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Biophys J, June 2002, p. 3254-3268, Vol. 82, No. 6
§§
*Dipartimento di Chimica, Università "La Sapienza,"
I-00185 Roma, Italy; General Purpose Italian
Beam Line for X-ray Diffraction and Absorption Collaborating Research
Group, European Synchrotron Radiation Facility, F-38043 Grenoble,
France; Dipartimento di Biologia, and
§Consiglio Nazionale delle Ricerche, Center for
Metalloproteins, Università di Padova, I-35121 Padova,
Italy
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
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The binuclear copper sites of the met and met-azido
derivatives of Octopus vulgaris and Carcinus
aestuarii hemocyanins at pH 7.5 were characterized by
high-resolution x-ray absorption spectroscopy in the low energy region
(XANES) and in the higher region (EXAFS). The accuracy of the analysis
of the data was tested with two mononuclear and six binuclear
copper(II) complexes of the poly(benzimidazole) ligand systems 2-BB,
L-5,5 and L-6,6 (Casella et al., 1993
, Inorg. Chem.
32:2056-2067; 1996, Inorg. Chem. 35:1101-1113). Their
structural and reactivity properties are related to those of the
protein's derivatives. The results obtained for those models with
resolved x-ray structure (the 2-BB-aquo and azido mononuclear complexes, and the binuclear L-5,5 Cu(II)-bis(hydroxo) (Casella et al.,
unpublished)), extends the validity of our approach to the other
poly(benzimidazole)-containing complexes and to the hemocyanin
derivatives. Comparison between the protein's and the complexes'
data, support a description of the met-derivatives as a
five-coordinated O-bridged binuclear copper(II) center and favors, for
both species, a bis(hydroxo) structure with a 3-Å Cu-Cu distance. For
O. vulgaris met-azido derivative a µ-1,3
bridging mode for the ligand appears the most likely. The structural
situation of C. aestuarii met-azido-derivative is less
clear: a µ-1,1 mode is favored, but a terminal mode
cannot be excluded.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
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Why comparing molluscs and arthropods
Hemocyanins (Hcs) are the oxygen carriers and
storage proteins of several species of molluscs and arthropods. Their
active site is of a binuclear coupled type with copper ions that are directly bound to six histidine nitrogen atoms of the protein chain.
Oxygen is reversibly bound to the active site in a
2Cu:O2 ratio as
µ-
2:2 peroxide
(Solomon et al., 1992; Solomon and Lowery, 1993).
The crystal structures that are available for a 50-kD subunit
Odg from Octopus dophlini (Cuff et al., 1998),
for Panulirus interrupts (Volbeda and Hol, 1989), and for
subunit II of Limulus polyphemus (Hazes et al., 1993), allow
for a comparison of Hcs from these two phyla. In both cases, the two
copper ions in the active site, labeled CuA and
CuB (Ling et al., 1994), are not equivalent and
are likely to play a different role in the biological function. The
CuA copper(II) center that seems to be more
accessible to the solvent in the binuclear site controls the reactivity
of the site, whereas the second copper(II) center
(CuB) plays a complex role in controlling the
local conformation and electrostatic effects in the oxygenation cycle.
An additional difference is observed in the secondary structure
elements that include the ligand histidine residues. In the arthropod
Hcs, both CuA and CuB are
coordinated by the histidines belonging to a two 
-helices motive. In
contrast, in the Hcs from molluscs, only the CuB
is coordinated by a two -helices motive, whereas the
CuA is coordinated to one histidine of an
-helix and to two histidines of a loop region, one of which is
involved in a unusual thioether bridge.
Also, the comparison of the functional properties of arthropods and
mollusc Hcs point out some significant differences that have been
documented (van Holde and Miller, 1995 and references therein; Zlateva
et al., 1998). The functional properties of the active site are
influenced by the protein matrix, which presents different aggregation
patterns and different architectures in the two phyla. The arthropod
proteins active site is more rigid and less accessible than in
molluscan proteins. The greater accessibility in the case of mollusc
Hcs allows the protein to exhibit a low-efficiency tyrosinase-like
activity involving oxidation of cathecol to quinone.
Chemical reactivity of the Hcs
The binuclear site of the Hcs undergoes, with complex redox
chemistry, reactions of exogenous ligands. Small anions and neutral molecules bind to the type 3 Cu site of the Hcs by producing a series
of derivatives in which the metallic ions of the active site assume
different oxidation states and different coordination geometries. The
two copper ions show a different reactivity and the equilibrium
position of the ligand substitution reactions depends upon the type of
ligands involved and the pH (Salvato and Beltramini, 1990; Beltramini
et al., 1992).
The ligand-binding chemistry occurs with greater affinity in mollusc
than in arthropod Hcs and the effects are different. The hydrogen
peroxide dismutase activity exhibited by the mollusc Hcs only appears
in line with the chemical differences that have been noted between Hc
from the two phyla (Himmelwright et al., 1980).
Why met-Hc
The met-Hc form, characterized by an electron paramagnetic
resonance-silent [Cu(II) Cu(II)] site, is an important derivative for
defining the structural characteristics of the active site in the
native protein and to diversify the chemical reactivity of the two Cu
sites. The spontaneous, but very slow, reaction of conversion of oxy-Hc
to the met-derivative can be stimulated by various anions including
fluoride, azide, and acetate (Beltramini et al., 1995 and references
therein). The proposed active site model for the met-Hc form assumes a
Cu(II) binuclear structure with a di-µ-hydroxo bridge. The pH
dependence of the CD features of the met-Hcs and the pH dependence of
the azide interaction is suggesting a partial protonation of these
bridges at low pH (Beltramini et al., 1995; Alzuet et al., 1997). An
extended x-ray absorption fine structure (EXAFS) study (Woolery et al.,
1984) shows that there are no differences in the coordination number between the oxy-Hc and met-aquo-Hc forms, however the fundamental question of the origin of the diamagnetism in the met-Hc form is still unresolved.
