(Received for publication, September 2, 1994; and in revised form, October 26, 1994)
From the
The geometries of the metal sites in cadmium-substituted azurins
have been investigated by Cd perturbed angular
correlation (PAC). The study includes wild type azurin as well as
Met
mutants of azurin, where methionine has been
substituted by Ala, Asn, Asp, Gln, Glu, and Leu.
The nuclear
quadrupole interaction of wild type azurin analyzed in the angular
overlap model is well described as coordination of His,
His
, and Cys
and cannot be described by
coordination of Met
and/or Gly
.
For most of the mutants, there exist two coordination geometries of the cadmium ion. With the exception of the Glu and Asp mutants, one of the conformations is similar to the wild type conformation. The other coordination geometries are either best described by a coordinating water molecule close to the original methionine position or by coordination by the substituting amino acid. These experiments show that even though the methionine does not coordinate it plays an important role for the geometry of the metal site.
The nuclear
quadrupole interaction of stellacyanin was also measured. The value
resembles the most prominent nuclear quadrupole interaction of the
Met
Gln mutant of Alcaligenes denitrificans azurin, indicating that the structures of the two metal sites are
similar.
Azurin and stellacyanin belong to the family of blue copper proteins with copper in a type 1 geometry. From x-ray diffraction (1, 2, 3) , it is known that in azurin 2 histidines and 1 cysteine are coordinating to the copper ion with the copper ion in the plane formed by the three ligands. This geometry is strained for Cu(II) as well as for Cu(I). Two additional amino acids are relatively close, namely methionine 121 with its sulfur at a distance of 3.1 Å and glycine 45 of which the backbone oxygen is at a distance of 3.0 Å.
The methionine residue is conserved not only in the azurins, but also in pseudoazurins, plastocyanins, and cucumber blue protein (see, e.g., (4) ). It is therefore believed to play an important role in the function of the blue copper proteins. One possible function of the methionine could be a fine tuning of the redox potential(5) . Stellacyanin is interesting in this context because it does not contain a methionine residue at all. The present paper addresses the question as to whether any conformational changes occur at the metal site in cadmium-substituted azurin upon substitution of the methionine residue with other amino acids.
The structure of the metal site in wild type
azurin from Pseudomonas aeruginosa and Alcaligenes
denitrificans does not change when the copper ion is
reduced(6, 7) . In contrast, the structure of the
metal site in the Met
Gln mutant of (A.
denitrificans) azurin depends strongly on the oxidation state of
the copper ion(8) . For this azurin mutant Cu(II) is
4-coordinate (His
, His
, Cys
,
and Gln
), whereas Cu(I) is almost linearly coordinated
with Cys
and His
as strong ligands and two
weak interactions with Gln
and His
(both at
a distance of 2.7 Å). This indicates that part of the function of
the methionine residue is to stabilize the conformation around the
copper and thereby to facilitate the exchange of electrons(8) .
The effect of substitution of copper with zinc in azurin from P.
aeruginosa has been investigated previously(9) . The
movement of the atoms upon substitution was minor, but resulted in a
distorted tetrahedral structure, where the zinc ion is now coordinating
to the carbonyl oxygen from Gly with a binding distance of
2.3 Å. The distance to the Met
S
is 3.4
Å, which means that a bonding interaction of methionine with zinc
is negligible. The effect of substitution by cadmium has been studied
for A. denitrificans azurin only. (
)The distance
between cadmium and glycine was 2.8 Å and between cadmium and
methionine 3.2 Å. This means that cadmium behaves more like
copper than zinc in this protein.
It should be noted when comparing
cadmium to copper that cadmium has the same valence as Cu(II) but a
closed shell d configuration like that of Cu(I).
Therefore, it is possible that cadmium in some situations will
coordinate in a way similar to Cu(I), while in other situations it will
coordinate like Cu(II).
The technique of perturbed angular
correlations of -rays (PAC) (
)is described in detail in
the review by Frauenfelder and Steffen (10) and with special
emphasis on biological application by Bauer(11) . An
introduction to the technique applied to metal proteins is given in (12) . Here, only a very schematic introduction will be given.
