From the
Biological nitrogen fixation catalyzed by purified nitrogenase
requires the hydrolysis of a minimum of 16 MgATP for each N
The hydrolysis of a minimum of 16 MgATP to 16 MgADP and 16
P
While only the
FeP
In an attempt
to elucidate the mechanism of nucleotide utilization by nitrogenase, we
are defining the nucleotide binding site on the FeP, residues involved
in hydrolysis, and residues responsible for the conformational change.
In recent work, we identified an amino acid sequence in the
Azotobacter vinelandii FeP that is involved in nucleotide
interaction
(18) . This sequence, located near the N terminus of
the FeP, is similar to motifs known to function in nucleotide binding
in other proteins (i.e. P-loop or Walker A motif)
(19) .
Specifically, we demonstrated that the highly conserved lysine at
position 15 of this motif plays a major role in both the FeP
interaction with the
Like the
Gln substituted FeP (K15Q), the arginine substituted FeP (K15R) was
found to be inactive in electron transfer, MgATP hydrolysis, and
substrate reduction. More detailed analysis of the properties of the
K15R FeP revealed, however, several differences from the K15Q FeP which
suggested a much broader role for Lys-15 in nucleotide interaction.
Previous reports on the affinities of MgATP and MgADP binding to the
reduced wild type FeP have suggested apparent dissociation constants
ranging from 220 to 1710 µM for MgATP and 44 to 91
µM for MgADP
(32) . The disparity in these reported
values probably reflect limitations in the various methods that have
been used to determine these values. We have found that particular care
must be taken in the use of equilibrium dialysis methods. During the 6
or more hours needed to reach equilibrium, we have found significant
hydrolysis of MgATP (up to 20%) to MgADP. The presence of MgADP in the
binding assay would have a significant effect on the apparent binding
constant for MgATP. In addition, because A. vinelandii FeP can
self-oxidize dithionite
(33) , the oxidized state of the FeP must
be used. Advantages of the equilibrium column binding technique
described in this work include the short time required for the
attainment of equilibrium. This allows the maintenance of dithionite in
the column buffer and thus the reduced state of the FeP. In addition,
the short time reduces the amount of MgATP hydrolysis. The high
performance liquid chromatography method used to detect the nucleotide
concentrations provides a sensitive way to detect any hydrolysis of
MgATP to MgADP or MgAMP. Using the column binding technique, nucleotide
hydrolysis was determined to be less than 4%.
Fig. 3
(panel
A) shows reduction curves for wild type FeP in the absence and
presence of either MgATP or MgADP. From the fits of the data to the
Nernst equation, midpoint reduction potentials of -290,
-420, and -430 mV versus the standard hydrogen
electrode were determined, respectively. In all cases, the maximum
number of electrons per protein were found to be between 0.9 and 1.0,
consistent with a single electron reduction of the [4Fe-4S]
cluster from the 2
MgATP binding and hydrolysis are an integral part of the
substrate reduction mechanism of nitrogenase
(4, 16) .
The results presented in this work support a model in which Lys-15 of
A. vinelandii FeP plays a central role in nucleotide
interactions with nitrogenase, functioning in binding of both MgATP and
MgADP and in the conformational changes induced by both nucleotides.
MgATP interactions with nitrogenase include MgATP binding to the FeP
followed by MgATP hydrolysis by the FeP
In our previous work, the function of Lys-15 was probed by analysis
of an FeP in which this residue was changed to the neutral amino acid
glutamine (K15Q). The K15Q FeP was found to bind MgADP with normal
affinity, clearly suggesting that the side chain of Lys-15 did not
participate in MgADP binding
(18) . In contrast, MgATP binding
was affected, decreasing to 35% of wild type. This suggested that
Lys-15 functioned in interaction with the
In conclusion, this
study provides evidence for a central role of Lys-15 of A.
vinelandii FeP in interaction with both MgADP and MgATP and
suggests a possible mode of interaction between Lys-15 and Asp-125.