The binding of azide to met-Hc derivative
Azide is a suitable exogenous ligand for probing the
characteristics and accessibility of the active site in various met-Hc derivatives, because, by coordinating with Cu(II), azide produces a
complex with a moderate absorption in the 350-440 nm range (
~ 1500-2000 M
1cm1)
caused by the ligand-to-metal charge transfer (LMCT) transition N3
Cu(II). The absorption and CD LMCT features
of this transition (position and intensity) are strongly dependent on
the mode of coordination of the ligand anion, thus allowing
discriminating between terminal and bridging binding modes of the
ligand (Solomon et al., 1992; Solomon and Lowery, 1993).
Concerning azide coordination, Octopus vulgaris and Carcinus aestuarii met-Hcs differ from each other. The affinity toward azide, the stoichiometry of binding, and its coordination mode show pH dependence. The two met-Hcs exhibit, at pH 7.0, the same 1:1 stoichiometry of the azide adducts. However, the absorption and CD LMCT features suggest that azide binds in a bridging mode in the case of O. vulgaris met-Hc active site in contrast to C. aestuarii met-Hc where azide binding, probably, occurs on the Cu(II)A center in terminal mode. The stoichiometry of binding of the azide ligand between the proteins of two phyla differs at pH 5.5. At this pH, a second azide binds to O. vulgaris met-Hc. The LMCT features are indicative of a bridging binding mode for the first azide (with greater affinity compared to pH 7.0) and a terminal binding mode for the second azide to copper CuA. In contrast, arthropod met-Hc at pH 5.5 binds one azide molecule only.
Assuming the same bis-hydroxo adducts of the binuclear site for both
met-Hcs, different reaction models for the binding of azide to O. vulgaris and C. aestuarii met-Hcs have been proposed (Beltramini et al., 1995; Alzuet et al., 1997). The substitution of the
hypothetical exogenous bridging ligands with azide allows for
disturbing, in a controlled manner, the structural properties of the
binuclear site on the met-Hc forms and for evaluating the effect of
different coordination modes of the ligand on the Hcs from the two phyla.
Why x-ray absorption spectroscopy
The synchrotron x-ray absorption spectroscopy (XAS) is an
effective technique for selectively investigating the local
coordination environment around the metal active site of
metalloproteins. The analysis of the oscillations that occur at ~50
eV above the threshold of the x-ray absorption spectrum (EXAFS)
provides information on the distances, the number, and the types of
atoms surrounding the metal center. The analysis of the threshold
region of the spectrum (x-ray absorption near-edge spectroscopy, XANES)
provides information on the geometry of the metal-ligand complex. The
technique is applicable to samples in any physical state, including
liquid or frozen solutions (Hasnain and Hodgson, 1999) and has been
successfully used to obtain structural information on the deoxy and
oxy-Hc forms, and on other binuclear copper proteins and complexes
(Feiters et al., 1999, and references therein; Sabatucci et al., 2002).
A comparative XAS investigation was undertaken to define a more precise structural model for the met- and met-azido oxidized derivatives of O. vulgaris and C. aestuarii Hcs at pH 7.5 and 5.5. The results presented in this work are restricted to the derivatives at pH 7.5 and to some related mononuclear and binuclear models. Because x-ray data are deposed only for the mononuclear compounds, the XAS characterization of all the related binuclear compounds is of importance. The results concerning the different derivatives at pH 5.5 will be published subsequently (E. Borghi, P. L. Solari, M. Beltramini, L. Bubacco, P. Di Muro, and B. Salvato, unpublished).
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MATERIALS AND METHODS |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
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Preparation of the native Hcs and of the Hc derivatives
Native Hcs were isolated from the hemolymph of O. vulgaris and C. aestuarii and were prepared as
described, respectively, in Beltramini et al. (1995) and Bubacco et al.
(1992). The protein solutions were stored at 
20°C in the presence
of 20% (w/w) sucrose as cryoprotectant.
Native Hcs are in equilibrium between the oxygenated and the deoxygenated state that depend on O2 partial pressure. The conversion to a fully deoxygenated form was achieved under argon atmosphere and was evaluated in a tonometer equipped with a quartz cuvette from the complete disappearance of the band in the absorption spectrum at 345 nm (O. vulgaris) and at 340 nm (C. aestuarii). A solution of fully oxygenated Hcs exhibits absorbance ratios A345/A278 = 0.25 (O. vulgaris) and A340/A278 = 0.21 (C. aestuarii).
