure of the protein to be able to adopt the proper conformation,
hence the inactivity seen in the A157S Fe protein.

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Fig. 8.
A view of the interactions between Glu-154
and Arg-213 that help to stabilize the binding of
MgADP·AlF4 . Also shown are other
residues in the region around Ala-157 which may be important for proper
Fe protein function.
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Replacement of Ala-157 with Gly would not cause this steric
interference. The helix would be free to move and would be able to
attain the correct orientation for the MgATP-induced conformational change and subsequent complex formation to occur. This is supported by
our observations that A157G Fe protein does undergo the conformational change. This portion of the protein sequence is highly conserved across
all Fe proteins, suggesting that the structure observed at the
NH2-terminal loop region of helix
5 is very important to
the proper function of the protein. This is further supported by the
"tightening up" of this pocket when comparing the complex structure
with the noncomplex structure as discussed above and illustrated in
Table II. The removal of steric constraints imposed by the presence of
the alanine side chain would affect this region by failure to force the
proper conformation of the residues around Ala-157. Furthermore, the
removal of this side chain would allow the formation of alternate
conformations in this region. In the latter case, the proper
orientation of helix
5 for the conformational change and subsequent
MoFe protein·Fe protein complex formation would not be impossible but
might be less likely given the increased opportunity for alternate
conformations. This would explain the lowered activity observed in the
A157G Fe protein. It is important to emphasize the fact that this
residue appears to be critical only for the initial MgATP-induced
conformational change. Its ability to support substrate reduction
indicates that once the initial conformational change has occurred, the
protein is able to proceed normally with MoFe protein binding, MgATP
hydrolysis, electron transfer, and complex dissociation.
The results presented in this paper suggest that the role of Ala-157
lies in the stabilization of the proper conformation of helix
5 in
the protein. The proper movement of helix
5 from its orientation in
the noncomplex structure to that seen in the Fe
protein·ADP·AlF4
·MoFe
protein complex structure is essential for proper Fe protein activity.
Specifically, it is important for the initial conformational change.
Replacement of Ala-157 with other residues destabilizes the proper
conformation of the NH2-terminal end of helix
5 and has
profound effects on the ability of the Fe protein to function normally.
We acknowledge Dr. Narasiah Gavini for work
on the A157S Fe protein; Dr. Doug Rees for kindly providing the
coordinates of the Fe protein·MoFe protein complex crystal structure;
and Dr. Hayley C. Angove for help, comments, and suggestions throughout the course of this project.