reduced. In the present study, we demonstrate a central function
for Lys-15 of Azotobacter vinelandii nitrogenase iron protein
(FeP) in the interaction of nucleotides with nitrogenase. Changing
Lys-15 of the FeP to Arg resulted in an FeP with a dramatically reduced
affinity for both MgATP and MgADP. From equilibrium column binding
experiments at different nucleotide concentrations, apparent
dissociation constants (K
) for wild type
FeP binding of MgADP (143 µM) and MgATP (571
µM) were determined. Over the same nucleotide
concentration ranges, the K15R FeP showed no significant affinity for
either nucleotide. This contrasts sharply with previous results with an
FeP in which Lys-15 was changed to Gln (K15Q) where it was found that
the K15Q FeP bound MgADP with the same affinity as wild type FeP and
MgATP with a slightly reduced affinity. Analysis of K15R FeP by EPR,
circular dichroism (CD), and microcoulometry revealed that the
[4Fe-4S] cluster was unaffected by the amino acid change and
that addition of either MgADP or MgATP did not result in the protein
conformational changes normally detected by these techniques. These
results are integrated into a model for how MgATP and MgADP bind and
induce conformational changes within the FeP.
is required for the reduction of each N
to 2
NH
by purified nitrogenase (for reviews, see Refs.
1-5). The exact mode of nucleotide binding, the mechanism of
MgATP hydrolysis, and the mechanism of coupling of this hydrolysis to
substrate reduction within nitrogenase remains largely undefined.
Nitrogenase is a complex metalloenzyme consisting of two separable
metalloproteins, the molybdenum-iron protein (MoFeP) and the iron
protein (FeP). The MoFeP contains the site of substrate binding and
reduction which is thought be at its molybdenum-iron containing
cofactor (FeMoco)
(6, 7) . The MoFeP also contains a
unique [8Fe-(7-8)S] cluster (8Fe or P cluster) which is
proposed to mediate the transfer of electrons from the FeP to
FeMoco
(6, 8, 9) . The FeP is a homodimeric
protein which contains a single [4Fe-4S] cluster bridged
between its subunits
(10) . The consensus model suggests that the
FeP functions to transfer a single electron from its [4Fe-4S]
cluster to the MoFeP (probably the 8Fe cluster) along with the
concomitant hydrolysis of 2 MgATP for each electron transferred. The
resulting oxidized FeP, with 2 MgADP bound, is released from the MoFeP,
is reduced by electron carrier proteins and the MgADP is replaced by
MgATP
(11) . The above cycle is repeated to provide sufficient
electrons for substrate reduction on the MoFeP.
MoFeP complex will hydrolyze bound MgATP, the FeP alone is
known to bind 2 MgATP or 2 MgADP per dimer. The binding of these
nucleotides to the FeP induces conformational changes in the FeP which
affect the properties of its [4Fe-4S] cluster
(4) .
Significant among these changes is the reduction of the midpoint
potential of the cluster from -310 to -430 mV
(12, 13) and the increased accessibility of the cluster to
chelators
(14, 15) , suggesting greater solvent exposure.
These conformational changes have thus been suggested to be a
prerequisite for the intercomponent electron transfer between the FeP
and the MoFeP. While the nucleotide induced conformational change of
the FeP is required for intercomponent electron transfer, it is not
sufficient. Rather, the hydrolysis of MgATP appears to be an absolute
requirement for electron transfer. The mechanism of coupling of these
two processes is complex and probably involves multiple conformational
changes in both FeP and MoFeP
(16, 17) .
-phosphate of MgATP and in the MgATP induced
conformational change. We found that changing Lys-15 to the neutral
amino acid Gln by site-directed mutagenesis resulted in an FeP that
still bound MgADP with normal affinity, but showed a slightly reduced
affinity for MgATP binding
(18) . In the present work, we extend
the function of the A. vinelandii FeP Lys-15 by analysis of a
protein in which Lys-15 has been changed to Arg. Analysis of the K15R
FeP reveal significant differences in nucleotide interactions when
compared to the wild type orK15Q FeP. A model is presented explaining a
central function for Lys-15 in nucleotide interactions with the FeP.