The met-Hc derivative of C. aestuarii was prepared by
incubating deoxy-Hc (1 mM) with an excess (5 mM) of hydrogen peroxide in 50 mM potassium phosphate buffer at pH 8 at 20°C (Felsenfeld and
Prinz, 1959). The preparation of the met-derivative of O. vulgaris Hc was also carried out using hydrogen peroxide as
described in Zlateva et al. (1998). Azide was added to an O. vulgaris deoxy-Hc solution (1 mM) in 50 mM potassium phosphate
buffer at pH 6.0 at 20°C, resulting in a final concentration of 100 mM; then the sample was treated with hydrogen peroxide (3 mM) for 20 min. To remove excess reactants, the protein samples of both phyla were dialysed against 50 mM phosphate buffer at pH 7.5. To evaluate the
yield of met-Hc, the absorption spectrum of the protein solution was
measured. The region around 340 nm is contributed by both the LMCT
transitions of met-Hc, whose intensity does not depend on oxygen
concentration, and by the peroxide-to-Cu(II) transitions of unreacted
oxy-Hc (see above). Thus, recording the spectrum in oxygen and in argon
allows the determination of residual oxy-Hc (Zlateva et al., 1998). The
met-azido-derivatives of both phyla were prepared by adding mM aliquots
of a 1 M buffered solution of the ligand to the met-Hc solution at pH
7.5. For both proteins, the yield of the reaction was evaluated to be
more then 95%, according to the residual intensity of the 345-nm band
assigned to the unreacted oxy-Hc.
The model compounds
The model compounds used in this study are the mononuclear
(Casella et al., 1996) and binuclear (Casella et al., 1993) copper(II) complexes with poly(benzimidazole) ligands, modeling the features of
the Cu2 cores in active site of Hcs.
Two mononuclear complexes of known crystal structure have been considered. These mononuclear compounds are [Cu(II)(2-BB)(H2O)2](PF6)2 and [Cu(II)(2-BB)(N3)]ClO4. The ligand bis[2-(1-methylbenzimidazol-2-yl)ethyl]amine (2-BB), a model of the tris(imidazole) array with different coordination numbers and stereochemistries, is related to the L-6,6 ligand (see below).
Six binuclear model complexes
[Cu(II)2(L)(X)2](ClO4)n
with L = L-5,5, L-6,6 and X = OH,
H2O, N
). The two poly(benzimidazole) ligands
,'-bis[[bis(1-methyl-2-benzimidazolyl)methyl]amino]-m-xylene (L-5,5) and
,'-bis[bis[2-(1-methyl-2-benzimidazolyl)ethyl]amino]-m-xylene (L-6,6) have identical donor groups, one tertiary amino and two benzimidazole nitrogen donors, but provide metal coordination sites
with different chelate ring size: 5-membered for L-5,5 and 6-membered
for L-6,6. Of the six models considered, only the analogous [Cu(II)2(L-5,5)(OMe)2](ClO4)2
compound has been structurally resolved by x-ray crystallography
(Casella, Univ. Pavia, Italy, personal communication).
The XAS measurements
The XAS fluorescence experiments have been carried out at 77 K,
in the XANES and EXAFS approaches, on the Italian Collaborating Research Group, General Purpose Italian Beam Line for Diffraction and
Absorption at the European Synchrotron Radiation Facility in Grenoble,
France. The beam was monochromatized dynamically with two independent
Si(311) crystals (
E/E = 104) (Pascarelli et al., 1996). The dynamical
focusing mode avoids solid angle effects during the collection of the
fluorescence spectra and produces an intense focal spot on the sample,
whose size (~2 mm) is kept constant during each scan. The incident
photon flux was measured with an ionization chamber, and the
fluorescence photons for each spectrum were collected by using a
high-purity germanium multi-element detector. Two Pd mirrors were used
for harmonic rejection. To avoid radiation damage of the samples, the
intensity of incident beam was attenuated with an Al filter of
100 µm.
The energy range used for the detection of the
Cu-K
fluorescence line was 8700-9800 eV for
all the spectra. The energy calibration was obtained by measuring,
contemporary to the fluorescence signal of a metal copper foil placed
after the sample. We collected an average of three to six single scans
for each sample with an average integration time of 15 s/point.
The met- and met-azido-derivatives of O. vulgaris and
C. aestuarii Hcs at pH 7.5 were measured in solution in the
presence of sucrose. As recently described (Ascone et al., 2000),
sucrose provides an excellent protection against x-ray damages,
allowing for longer exposure to the x-ray beam. The sucrose was added
to the protein solution in a sucrose-to-protein ratio of 50% w/w. The
binuclear complexes of the poly(benzimidazole) ligands L-5,5 and L-6,6,
and the mononuclear 2-BB model compounds were measured as pellets in
50% w/w sucrose matrix.
Data analysis
The EXAFS first-shell analysis was carried out using the
complete package of Michalowicz (1990). To extract the experimental modulation function 
(k), the free atomic background was
reduced by cubic splines using the Heilter formula (Lengeler and
Eisenberger, 1980) and normalized to the edge jump.
The (k) function was then multiplied by a Kaiser window
before being transformed into real space. The Fourier transforms were
obtained by integration of cubic k-weighted EXAFS function over the ~3- to ~11-Å1 range. Structural
information was obtained by back-transforming the first peak of the
Fourier transform (corresponding to the range 0.8-2.0 Å of the main
distance R) and by fitting the resulting function with the
appropriate theoretical function defined by Lee and Pendry (1975). This
function is expressed in terms of the coordination number N
of the first-shell atoms around the photoabsorber atom, in terms of
their mean interatomic distance R (Å), and in terms of the
variance 
2 (Å2)
(Debye-Waller (DW) factor), which accounts for their structural and
thermal disorder. The phase shifts and amplitudes for the photoabsorber
and the back-scatterer atoms that were used in the fits were taken from
the EXAFS analysis of the Cu K-edge spectra of appropriate reference
compounds and from the theoretical data (Mc Kale et al., 1988).