PAC provides a means of measuring the nuclear quadrupole interaction
at the site of the nucleus. The NQI is the interaction of the electric
quadrupole moment of the nucleus and the electric field gradient
tensor, V, due to the surrounding charge
distribution. For samples of identical, randomly oriented molecules,
with no rotational diffusion, two parameters can be measured: the
magnitude of the electric field gradient,
V
, and the asymmetry of
the electric field gradient,
. (A more detailed description of
the different parameters derived from the PAC spectrum is given under
``Materials and Methods.'')
PAC requires an isotope that
decays by emitting two successive -rays. (The principle of the
technique is illustrated in Fig. 1.) The detection of the first
-ray in a specific direction selects an ensemble of nuclei with
non-isotropic orientation of nuclear spin. The direction of the
emission of the second
-ray depends on the orientation of the
nuclear spin, and the second
-ray can therefore be emitted in a
non-isotropic way relative to the first. For
Cd this
intrinsic probability of detecting the second
-ray at an angle of
180° relative to the first
-ray is about 26% higher than at an
angle of 90°. If, however, the nucleus is under the influence of
external forces (for example from ligands) during the time between the
emission of the two
-rays, this ``angular correlation''
is ``perturbed.'' Classically the interaction of the electric
field gradient with the nuclear quadrupole moment causes a torque on
the nucleus resulting in a precession of the nuclear spin. Since the
emission direction of the second
-ray is related to the direction
of the spin, this causes oscillatory variations in the angular
correlation between the two
-rays. Quantum mechanically, this
interaction causes an energy splitting of the intermediate energy of
the nucleus. This energy splitting of the intermediate level is
reflected in a PAC spectrum. In the case of
Cd, the
intermediate level has spin 5/2 and the energy splits into three levels
under the influence of an electric field gradient. Between these three
levels, three energy differences exist. Each energy difference,
E
, gives rise to an oscillation in
the PAC spectrum,
=
2
E
/h, where h is Planck's constant. In the case of identical, static, and
randomly oriented molecules, the perturbation function measured by PAC, G
(t), is as shown by .
Figure 1:
Schematic illustration of a PAC
experiment. The top right illustrates the decay of Cd. In the top left the effect of the electric
field gradient on the intermediate energy level (I =
5/2) is illustrated. The electric field gradient causes the energy to
split into three (double degenerate) levels depending on the quantum
number for the z component of the nuclear angular momentum, m. (
denotes Planck's
constant divided by 2
.) The bottom part illustrates the
principle of the experiment. The present work was carried out with six
detectors (only three are shown) in an octahedral arrangement whereby 6
combinations of 180° and 24 combinations of 90° are achieved.
For each pair of detectors, the probability of detecting the second
-ray at the angle
is detected as a function of the time
elapsed between the two
-rays.
Each frequency is proportional to
V
with a proportionality
constant depending on
. The four amplitudes, a
to a
, also depend on
. Thus, from G
(t), V
and
can be determined(11) . An example of a
PAC spectrum (G
(t)) and the Fourier
transform are shown in Fig. 2. In general, it is necessary to
include rotational diffusion and frequency distributions in the
description of the spectra. Further details can be found
elsewhere(13) .
Figure 2: PAC spectrum (top) of cadmium-substituted wild type azurin from P. aeruginosa and Fourier transform (bottom). The solid line in the PAC spectrum represents the result of a least squares fit of a theoretical perturbation function to the data points. The solid and dashed lines in the Fourier transform represent the theoretical and experimental PAC spectra, respectively.
It is the fact that the surrounding charge
distribution is reflected in the electric field gradient, and in
particular the possibility through the angular overlap model (AOM) (14) to calculate the electric field gradient for a given
particular coordination sphere, that makes Cd PAC a
useful tool for studying metal sites in proteins.