Site-directed Mutagenesis, Expression, and Purification
of Altered FeP
Oligonucleotide directed mutagenesis of the
A. vinelandii nitrogenase FeP gene, nifH, was carried
out as described previously
(18, 20) . The mutated
nifH gene was incorporated into the A. vinelandii chromosome in place of the wild type nifH gene for
expression
(20, 21) . Altered FeP was expressed in A.
vinelandii cells and purified as described
(20) . Protein
concentrations were determined by using the Biuret assay
(22) with bovine serum albumin as the standard.
SDS-polyacrylamide gels were prepared as described
(18) and were
stained with Coomassie Blue.
MgATP-dependent Chelation of Fe
The chelation of Fefrom Wild Type and K15R FeP
from both the wild type and K15R FeP by
,
`-dipyridyl
was performed as described previously
(18) . Spectrophotometric
measurements were done on a Hewlett Packard 8452A diode array
spectrophotometer.
Equilibrium MgADP and MgATP Binding to Wild Type and K15R
FeP
The binding of MgATP or MgADP to the reduced wild type or
reduced K15R FeP was determined by a modification of an equilibrium
column binding technique
(18, 20) . A series of Sephadex
G-25 columns (0.7 15 cm) were equilibrated with 50 mM
Tris buffer, pH 8.0, containing 2 mM dithionite and various
concentrations of either MgADP (0-300 µM) or MgATP
(0-1500 µM). A fixed amount of FeP was loaded onto
each column and the columns were developed. The protein containing
fractions were collected and the concentration of protein was
determined by a modified Biuret method
(22) and the
concentration of nucleotide was quantified by separation and analysis
by high performance liquid chromatography
(20) . Midpoint Potential of the [4Fe-4S] Cluster in Wild Type and
K15R FeP-The midpoint potentials for the
[4Fe-4S]
/[4Fe-4S]
couple in wild type or K15R FeP were determined by
microcoulometry as described previously (12, 23). In an argon filled
glove box, reduced FeP containing 2 mM
Na
S
O
was oxidized by adding an
excess of 5.6 mM indigo disulfonate just until a blue color
remained. The oxidized FeP (FePox) was separated from small molecules
by passage through a Sephadex G-25 column (0.7
10 cm)
equilibrated with 50 mM Tris buffer, pH 8, under argon. The
concentration of FePox was determined from its absorption spectrum and
an absorption coefficient of 13.3 mM
cm
at 400 nm
(24) . Electrochemical
reductions were determined for 150-µg aliquots of FePox in 50
mM Tris buffer, pH 8.0, with 200 mM NaCl including
0.1 mM methyl viologen, 0.1 mM benzyl viologen, and
0.1 mM flavin mononucleotide as mediators. For determination
of midpoint potentials with nucleotides, either MgATP or MgADP was
added to a final concentration of 3 mM. The number of
electrons per protein was plotted against the applied potential ranging
from -150 mV to -450 mV at 30 mV increments. The data were
fit to the Nernst equation to determine the midpoint reduction
potential and the number of electrons per protein. All potentials are
relative to the standard hydrogen electrode.
EPR
Samples to be analyzed by EPR were frozen in
liquid nitrogen as described previously
(18) . MgATP was added as
a 5-fold molar excess over protein from a stock solution prior to
freezing. All spectra were recorded on a Bruker ER200D spectrometer
with an Oxford ESR9 cryostat.
Circular Dichroism Spectra of FeP Samples With or Without
Nucleotides
FeP samples (wild type or K15R; 550 µl of 69
mgml
) were desalted on a Sephadex G-25 column
(0.5
10 cm) equilibrated with 100 mM Tris buffer, pH
7.5. The FeP samples were oxidized by the addition of 20 µl of an
anaerobic 20 mM indigo disulfonate solution. The oxidized FeP
was separated from small molecules by passage through a Sephadex G-25
column (0.5
10 cm) as above. The FePox was diluted to 4 ml with
100 mM Tris buffer, pH 7.5, and 2 ml of this solution was
transferred into a 1-cm path length, argon-purged quartz cuvette fitted
with a butyl stopper. MgATP or MgADP were added as a 5-fold molar
excess over protein from a stock solution. All spectra were recorded
using a Jasco model J-500 spectropolarimeter and were baseline
corrected.