A more detailed quantitative analysis of the EXAFS data has been
performed using the GNXAS set of programs (Filipponi et al., 1995; Filipponi and Di Cicco, 1995). The approach with the GNXAS package involves an ab initio calculation of the absorption coefficient cross-section using the multiple-scattering theory, starting from a
given geometrical atomic configuration around the absorber. The
theoretical structural signal (k) is calculated as a sum of the two-body single scattering signal (SS) and the three-body and
four-body multiple scattering signals (MS) associated with the
n-atom configurations including the photoabsorber. The
two-body signals are associated with pairs of atoms and probe their
distances and variances. The three-body signals are associated with
triplets of atoms and probe angles and their variances. The four-body
signals are associated with quartet of atoms and probe the presence of the imidazole rings of a histidine residue (protein case) or of a
benzimidazole residue (model case) (Meneghini and Morante, 1998). Then,
by comparison with experimental data, the model signal is refined with
a fitting procedure.
In both approaches, the statistical errors associated with the
parameters were calculated by assuming that the residual is a
2-distributed variable and by considering a
critical value corresponding to the 95% confidence level (Michalowicz,
1990; Filipponi and Di Cicco, 1995).
The near-edge structure (XANES) spectra were normalized to the total jump after removal of a linear background. The background was fitted in the pre-edge region and extrapolated to the whole spectrum. In all spectra, the zero on the energy scale was fixed at 8979.0 eV, corresponding to the first inflection point of the absorption threshold of metal copper.
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
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The XAS investigation of binuclear copper sites as those found in Hcs poses some severe problems that mainly derive from the presence of two scattering atoms and from the fact that the metal-metal contribution in the spectra is superposed to the Cu-His signals. The determination of both a correct value of the Cu-Cu distance and a measure of the apical distortion at the copper site, considering that the apical histidine movement is certainly involved in the mechanism of ligand association, are the two fundamental aspects that we are aiming to resolve with this investigation.
To evaluate the accuracy of our analysis, we considered, as a first test, the two mononuclear model complexes with resolved x-ray crystallographic structures. In the aquo complex, the metal center is five-coordinated in a compressed trigonal bipiramidal stereochemistry. In the azido complex, the metal center is four-coordinated in a stereochemistry intermediate between square planar and tetrahedral. The azide ligand is bound in an end-on coordination mode with the two N-N bonds statistically equal.
The following step has been to consider the L-5,5-(OH)2 binuclear model. Also, for this complex, a resolved x-ray structure is available, and so it constitutes a test for the accuracy of our analysis in the presence of a binuclear metal site. The L-5,5 complex is a good model for the proposed bis(hydroxo)-bridge structure of the binuclear copper(II) center in the met form of the protein. In fact, the L-5,5 ligand complex, which constrains the chelation on each Cu(II) center with two five-term rigid rings, presents an antiferromagnetic coupling as in the met-Hc form. The L-6,6 bis(hydroxo)di-Cu(II) complex, which has also been considered, has a higher flexibility that is more apt to mime the redox reactivity of the protein. Thus, the study of this complex is useful to evidence small differences of the coordination geometry that occur in the site and, in particular, the movements of the apical ligand. Of the other models that we have considered, the two bis(aquo)di-Cu(II) complexes may provide the basis to identify the differences between a bis(hydroxo)-bridge and a bis(aquo)-bridge structure to test the possibility that the bridging ligand in the Hcs derivatives is a water molecule. The two bis(aquo) complexes show a high affinity for the azide and differentiate the mode in which the azide ligand is coordinated. Finally, the two monoazide adducts of the binuclear derivatives have been considered because they show a µ-1,3 bridging coordination for L-6,6 and a µ-1,1 for L-5,5. Their features, and those of the azido-mononuclear complex, provide information on the three possible coordination modes of the azide ligand. The complete MS analysis of all the models considered will be published in a separate article. In this study, we are reporting only the XAS results of the model compounds that are important for the comprehension of the analogous characteristics of the related biological samples. Confident in the validity of this approach, which gives results that are in agreement with previous spectroscopic studies (see below), we have extended the XAS analysis to the Hcs derivatives.
EXAFS approach
In Figs. 1-4, we show the cubic k-weighted Cu K-edge EXAFS (A parts) and Fourier transforms (FTs) (B parts) for the met- and met-azido Hc forms of the two species considered (Fig. 1), for the binuclear L-5,5 models (Fig. 2), for the binuclear L-6,6 models (Fig. 3), and for the mononuclear 2-BB models (Fig. 4).
In the FTs (part B of Figs. 1-4), the intense first peak is assigned to the scattering from the nearest neighbor atoms. The following secondary peak of variable amplitude is assigned primarily to the scattering from the second shell of atoms of the histidine imidazole rings and partially to the Cu-Cu scattering. The third peak is due primarily to the external shell of atoms of the imidazole rings. By comparing the different FT spectra, we observe significant differences among the various compounds on going from the hydroxo-, to the aquo-, and to the azide form. In the L-5,5 FT spectrum (Fig. 2 B), the first and second peak decrease. In the L-6,6 FT spectrum (Fig. 3 B), the decrease of the first peak is more pronounced and the following peaks increase. The same trend is observed with the mononuclear compounds (Fig. 4 B). The decrease of the FT first peak is much smaller in the case of met- and met-azido-Hcs as compared to the models (Fig. 1 B). The binding of azide to met-Hc produces little changes at ~3.0 Å (Carcinus case) and between 2.0 and 3.0 Å (Octopus case).