The binding properties of cadmium
to the apoprotein were investigated using Cd with a
half-life of 1.3 years, with nonradioactive cadmium acetate added as
carrier. These experiments showed that the binding kinetics for cadmium
are much slower than for copper, particularly if the cadmium was added
at about pH 6. However, if the binding was performed at a pH of about
7.5, more than 90% of the metal sites were reconstituted by cadmium
after 2 h of incubation time. Due to the slow binding kinetics of
cadmium to the apoprotein and the short half-life of
Cd
(49 min), most of the PAC experiments were carried out without cadmium
carrier. In the cases where cadmium carrier was used,
Cd
was added to cadmium chloride or cadmium acetate in a 1:1 stoichiometry
of Cd
to protein. Protein-bound cadmium was separated
from free cadmium by G25 chomatography. For the experiments without
carrier, the G25 chomatography was omitted. In all cases the metal
incubation was carried out at room temperature for 5-60 min.
In order to slow down the rotational diffusion, which would
otherwise damp the oscillations in the perturbation function, sucrose
was added at a concentration of 55 weight % and the sample was cooled
to 4 °C. Buffers used were either Tris or HEPES, or a mixture of
both, with a molarity between 10 and 200 mM. The pH was
measured at room temperature and was between 7.2 and 7.9, except for
three experiments where the pH was varied on purpose: Met
Asp, pH 10.0; and Met
Leu, pH 5.8 and
pH 9.0. The actual pH values at 4 °C are about 0.7 pH units higher
(estimated from the known temperature dependence of the buffers). For
the experiments with pH 9 and 10, additional corrections for the
presence of sucrose were made. The sample volumes varied from 25 µl
to 2 ml.
Powder x-ray diffraction analysis of Cd(tda)HO was
carried out with a Phillips x-ray diffraction unit using Co-K
radiation and a XPLOT software program packet (CSIRO 1990).
Analysis of the resulting pattern obtained from crystals of
Cd(tda)H
O used in the PAC experiment revealed 26 lines in
the interval from 10° to 50°. All lines were indexed using the
computer program TREOR giving a monoclinic unit cell with the dimension a = 8.008(7), b = 5.361(4), c = 9.143(7) (all in Å), and
= 115.92(6).
This is in agreement with the unit cell determined previously in a
single crystal x-ray study(17) .
Cd was
produced by bombarding
Pd with
-particles at the
cyclotron at either the Niels Bohr Institute or at the National
Hospital, both located at Copenhagen. The sample was then transported
to the Royal Veterinary and Agricultural University, where all
chemistry was carried out. Palladium and cadmium were separated by the
procedure described elsewhere(12) . This reduces the amount of
palladium to about 10
mol.
The Met
Glu experiment was repeated at the ISOLDE facility at
CERN, Geneva, Switzerland. The sample preparation was similar to the
one at Copenhagen. However, at CERN the solution was saturated with
sucrose and the temperature was 0 °C. These differences are not
expected to affect anything but the rotational diffusion time.
The
PAC spectrometer consists of six BaF scintillator detectors
with conical fronts arranged such that each detector is situated at the
face center of an imaginary cube. The sample is positioned at the
center of this cube. It is a built-out version of the ``PAC
camera'' described previously(18) . In addition, the
instrument has a facility for automatic adjustment of detector-sample
distance, thus adjusting to an optimal counting rate until the
sample-detector distance reaches its minimum value.
The temperature of the sample was controlled by a Peltier element in thermal contact with the sample. The temperature can be set between -10 °C and 40 °C with an accuracy of ±2 °C.
Six combinations of 180° coincidence spectra and 24 combinations of 90° coincidence spectra were collected simultaneously. In each coincidence spectrum, the background due to accidental coincidences is subtracted and the perturbation-function is formed according to
where W(180°,t) denotes the sixth root of
the product of the six 180° spectra after background subtraction
and zero-point adjustment, and W(90°,t) denotes
the 24th root of the product of the 90° spectra. The time
resolution was about 850 ps full-width at half-maximum at the energies
of Cd.
is the magnitude of the
nuclear quadrupole interaction and is proportional to the numerically
largest diagonal element of the electric field gradient,
V
, by the relation shown in .
is the asymmetry parameter and is defined as shown in .
(In the two equations, above the electric field gradient tensor
is assumed diagonal, and V
V
V
.)
and
depend on the coordinating ligands as well as their positions.