Lys-15 of FeP
Most nucleotide binding proteins
contain a series of conserved amino acids which are involved in
nucleotide interaction
(19) . The most common of these motifs is
the sequence GXXXXGKS/T (Walker A or P-loop) where several of
these amino acids have been shown to interact with the phosphate
portion of the bound nucleotide
(25) . It has been previously
shown that nitrogenase FeP from a range of bacteria all contain this
conserved nucleotide binding motif near the N terminus
(26) .
Substitution of the strictly conserved Lys of this sequence in A.
vinelandii FeP (Lys-15) with the neutral amino acid Gln resulted
in an FeP that was inactive in both electron transfer to the MoFeP and
MgATP hydrolysis
(18) . Analysis of the K15Q protein suggested
that this Lys interacted specifically with the -phosphate of MgATP
and not with the
- or
-phosphates of MgADP and was essential
to the nucleotide-induced conformational changes
(18) .
Subsequent solution of the x-ray crystal structure of the FeP revealed
that Lys-15 is located in a loop analogous to the phosphate binding
loop in other nucleotide binding proteins and that Lys-15 was located
near the phosphates of an adventitiously bound ADP in the
structure
(10) . The x-ray structure also suggested that Lys-15
might form a salt bridge with Asp-125, which has subsequently been
shown to be part of a nucleotide-induced conformational
switch
(27) . To probe the possible significance of this salt
bridge, it was of interest to change Lys-15 to the positively charged
amino acid arginine. This substitution would have the effect of
maintaining the positive charge, but would change the length of the
side chain and the possible hydrogen bonding to Asp-125.
MgATP-induced Conformational Changes in the K15R
FeP
Given the role of Lys-15 in nucleotide interaction, it was
of interest to examine the nature of MgATP interactions with K15R FeP.
MgATP binding to wild type FeP results in changes in the environment of
the [4Fe-4S] cluster which can be monitored by several
techniques. The EPR spectrum of the reduced [4Fe-4S] cluster
in wild type FeP has a rhombic line shape with signals arising from
both spin 1/2 (g = 2 region) and spin 3/2 (g = 5 region)
states
(28, 29) . Addition of MgATP to the FeP results in
a change in both the proportion of spin 1/2 and spin 3/2 signals and in
a change in the spin 1/2 signal line shape to axial (Fig. 1).
Analysis of the K15R FeP by EPR revealed a normal rhombic EPR spectrum
in the absence of nucleotide (Fig. 1), confirming that changing
Lys-15 to Arg did not result in conformational changes in the protein
that might result in a perturbation of the [4Fe-4S] cluster.
Significantly, the addition of MgATP to the K15R FeP did not result in
any changes in the EPR spectrum, suggesting a lack of
nucleotide-induced conformational changes.
Figure 1:
EPR
spectra of purified wild type FeP and K15R FeP with and without MgATP.
Purified wild type (69 mgml
) and K15R (69
mg
ml
) FeP were prepared as described under
``Materials and Methods.'' All samples contained 2
mM dithionite and were prepared and frozen under argon. All
spectra were recorded at 4 K, 9.44 GHz, and 15 milliwatts microwave
power. Where noted, MgATP was added as a 5-fold molar excess over
protein prior to freezing.
MgATP-induced
conformational changes in the FeP have also been shown to result in a
time-dependent chelation of the Fe from the cluster
by iron specific chelators
(14, 15) . This has been
interpreted to indicate an exposure to solvent of the
[4Fe-4S] cluster as a result of nucleotide binding, allowing
chelator access. The kinetics of MgATP-induced Fe
chelation from both wild-type and K15R FeP revealed two points.
First, the K15R FeP does not show any Fe
chelation
prior to nucleotide addition, confirming that changing Lys-15 to Arg
does not result in conformational changes that expose the
[4Fe-4S] cluster to the chelator. Second, the addition of
MgATP does not result in Fe
chelation from K15R FeP,
consistent with a lack of nucleotide-induced conformational changes.