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The first-shell analysis
The FT first-shell peaks were filtered, back-transformed, and analyzed for nearest neighbors scattering atoms. At this stage of the analysis, it was not possible to separate the contributions of the different low Z atoms (N/O) due to the high correlations that exist between the parameters of the subshells. The values obtained in the fits are shown in the left side of Table 1. The errors are given in parentheses and refer to the last digit of the parameters. These results indicate that, for all the samples, a single shell of low Z atoms at an average distance of 1.94-1.98 Å can be considered.
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; Casella et al.,
1996; L. Casella, Univ. Pavia, Italy, personal communication).
The values obtained for the models of both families indicate that the
decrease observed in the first FT peak, on going from the hydroxo- to
the aquo- and to the azide-form (Figs. 2 B and 3 B), is the result of a combined decrease of the
coordination number and increase of the Debye-Waller factor. The
decrease of the coordination number can be explained, at least in part,
by the displacement of some coordinating atoms to distal positions. The
trend seen for the DW factors is in agreement with an expected increase
of the static structural disorder around the metal center.
The results obtained for the Hcs indicate fine differences between the
met- and met-azido-forms (see Table 1, left side). The
O. vulgaris Hc appear to have a slightly longer first shell average distance and a slightly greater DW factor with respect to the
C. aestuarii Hc, thus having a greatly disordered active site.
The multiple-scattering approach
Although the Fourier filtering in the single-scattering approach is a suitable method to investigate the first shell, it gives unreliable results for the analysis of the subsequent shells. Indeed, outside the first-shell range, the multiple scattering contributions from the imidazole rings that surround the metal center become particularly important. Therefore, we have started an analysis of the whole spectra with the multiple-scattering approach using the GNXAS package programs (Filipponi et al., 1995; Filipponi and Di Cicco,
1995).
From the MS analysis performed on the compounds and on the Hcs
derivatives, the trend described above for the first-shell analysis
seems to be confirmed. In the right side of Table 1, we report the
results (with errors in parentheses) obtained by distinguishing two,
three, or four different waves for the first shell (the Cu-O, the
equatorial Cu-N, the apical Cu-N', and a second Cu-O or Cu-N shell
for the aquo- and the azido-compounds, respectively). The low
coordination numbers observed in the Fourier-filter first-shell
analysis appear effectively to be a consequence of the longer distances
of some of the first neighbors or of the greater value of the
Debye-Waller factors associated with some of the subshells.
In Table 1, we also report the first results obtained for the Cu-Cu
shell. It is known (Scott and Eidsness, 1988) that, in biological
systems and biomimetic models involving heterocyclic aromatic ligands
(imidazole, pyridine), it may be a problem to distinguish the Cu
contribution from low-Z (C/N in second and third shells) contributions
at the same distance, because the contributions are different only in
the envelope of the backscattering amplitude, while the phase
relationships between EXAFS and FT are the same. By using a multiple
scattering-fit approach, it becomes possible to establish the Cu-Cu
distance (Lynch et al., 1994; Feiters et al., 1999), but, nonetheless,
this remains a difficult task.
The results obtained for the mononuclear compounds and for the
L-5,5-(OH)2 are in agreement with the structural
data. Only for the 2-BB-aquo is there a slight increase of the average
distance of the coordinating waters, which can be partly attributed to the strong disorder associated with these atoms.
The results obtained for the L-6,6-(OH)2 are
comparable to those of the L-5,5-(OH)2. The L-6,6
bis(hydroxo) complex is characterized by a shorter apical ligand
distance. The shorter distance obtained for the apical nitrogen and the
longer average distance obtained for the equatorial nitrogens are in
agreement with the lower conformational rigidity imposed by the L-6,6
poly(benzimidazole) ligand. The XAS values indicate that the binuclear
copper(II) centers with the two ligands show a similar
bis(hydroxo)-bridged structure (i.e., similar Cu-Cu distance, similar
coordination number in the first shell) so that a similar geometry of
the overall cluster is expected. This agrees with previous
spectroscopic studies (Casella et al., 1993).
For the L-5,5-(H2O)2, the
data can be fitted correctly only by separating the contribution of the
oxygens of the two water molecules. We find that one of the oxygens is
displaced at a larger distance with respect to the bis(hydroxo) form.
This agrees with the Fourier filter analysis for which a reduction of
coordination number of the first neighbors was observed. The observed
variation in the Cu-Cu distance, besides the Cu-O/O' and the Cu-N/N'
distances, comports a perturbation in the Cu-O-Cu angle, so a
different symmetry of the Cu cluster is expected, as compared to the
bis(hydroxo) complex. The same effect is seen for the
L-6,6-(H2O)2 compound with
respect to the L-6,6-(OH)2 one. The shorter
Cu-Cu distance in this case reflects the presence of a more ordered
geometry consistent with the L-6,6 ligand-forming six-membered chelate rings instead of five-membered as with L-5,5 ligand.
The L-5,5-N3 spectra can be adequately fitted,
including one oxygen and three equatorial nitrogens (one is coming from
the azide) at an average distance of ~1.9 Å. An additional axial
nitrogen at 2.2 Å, which takes into account the contribution of the
apical Cu-N' of the L-5,5 ligand, has to be considered. The best fit was obtained by starting the calculations from a theoretical model, which accounts for the L-5,5 geometry of the ligand and for the µ-1,1
bridging coordination of the azide (as in the model by Kahn et al.,
1983). The first-shell coordination, the MS results obtained and the
average Cu-Cu distance (~3.20 Å) indicate a different geometry of
the copper cluster as compared to the bis(hydroxo) complex and are
compatible with the presence of the µ-1,1 azide-bridged structure.
For the L-6,6-N3, the best fit is obtained by
considering an azide contribution in the µ-1,3 bridge coordination.