If the partial nuclear quadrupole interaction is known for each ligand
as well as the geometry of the metal site, they can be calculated in
the AOM(14) .
/
is
used for describing small inhomogeneous frequency distributions due to
variations from one molecule to another with respect to conformations
of the probe sites. It is assumed that the variations can be described
by identical asymmetry parameters,
, and a Gaussian distribution
of
with the width
/
. Thus, deviations from zero
indicate that the
Cd nuclei are subjected to a
distribution of surroundings. In general, shifts in angular position of
a ligand by only a few degrees will be quite enough to show up as a
frequency distribution of a few percent.
is the
correlation time of the rotational diffusion induced by the Brownian
motion. For a rigid spherical molecule with volume V, embedded
in a solution with viscosity
, the correlation time is:
=
V/(kT), where k is Boltzmann's constant and T is the absolute
temperature (19) .
A is the amplitude
of the perturbation function. It is a property of the radioactive
nucleus, and for
Cd it has a maximum value of
+0.16(20) . The experimental value is normally
significantly lower due to solid angle correction factors (sample
volume, detector sizes, and sample-detector distance). In the case
where inequivalent sites are present, each NQI must be included in G
(t) with the relative populations of the
nuclear quadrupole interactions.
All PAC spectra were analyzed with
both one and two nuclear quadrupole interactions, respectively. A
single nuclear quadrupole interaction was chosen unless the reduced
of the fit with two NQIs was significantly better
(the requirement was that the probability of the one-component fit was
less than 5% based on an F-test). This nuclear quadrupole interaction
was then analyzed in the angular overlap model.
The rotational diffusion time for the mutants was taken from the fit of wild type azurin and subsequently fixed.
In the case of the Met
Glu experiments, the ISOLDE experiment was used to
establish all parameters except the amplitude, the relative
populations, and the rotational diffusion time. The result of the
experiment carried out at the Veterinary and Agricultural University
was then analyzed with these parameters fixed (see Table 1).
The two parameters
describing the experimental nuclear quadrupole interactions
( and
) of the proteins were analyzed in the
following way. The theoretical values of
and
were calculated using the published crystal structure and the partial
nuclear quadrupole interactions of the ligands. If the calculated NQI
of one of the tested structures lies within the uncertainty of the
experimental
and
, then this structure is
considered probable for the cadmium coordination. If this is not the
case, either the copper protein structure is different from the cadmium
protein structure or another possible explanation is that the limited
resolution of the x-ray structure and/or the limited accuracy in the
partial nuclear quadrupole interactions leads to a small but
significant difference. Since such differences between theory and
experiment should not exclude a structure, they were taken into account
by performing a least
AOM fit minimizing
defined as shown by .
In the equation above the indexes ``m'' and
``f'' refer to ``measured'' and
``fitted'', respectively. ``c'' refers to
values ``calculated'' from the fitted parameters.
refers to the standard deviation of the parameter (see below).
and
are the polar and azimuthal angles of the
different ligands. The measured values are taken from the x-ray
structure of native azurin from P. aeruginosa at pH 5.5 and
9.0(3) , except for the experiment on Met
Gln from A. denitrificans where the structures published for
Cu(I) as well as Cu(II) were used(8) .
The coordinate system
was chosen such that the coordinating nitrogen of His was
positioned on the z axis (
= 0° and
= 0°) and N
of His
was lying in the z-x-plane (
= 0°). The rest of the
polar and azimuthal angles were calculated from the average of the high
pH and low pH structure (3) with an estimated standard
deviation of 5° (
(
) and
(
)). When a water
molecule was placed at the position of the mutated amino acid, the
standard deviations of the angles were chosen to be 10°. This value
was also chosen as standard deviation for calculations including a
coordinating atom from the substituting amino acid. The Met
Gln mutant of A. denitrificans azurin was
analyzed based on the x-ray structure of this mutant with copper in the
reduced and oxidized state, respectively(8) . The coordinate
system was chosen as described above, and
(
) and
(
) were chosen as 5°.
refers to
the partial nuclear quadrupole interaction of the ligands. Here, we
used the measured values published in (14) (water, imidazole,
carbonyl, and carboxylate) and (22) (cysteine) and the partial
nuclear quadrupole interaction of methionine determined from
thiodiacetate. The values used are: (in Mrad/s): carbonyl oxygen, 161;
monodentate carboxylate oxygen, 245; bidentate carboxylate oxygen, 175;
water oxygen, 207; methionine sulfur, 102; cysteine sulfur, 300;
imidazole nitrogen, 95; amine nitrogen, 139.