MgATP and MgADP Binding to the K15R FeP
The lack
of changes in the EPR line shape and the lack of a nucleotide
stimulation of Fe chelation for the K15R FeP is
similar to results obtained with the K15Q FeP, suggesting in both cases
that MgATP binding does not result in conformational changes
communicated to the [4Fe-4S] cluster. The lack of a
conformational change in the K15Q FeP was not the result of a lack of
MgATP binding since K15Q FeP was found to still bind MgATP, although
with a somewhat reduced affinity compared to wild-type FeP (33% of
wild-type)
(18) . It was, therefore, necessary to examine the
affinity of MgATP binding to the K15R FeP. Fig. 2(panel
A) shows the ratio of MgATP bound per FeP at different MgATP
concentrations determined by an equilibrium column binding method.
Fitting the wild type FeP data to the Hill equation revealed an
apparent dissociation constant (K
) of 571
µM. Under the same conditions, the K15R FeP showed no
detectable binding of MgATP (Fig. 2, panel A). The
sigmoidal nature of MgATP binding to the wild type FeP suggests
cooperativity between the binding of nucleotides to the two binding
sites, as has been previously suggested (15, 30, 31). The fit to the
Hill equation resulted in a cooperativity factor of 2.1, supporting
strong cooperativity in the binding of the two nucleotides. Given the
strong cooperativity, the observed apparent dissociation constant
(K
) represents the product of the two
true dissociation constants.
Figure 2:
MgADP and MgATP binding to wild type and
K15R FePs. Equilibrium column binding of nucleotides to wild type FeP
or K15R FeP were performed as described under ``Materials and
Methods.'' Panel A, MgATP binding to wild type ()
or K15R (
) FeP. Panel B, MgADP binding to wild-type
(
) or K15R (
) FeP. The ratio of the concentration of
nucleotide bound divided by the FeP concentration was plotted against
the concentration of free nucleotide. In each case, 4 mg of FeP was
loaded onto the column and collected into one fraction. The data were
fit to the Hill equation (F =
M
[S]/(K + [S])),
where F is the fraction of nucleotides bound per FeP, M is the maximum number of nucleotides bound per FeP, [S]
is the free nucleotide concentration, and n is the
cooperatively factor. For wild type FeP, apparent dissociation
constants (K) of 143 µM for MgADP and 571
µM for MgATP and values of n of 2.0 and 2.1 were
determined.
We previously found that the affinity
of the K15Q FeP for MgADP binding was nearly identical to that observed
for wild-type FeP
(18) . Fig. 2(panel B) shows
the ratio of MgADP bound per FeP at different MgADP concentrations
determined by an equilibrium column binding method. Fitting the wild
type FeP data to the Hill equation revealed an apparent
K of 143 µM. Again, the
sigmoidal nature of the data suggested cooperativity in the binding of
MgADP to the two binding sites, which was supported by a value of n = 2.0 for the fit to the Hill equation. In contrast to the
binding of MgADP to the wild type FeP, the K15R FeP showed an extremely
low affinity for MgADP even at concentrations as high as 300
µM. This extremely low affinity for MgADP contrasts with
the normal affinity observed for the K15Q FeP
(18) . Control
binding experiments in which Mg
was omitted revealed
no detectable binding for either the wild type or K15R FePs.
MgADP-induced Conformational Changes in the K15R
FeP
Binding of MgADP to the FeP has been shown to induce changes
in its [4Fe-4S] cluster
(12, 34) . These
changes are clearly different from those induced by MgATP. For example,
addition of MgADP does not result in EPR line shape changes or in
chelation of the iron from the cluster. MgADP binding does result in
changes in the circular dichroism (CD) spectrum of the oxidized FeP
which is different from changes induced by MgATP binding. Likewise,
MgADP binding results in a change in the midpoint reduction potential
of the [4Fe-4S] cluster from -310 to -430 mV
(12). We examined the K15R FeP by both CD spectroscopy and
microcoulometry as additional ways to probe both the integrity of the
[4Fe-4S] cluster and as more sensitive ways to detect
MgADP-induced conformational changes.
to the 1
state.
The results for reduction of the K15R FeP are shown in Fig. 3,
panel B. In the absence of nucleotides, the midpoint reduction
potential for the K15R FeP was found to be -290 mV, similar to
the wild type FeP. This data confirms that changing Lys-15 to Arg does
not result in detectable changes in the [4Fe-4S] cluster. In
contrast to the results for wild type FeP, the inclusion of MgATP or
MgADP to K15R FeP was found to have no effect on the midpoint potential
of the K15R FeP (Fig. 3, panel B).