The first shell is correctly fitted by two equatorial nitrogens at ~2
Å and one oxygen. The contributions coming from the nitrogen of the
azide and from the ligand are found at larger distances. The long
distance obtained for the Cu-Cu shell is compatible with the
(µ-hydroxo)(µ-1,3-azido) model of Kitajima et al. (1993), which
shows similar distances (average Cu-N at 2.02 Å, Cu-N at 2.21 Å).
The distance obtained for the oxygen atom is compatible with the second
aquo bridge expected in this adduct (Casella et al., 1993). As we
already stressed, the results obtained on the model compounds that are
presented in this work are used as a proof of the goodness of our
approach, and they will be discussed in detail elsewhere.
The results of the fits of the EXAFS of the Hcs forms, obtained with
the multiple scattering approach are shown in Fig.
5. The best fit value of the residual,
Rexp, and the calculated expected value, Rth, (Filipponi and Di Cicco,
1995) are also shown in the figure as a measure of the goodness of the
fits. The differences between the met- and met-azido Hcs forms of the
two phyla, which are less evident in respect to those noted for the
models when considering the first-shell analysis, can be partly
clarified by the MS approach. In all cases, the two derivatives of both species are five-coordinated and the distances of the first-shell atoms
are included between 1.9 and 2.0 Å (see Table 1).
|
). In effect, by distinguishing between the Cu-O
wave and the Cu-N equatorial and Cu-N' apical waves coming from the
imidazole, the met-Hc forms appear three-coordinated with the nitrogen
atoms and two-coordinated with the oxygen. The apical nitrogen is found
for both proteins at an average distance of 2.3-2.4 Å, which is close
to the value found for the L-6,6-(OH)2 compound.
For both species, a shorter Cu-Cu distance, of ~3 Å, is obtained
with respect to the oxy-Hc, in which the oxygen is coordinated in the
µ-2:2 mode, and the
Cu-Cu distance is ~3.5 Å. By itself, the shorter Cu-Cu distance
could account for the magnetic coupling of the type 3 site of the met
derivatives without the necessity of invoking additional bridging
ligands. Nevertheless, the results obtained with the model compounds
show that a double O coordination at a distance of 1.95 Å or lower,
and a short Cu-Cu distance at ~3.0 Å, are expected in the case of a
bis(hydroxo) structure, whereas the results seem to be incompatible
with bis(aquo) structure, for which longer distances are obtained for
one of the two water molecules. Thus, our results, in which the
Cu-O-Cu multiple-scattering path has been considered, seem to assert
the presence of a bis(O)-bridged structure and are consistent with a
perturbation of the bending angle of the
Cu2O2 unit to a value of
~100-102°. (Note that it is ~137° in the oxy case [Cuff et
al., 1998 and references therein]). Also the EXAFS calculations by
Feiters et al. (1999) for O2 complexes with the
Cu-Cu parameters at the same distance indicate a similar bending. A
Cu-Cu distance ~3 Å in the met-Hcs forms is also concordant with
the trend that is seen for Cathecol oxidases, in which the Cu-Cu
distance decreases from 3.8 to 2.9 Å upon going from the oxy- to the
met-form, though, in this later case, a decrease in the coordination
has also been observed (Eicken et al., 1998). Compared to the model
compounds that we have considered, the results for the met-Hcs forms
appear to be similar to those obtained for the L-6,6-bis(hydroxo)
complex. This similarity and the differences obtained with respect to a
previous work on different Hc phyla, in which met-aquo forms were
considered (Woolery et al., 1984), favors the hypothesis of the
hydroxide atoms as exogenous ligands for the met-Hc forms.
Using the MS approach, the results obtained for met-azido-Hcs bring up
differences between the derivatives of the two species that were not
detectable by a simple first-shell analysis (Fig. 5, B and
D). The azide-derivatives are also different with respect to
their met-correspondent, though, maybe, to a lesser extent than what is
observed for their homologous model compounds.
The starting spectroscopic hypothesis for the azide-binding mode is an
end-on geometry for C. aestuarii derivative (Alzuet et al.,
1997) and a µ-bridge geometry for O. vulgaris, with a preferred µ-1,3 bridging mode (Beltramini et al., 1995). For the O. vulgaris met-azido-Hc, the best fits were obtained by
considering a structure with a coordinated µ-1,3 azido bridge as the
starting model for the calculations of the theoretical signal (Fig.
5 B). The results indicate that the protein is
five-coordinated with a longer Cu-Cu distance, of ~3.8 Å. The
shorter value obtained for the Cu-O distance may indicate a severe
distortion of the Cu-O-Cu bridge or probably the breaking of this
bridge, each copper being coordinated to a different oxygen atom.
Nonetheless, the proposed µ-1,3 bridging coordination mode for the
azide is the most favorable, because alternative fits starting from
µ-1,1 bridging coordination were unsatisfactory. So, in this
case, the spectroscopic hypothesis seems to be confirmed.
For the C. aestuarii met-azido-Hc, the best fits were
obtained by considering a µ-1,1 bridging structure as the starting
model for the calculations (Fig. 5 D). In this case, the
differences in the first shell between the azide derivative and the met
derivative are less important. The protein is five-coordinated and the
Cu-Cu distance is ~3.2 Å as for the L-5,5-N3
complex. However, the starting hypothesis for the Carcinus
case is an end-on geometry, the first shell and Cu-Cu shell results
seem to favor a µ-1,1 bridging mode. The results obtained in a
previous EXAFS study, in which the MS contributions where not
considered (Woolery et al., 1984), suggested a µ-1,3 geometry, with a
Cu-Cu distance of ~3.7 Å, for both molluscan and arthropodan Hcs.