The experimental
standard deviations of are between 0.2 and 0.9
Mrad/s, and the experimental standard deviations of
are between
0.001 and 0.02. If these values had been used in the AOM fit, too much
emphasis would be put on the experimental nuclear quadrupole
interactions compared to the x-ray data. This is related to
contributions to the nuclear quadrupole interaction that are not taken
into account by the AOM model, such as more distant contributions to
the NQI. A unit point charge at a distance of 5.4 Å, for example,
will contribute with a partial nuclear quadrupole interaction of about
10 Mrad/s which is unaccounted for. Therefore, in the analyses, the
experimental values of
and
were used with
standard deviations of 10 Mrad/s and 0.05, respectively. For the same
reason, the partial nuclear quadrupole interactions are taken into
account with standard deviations of 10 Mrad/s, although the published
standard deviations are generally on the order of 4-8 Mrad/s.
These choices will, of course, affect the absolute values of the
. However, we do not believe that a different choice
of standard deviations will have any major effect on the relative
values of
.
The AOM fits are a way of fitting the
x-ray structure and partial nuclear quadrupole interactions to the
measured nuclear quadrupole interaction, but at the same time ensuring
that the structure and partial nuclear quadrupole interactions cannot
deviate too much from the starting point. The number of degrees of
freedom is 2, and so the reduced is defined as
/2.
The PAC spectrum and the Fourier transform of wild type
azurin from P. aeruginosa are shown in Fig. 2. The
Fourier transforms of the PAC spectra of stellacyanin and Met
Gln from A. denitrificans are shown in Fig. 3. Fig. 4shows the Fourier transforms of the PAC
spectra measured for the Met
mutants of azurin from P. aeruginosa. For the mutants Met
Asp
and Met
Leu, the pH was varied (Met
Asp pH = 7.2 and 10.0; Met
Leu
pH = 5.8, 7.2, and 9.0). Lower pH values were not applied due to
slow uptake of cadmium by the proteins at low pH. The pH was varied in
order to see whether the two different nuclear quadrupole interactions
displayed any variation in NQI or in relative population as a function
of pH. The experiments showed no detectable difference. Therefore
spectra collected at different pH were added before the final least
analyses were carried out. The results of the least
analyses are given in Table 1. All of the
analyses listed in this table gave a satisfactory
.
Figure 3:
Fourier transform of PAC spectra of
cadmium-substituted stellacyanin from Japanese lacquer tree (R.
vernicifera) (bottom) and of MetGln azurin
from A. denitrificans (top).
Figure 4:
Fourier transforms of PAC spectra of
various cadmium-substituted Met mutants of azurin from P. aeruginosa.
The total amplitude, A, was between 0.05 and
0.09. The rotational diffusion time of stellacyanin was 530(170) ns,
that of the Glu mutant in saturated sucrose at 0 °C was 444(6) ns,
and for wild type azurin in 55% sucrose at 4 °C the correlation
time was 200(20) ns. The rotational diffusion time of wild type azurin
was fixed in the fit of all other Met
mutants.
It should be noted that
the number of degrees of freedom is only 2 for the fits in Table 3and Table 4. This means that the probability
distribution is very wide. The median of reduced is
0.693 (5% will be above 2.996)(23) .
No satisfactory AOM
fits of the Met
Glu mutant could be achieved when
the structure of the metal site was based on the wild type structure
(see Table 3). The crystal structure of this mutant at pH
6-6.5 has, however, been determined recently. (
)This
shows that the Glu coordinates by one oxygen. This structure was then
used for an additional AOM analysis of one of the two NQIs determined
for Met
Glu. The result of this analysis are given
in Table 5. Here, the fitted angles are also listed, since a
rather significant change in structure is necessary compared to the
copper structure in order to get a satisfactory fit.