Figure 3:
Reduction curves for wild type FeP and
K15R FeP with or without nucleotides. Microcoulometric electrochemical
reduction of oxidized wild type or K15R FeP was performed as described
under ``Materials and Methods.'' Panel A, reduction
curves for wild-type FeP in the absence of nucleotides (), in the
presence of 3 mM MgATP (
) or 3 mM MgADP
(
). Midpoint potentials were determined to be -290,
-420, and -430 mV, respectively. Panel B,
reduction curves for K15R FeP in the absence of nucleotides (
),
in the presence of MgATP (
), or in the presence of MgADP (
).
All reduction midpoint potentials for K15R FeP were found to be
-290 mV. Potentials are reported relative to the standard
hydrogen electrode (SHE). Fits of the data to the Nernst equation are
shown. In all cases, the maximum number of electrons was between 0.9
and 1.0 electrons per FeP.
Previous studies
have shown that the circular dichroism (CD) spectrum of FeP in the
visible wavelength region is a sensitive measure of the environment
around the [4Fe-4S] cluster
(34) . The addition of
MgATP or MgADP to the FeP results in significantly different CD
spectra, indicating different conformational changes.
Fig. 4
(panel A) shows the CD spectra for oxidized,
wild-type FeP both with and without added MgADP or MgATP. As
reported
(34) , the binding of nucleotides results in
significantly different CD spectra. A similar experiment with K15R FeP
(Fig. 4, panel B) revealed that in the absence of added
nucleotides, the oxidized K15R FeP CD spectrum was nearly identical to
the oxidized wild-type FeP. These results support the conclusion that
changing Lys-15 to Arg does not change the environment around the
[4Fe-4S] cluster, even when the [4Fe-4S] cluster is
in the oxidized state. As predicted, the addition of MgATP or MgADP to
the K15R FeP did not result in significant changes in the CD spectrum.
This supports the conclusion that MgATP and MgADP do not induce
conformational changes within the K15R FeP. A small decrease in
ellipticity was observed at about 360 nm for the K15R FeP in the
presence of MgADP.
Figure 4:
Circular dichroism spectra of wild type
FeP and K15R FeP in the presence or absence of nucleotides. The visible
circular dichroism (CD) spectra of oxidized wild type FeP or K15R FeP
were recorded as described under ``Materials and Methods.''
Panel A, CD spectra of wild type FeP in the absence of
nucleotides (WTox) or in the presence of MgADP or MgATP.
Panel B, CD spectra of K15R FeP in the absence of nucleotides
(K15Rox) or in the presence of MgADP or MgATP. All spectra
were baseline subtracted.
MoFeP complex. The FeP
alone is known to bind 2 MgATP, one per subunit. This binding to the
FeP results in conformational changes which appear to make the FeP
competent for interaction with the MoFeP. The MgATP-induced
conformational changes have been monitored by several methods, most of
which detect changes in the [4Fe-4S]
cluster
(12, 29, 34, 35, 36, 37) .
From the x-ray crystal structure and previous site-directed mutagenesis
studies on FeP, it is known that the nucleotide-induced conformational
changes within the FeP must be communicated over a distance of at least
19 Å from the nucleotide binding site to the [4Fe-4S]
cluster
(10, 18) . These changes have been suggested to
be a prerequisite to the correct docking of the FeP to the MoFeP prior
to MgATP hydrolysis and intercomponent electron
transfer
(16, 17) . Once the FeP
MoFeP complex is
formed, the hydrolysis of MgATP is somehow linked to the transfer of
the electron from the FeP to the MoFeP and possibly directly to
substrate reduction
(16, 18) . The exact mechanisms for
how MgATP activates the FeP for binding to the MoFeP and how MgATP
hydrolysis is linked to electron transfer remains largely undefined. It
is becoming clear, however, that conformational changes within the FeP,
and probably within the MoFeP, play an integral part in this mechanism.