This cannot be in our case for the Carcinus derivative.
XANES approach
Figures
6-9
show the normalized XANES spectra (upper panels) and the
relative first derivatives (lower panels) of the met- and met-azido-Hcs and of the poly(benzimidazole) compounds. The energy position of the Cu K-edge for all the considered model complexes and
protein derivatives is in the range of the 3d9
electronic configuration of Cu(II) with N/O donors coordination. All
the spectra show features in the pre-edge region, in the rising-edge region and in main-peak region, which have been detected and assigned for other Cu(II) clusters (Kau et al., 1987; Sano et al., 1992; Shadle
et al., 1993, Pickering and George, 1995). Because the XANES spectra
are expected to contain significant information on each copper-site
symmetry, the position and the intensity of the XANES features can be
correlated to general geometric properties of the metal site to
elucidate qualitatively some aspects of the copper-site conformational
changes. Our qualitative discussion of the low-energy region of XAS
spectra will refer to the meanings of the experimental XANES features
and to the information obtainable from the MS simulations of the XANES
spectra reported in literature for Cu(II) complexes with coordination
through N/O donors.
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|
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Meaning of the XANES features
The pre-edge P feature corresponds to the formally dipole-forbidden 1s3d transition (l = ±2, quadrupole
allowed, l being the azimuthal quantum number). The presence of the P
feature (the smallest resolved feature in the spectrum at the energy
absorption 
0 eV) marks the copper valence state as Cu(II). The
intensity of the P feature for Cu(II) complex depends, mainly, on the
centro-symmetric character of the average symmetry around the Cu(II)
metal. This peak is absent for centro-symmetric clusters in which the
1s3d transition is completely dipole forbidden, but has a nonzero
intensity for noncentro-symmetric clusters and raises to a strong
intensity for tetrahedral clusters due to the metal d-p orbital mixing
through the perturbation of the ligand field. So differences in the
pre-peak P intensity are reflecting different average symmetry around
the Cu(II) center (Sano et al., 1992).
The A, B, C, D features, labeled in order of increasing energy, and
their related , 
, 
, 
peaks in the first derivative spectra
are typical of the Cu K-edge and correspond to the electric dipole-allowed 1s4p transition (l = ±1). The two
higher-energy features E, F (and the related derivative peaks ,
), which intensities are relatively low in comparison to those of
the features at lower energies, may not be accurately estimated.
The 1s4p transition and the corresponding creation of the core hole
cause a relaxation of the valence levels. Two final states are
possible: the
(1s)1···(3d)94p1
configuration associated with the pure 1s4p transition (main resonance peak) and the
(1s)1···(3d)10L14p1
configuration (where L1 denotes a copper ligand
hole) associated with the two-electron 1s4p + LMCT
transition (shakedown resonance peak). The ligand-to-metal charge-transfer energy shifts the shakedown resonance to a lower energy. At present, the typical near-edge A-to-D structures are believed to be two replicas of the same one-electron and two-electron transitions, i.e., the A and C features are assigned to the shakedown transition and B and D are assigned to the main transition (Shadle et
al., 1993; Pickering and George, 1995).
The A and B features are the peaks in the rising-edge region. The A
feature (peak ) at the absorption edge (A, 4-7 eV) is originated by
the tetragonal distortion in the metal cluster, and its magnitude and
position depend on the degree of the distortion. This structure is
absent for centro-symmetric six-fold coordinated clusters, but it is
present as a shoulder for a moderate tetragonal distortion or as a
broad band for a square planar symmetry (Palladino et al., 1993;
Pickering and George, 1995). The A feature is also expected with
Td and C3v symmetry
clusters, and a not negligible presence of this feature has been
suggested to be an indicator of a five-fold square-pyramidal
coordination (Della Longa et al., 1993). It has been shown (Kau et al.,
1987) that its intensity is in the 0.15-0.5 values range for Cu(II)
clusters. Also, the B feature (peak ) (B, 8-14 eV) reflects
essentially the difference in the axial geometry of the Cu clusters.
The C and D features occur in the main peak region. The C feature (peak
) is at 14-19 eV, whereas the D structure (peak ) is at 18-22
eV. These features reflect the coordination number and the symmetry of
the Cu sites. The presence of well-distinct features in this region can
be an indicator of an anisotropic character due to inequivalence on the
coordination geometry of px,
py,
pz orbitals. The features at
higher energy E (20-30 eV) and F (
30 eV), when of observable
intensity, are probing small differences in the geometry of
coordination (i.e., variation on bonding angle or movement of axial
ligand around the Cu center). Their presence confirms the anisotropic
character of the symmetry.
Information provided by the simulations
Because it is difficult to rationalize the XANES features of these spectra with a qualitative analysis only, we have started simulations of the absorption coefficient in the XANES region, in the MS approach, with the G4XANES package (Durham et al., 1982) and the CONTINUUM code
(Tyson et al., 1992). In accordance with the previous MS calculations
of cobalt-substituted-Hc and oxy-Hc (Della Longa et al., 1993), we are
considering scattering clusters that include the three imidazole rings
around the copper site (E. Borghi, unpublished).