This is the first time a 3-coordinate cadmium complex has been measured by PAC. The existence of 3-coordinate complexes has, however, been demonstrated recently(25, 26, 27) .
The Met
Asn gives almost
the same NQI as wild type but with a significantly larger
/
, indicating a less rigid
structure. The NQI is interpreted as a 3-coordinate complex like the
wild type (Table 3). However, it cannot be excluded that the
asparagine or a water molecule coordinates (Table 3).
The
Met
Asp data can be fitted with one NQI only. This
NQI, however, is significantly different from the wild type NQI and
cannot give a satisfactory fit by assuming 3-coordination by the wild
type ligands. It is therefore assumed that cadmium in this case is
4-coordinate with aspartate or water as the fourth ligand. This is
further supported by the AOM analysis in Table 3and Table 4. For this mutant, a significantly higher
/
was also found, again
indicating a less rigid site.
For the Met
Gln (A. denitrificans), Met
Glu, Met
Ala, and Met
Leu mutants, it was
necessary to include two different NQIs in the data analysis. With the
exception of the Glu mutant, one of the NQIs could be described as a
wild type-like 3-coordinate complex.
For the Met
Gln mutant of P. aeruginosa, only one nuclear
quadrupole interaction was found. This NQI was analyzed based on the
wild type structure of (P. aeruginosa) azurin (Table 3).
The result of this analysis is that it is not possible based on this
NQI to discriminate between 3-coordinate cadmium as in wild type azurin
and 4-coordinate cadmium where the fourth ligand is either water or the
amide oxygen of Gln.
For the Met
Gln mutant of A. denitrificans azurin, two geometries with quite close NQIs
were found. The x-ray diffraction work (8) on the Cu(I) and
Cu(II) crystals showed no evidence of presence of more than one form in
the same crystal but the metal site geometry depends strongly on the
oxidation state of the copper ion. The two NQIs were analyzed based on
the structures of the Met
Gln mutant of A.
denitrificans (Table 4). The analysis shows that the cadmium
ion cannot be 4-coordinate in the same geometry as Cu(II). For the NQI
with
= 309.6 Mrad/s and
=
0.496, the fit with the lowest
is 3-coordinate with
the same angular distribution of the ligands as in the Cu(II) crystal.
Similarly, the NQI with
= 304.0 Mrad/s and
= 0.392 is best fitted with four ligands in the same
angular positions as in the Cu(I) crystal (Table 4). Other
explanations are also possible according to the table. It is important
to note that the AOM does not give any information on the bonding
distance of the ligands. We interpret the result of the AOM analysis as
follows. For cadmium there exist two conformations; one of them could
be 4-coordinate with 2 His, 1 Cys, and the amide oxygen or nitrogen of
Gln, and the other probably 3-coordinate with 2 His and 1 Cys, but it
cannot be excluded that a water molecule is binding in one of the two
forms. Partly due to the lack of information on the
of the amide nitrogen, the AOM analyses are not conclusive on
whether the nitrogen or oxygen of Gln is coordinating.
The
Met
Gln mutants of A. denitrificans and P. aeruginosa are significantly different. The difference is,
however, not bigger than what could be expected by comparing nuclear
quadrupole interactions based on the x-ray structure of wild type A. denitrificans azurin (not shown) with calculations based on
wild type P. aeruginosa azurin (Table 2). Thus the
difference might simply reveal differences in angular distribution of
the ligands and not necessarily differences in which ligands
coordinate.
The two mutants Met
Ala and
Met
Leu both have an NQI that is best described as
a wild type-like 3-coordinate complex; however, they can also be
described with an additional coordinating water molecule. The other NQI
is in both cases best described by a coordinating water molecule. The
NQI of Met
Leu that is best described by a
coordinating water molecule has a much higher
/
than wild type azurin. This
further supports the coordination of a water molecule, which could be
less restricted in its position than ligands from the protein.