-phosphate of MgATP. The
K15Q FeP did not undergo conformational changes as detected by EPR or
,
`-dipyridyl chelation. This lead to the conclusion that
Lys-15 played a central role in nucleotide-induced conformational
changes. Analysis of the x-ray structure of the FeP suggests that the
-amino group of Lys-15 forms a salt bridge with the carboxylate of
Asp-125 (Fig. 5A). Asp-125 is part of a second
nucleotide binding pattern often observed in nucleotide binding
proteins (Walker B)
(18, 27) . Changing the conserved
Asp-125 to Glu (D125E) was found to have dramatic effects on
nucleotide-induced conformational changes within the FeP, suggesting
that Asp-125 could provide a linkage from the nucleotide binding site
to the [4Fe-4S] cluster
(27) . This linkage has been
suggested to be mediated through the amino acids between Asp-125 and
Cys-132, a ligand to the [4Fe-4S] cluster
(27) .
Figure 5:
Stereoscopic view of the possible
interactions of FeP Lys or Arg at position 15 with Asp-125. Molecular
models were generated using the program Molecular Images (US Science,
San Diego, CA) from the x-ray coordinates of the A. vinelandii FeP (10). A, model of the possible interactions of Lys-15
with Asp-125 in the proposed phosphate binding site of the FeP.
B, substitution of Lys-15 with Arg showing possible
interactions of the Arg side chain with the carboxylate side chain of
Asp-125.
The
results presented in this work on the properties of an FeP in which
Lys-15 has been changed to an Arg can be integrated into the current
model of the phosphate binding site and the function of Lys-15 in this
interaction. In the absence of a bound nucleotide, Lys-15 and Asp-125
would be expected to form a salt bridge, neutralizing the charged
residues in the interior of the FeP and stabilizing conformational
changes mediated through Asp-125 (Fig. 5A). Upon binding
of MgATP to the FeP, the salt bridge between Lys-15 and Asp-125 would
be expected to be broken, with Lys-15 now interacting with the
negatively charged phosphate groups (- and
-) and Asp-125
with the Mg
bound to the
- and
-phosphates
of MgATP. The movement of Asp-125 to accommodate the bound nucleotide
could result in a conformational change in the [4Fe-4S]
cluster by changes in the relative position of Cys-132
(27) . The
K15Q FeP would not be expected to form this salt bridge to the Asp-125.
To probe the potential importance of this salt bridge, it was of
interest to change Lys-15 to the positively charged amino acid Arg.
This change would maintain the positive charge, but would change the
nature of the interaction. A significant finding of the present work is
that changing Lys-15 to Arg in the FeP greatly reduces K15R FePs
affinity for both MgATP or MgADP. In addition, changing Lys-15 to Arg
was found to prevent nucleotide-induced conformational changes. These
results can be integrated into the model outlined above. Arg-15 is
expected to form a salt bridge with Asp-125, as did the wild type
Lys-15 (Fig. 5B). The nature of the interactions of
Arg-15 with Asp-125 and with nucleotides is, however, clearly different
from Lys-15. One possibility is that Arg-15 forms a stronger salt
bridge with Asp-125, preventing the opening of the phosphate site to
accommodate the phosphate groups of the incoming nucleotide. Consistent
with this model, the guanidino group of Arg is more basic than the
-amino group of Lys and also has the potential to form a hydrogen
bond with the carboxylate of Asp as well as a charge interaction. The
net effect could be to ``lock'' the active site closed,
preventing either MgATP or MgADP from binding. Another related
explanation is that to maintain an optimal distance for the salt bridge
between Asp and the longer Arg, the protein backbone must shift to
accommodate the 1-Å increase in length. This movement could move
other amino acids critical for nucleotide binding out of place, thus
having multiple effects on binding energetics. It should be noted,
however, that no conformational changes in the EPR, CD, or midpoint
potential of the cluster could be detected for the K15R FeP, suggesting
that any conformational changes resulting from the change of Lys-15 to
Arg are either very small or confined to the environment around the
phosphate site. The formation of this tighter interaction between
Arg-15 and Asp-125 could stop the nucleotide-induced conformational
change proposed to be mediated through Asp-125.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.