The
Met
Glu mutant could not be analyzed
satisfactorily in the AOM model with any of the proposed structures ( Table 3and Table 4) if the structure was taken from the
wild type x-ray structure. This means that the structure must have
changed more than the ±5° allowed for in the fit (±
10 for the mutated amino acid) or that the right coordination sphere
(for example bidentate coordination by Glu) was not tested. The x-ray
structure of the Met
Glu mutant of P.
aeruginosa has been determined recently (28) . (
)The crystals contain four subunits, and the variation in
angles between pairs of ligands from one subunit to another subunit
makes it necessary to use a standard deviation in the polar angles of
±10°. This structure is 4-coordinate with the carboxyl group
of the Glu as the fourth ligand. The Met
Glu-NQI
with the highest
was analyzed in the AOM model and
the result is listed in Table 5. It shows that this NQI can
indeed be interpreted as 4-coordination by the 2 histidines, the
cysteine, and the Glu, but also that this fit gives a rather big change
in the angle between the two histidines, being the main reason for the
very high reduced
. The lower NQI measured could
possibly be the Glu coordinating in a bidentate fashion. This
hypothesis was not tested by AOM calculations.
Met
Asp has been studied by x-ray absorption fine structure
(XAFS), electron paramagnetic resonance, and optical
spectra(31) . The main conclusion by the XAFS is the occurrence
of an additional oxygen ligand at a distance of 2.26-2.23 Å
at pH 5 as well as pH 8. EPR, in contrast, shows no detectable
difference at pH 5 compared to wild type and at pH 8 shows a rather big
change in g-parameters as well as A
and A
. The PAC experiments were carried out at pH 7.2
and pH 10.0. There was no detectable difference between these two
experiments, and the nuclear quadrupole interaction is well described
by a coordinating carboxyl group from the aspartate but can also be
explained by a coordinating water molecule ( Table 3and Table 4). Thus, the results are in good accordance with the
results at pH 8 achieved by XAFS and EPR. The pH in the PAC experiment
was not lowered further due to the low affinity of cadmium ions to
azurin at low pH.
Met
Asn shows a significant
difference in EPR parameters as compared to wild type(30) .
This is in contrast to the PAC results. A possible explanation could be
a difference in the coordination chemistry between Cu(II) and Cd(II)
for this mutant.
Met
Gln from A.
denitrificans was studied by Romero et al.(8) .
Their study showed that the mutant had ultraviolet-visible and EPR
characteristics of a type I site but the spectroscopic details and
midpoint potential differ significantly from wild type. This is
generally in accordance with the changes observed by PAC. However,
Romero et al.(8) do not report more than one metal
site geometry with any of their techniques.
Met
Glu has the characteristic absorption spectrum of a blue copper protein
at low pH but not at high pH(16) . The pK of this
change is 4.9. This pH behavior is also found in the EPR spectrum,
where at low pH a rhombic EPR spectrum is observed, while at pH 7 the
EPR characteristics are in between those of a type 1 and a type 2
site(16, 30) . In particular, the hyperfine splitting
changes dramatically. This is in good accordance with the drastic
change in nuclear quadrupole interaction seen by PAC measured at pH
above 7.2. These changes are all in good accordance with the
coordination of the carboxyl group of Glu.
The use of Cd PAC on copper proteins has the advantage that
cadmium with its d configuration might give some
information on the behavior of Cu(I) not accessible by other
spectroscopic techniques. Whereas many other techniques require low
temperatures or crystals, PAC also has the advantage that it can be
applied to molecules in solution without loss of resolution. Thereby
the molecules studied by PAC can be in an environment that better
mimics the natural environment of the protein.
The present work
shows for the first time that detailed information on copper proteins
can be obtained with the technique of perturbed angular correlations of
-rays from
Cd. In spite of the chemical difference
between copper and cadmium, the active site of the wild type protein is
apparently unaffected by the substitution of copper with cadmium. In
contrast to this, the substitution of methionine 121 with other amino
acids is generally characterized by the presence of two different
conformations and/or wider linewidth, both characteristics indicating a
less rigid structure. The comparison of the PAC spectrum of
stellacyanin with the different Met
mutants further
supports that stellacyanin has a glutamine at this position.
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