The size of the scattering cluster is a key factor of XANES
simulations. In effect, by considering progressively increasing cluster
sizes starting from the first-shell atoms, the calculations show that,
although the A, B, C peaks arise from the first shell, the D peak
reflects the whole geometry of the copper site. So, a red shift of the
position of main peak D can reflect a major isotropic character of the
structure and a different geometry of coordination. A low-intensity
ratio between the peaks C and D (i.e., feature more intense) can
reflect a lowering of the coordination number in the cluster. A
different ratio between the and peaks should involve changes in
the first-shell coordination geometry (Bianconi, 1988).
The simulations on oxy-Hc (Della Longa et al., 1993) have been able to
clarify and to assign the polarization dependence of the XANES
features: peaks A and B are assigned to a final state 4pz, whereas peaks C and D to the
4pxy state. Furthermore, MS calculations starting from the oxy- and deoxy-forms of Hcs have been
able to reproduce the XANES spectra of alternative five-coordinated structures of this copper(II) site (Della Longa et al., 1993, 1996). In
particular, they enabled correlation of the red shift of the peak,
and the associated variation of the relative-intensity ratio of the and peaks, to a longer bond distance of the apical ligand, i.e.,
from 11.5 eV (d = 1.9 Å) to 1.5 eV (d = 2.7 Å), and thus to an apical distortion of the standard structure
of oxy-form (square-pyramidal copper site geometry). So, the resulting d (Å) versus E E0 (eV) correlation can provide a
qualitative criterion to understand the structural characteristics of
the Hcs derivatives and of the related model compounds considered in
our study.
Comparative analysis of model compounds and Hcs derivatives
In the case of the two monomeric models of the 2-BB ligand, the features of the XANES spectra (Fig. 9) are in agreement with the x-ray data. The peak P indicates the noncentro-symmetric character of the two metal centers. The shape and the energy position of the features of the same type differ for the two compound, indicating different coordination geometry: the spectrum of 2-BB azide compound presents clearly the A, B, C, D features and a major intensity of the peak. This reflects the four-coordination in the
2-BB-N3 case with respect to the
five-coordination for the
2-BB-(H2O)2 compound and
the difference of their symmetries.
All the XANES spectra of the binuclear model compounds are
complicated and show differences between the homologous of the two
ligands L-5,5 and L-6,6 (Figs. 7 and 8). A qualitative discussion is
possible only inside the same ligand family. The absorption shapes for
all the L-5,5 complexes are compatible with five-coordinated Cu
clusters in accordance with the x-ray results (L. Casella, Univ. Pavia,
Italy, personal communication) for the L-55 bis(hydroxo) complex. So all the L-5,5 complexes are showing a similar geometry. In
the case of the L-5,5-(OH)2, the form of the
derivative and the position of the peak suggest the presence of an
axial distortion inside a noncentro-symmetric Cu cluster (P feature).
An apical distance of ~2.5 Å is estimated in agreement with the
x-ray data of the metoxy analog. The shapes of
L-5,5-(H2O)2 and
L-5,5-N3 appear different in the edge and in the
main peak region (the C feature is more clearly present). This
indicates a different axial symmetry in these Cu site(s) with respect
to that of bis-hydroxo (see Fig. 7). Shorter apical distances can be
estimated from the position of the peaks, i.e., ~2.3 Å for
L-5,5-(H2O)2 and ~2.4 Å for L-5,5-N3.
The absorption shapes of the L-6,6 compounds, by comparison to the
homologous derivative of L-5,5 ligand, are compatible with an overall
five-coordination number at the Cu site(s) and similar noncentro-symmetric character (P feature). The L-6,6 ligand presents a
higher flexibility with respect to the L-5,5, so, in this
family, the structure of the Cu clusters are expected to reveal a more isotropic character with respect to that of L-5,5. The and derivative features, clearly evident as in the L-5,5 family, are an
indicator of the anisotropic character of the symmetry of these complexes. However, in the L-6,6 family, the shape and intensity of the
feature differ, indicating some differences inside the conformational details of the geometry of coordination. In the L-6,6 family (see Fig. 8) the major differences are in the region of
the main peaks, C and D, suggesting diversities in the axial geometry
at the Cu sites with respect to the L-5,5 analog. The form of the
derivatives and the position of the peaks propose the presence of a
minor distortion for which a value of ~2.3 Å can be estimated
similar within the L-6,6 family. The more intense features
suggest a smaller number of Z-donor in the first shell for
L-6,6-N3 and
L-6,6-(H2O)2 with respect
to the L-6,6-(OH)2.
By comparing the XANES features for the Hcs (Fig. 6) we see
significant differences between homologous derivatives. Evident changes
are present in the XANES spectra of the met-azido-Hcs with respect to
the corresponding met-Hcs forms. There are differences also with
respect to the model compounds (see Figs. 7 and 8). This implies
differences on chromophores of the met-forms and of the azide adducts
of the proteins with respect to the homologous models. The absorption
shape of all Hc derivatives indicates an overall five-coordination
number at the Cu site(s) with noncentro-symmetric character (P
feature). In any case, the met-Hcs spectra resemble those of the
L-6,6-(OH)2 and
L-6,6-(H2O)2 models,
suggesting a similar anisotropic character of the overall symmetry (see
shape and position of the and derivative features), but
different local conformation (see shape and position of the , ,
, peaks) with respect to the structure of these complexes. The
similarities seem to be major with the bis(hydroxo) complex. From the
position of the peaks, the presence of an apical ligand distance
between 2.3 and 2.4 Å can be proposed for the met- and met-azido-Hc
forms. The met-azido-Hcs spectra are different from those of both
L-N3 and 2-BB-N3. However,
the shapes of the derivatives indicate a greater similarity between the
met-N3 from O. vulgaris and the L-6,6-N3 model (µ-1,3 bridging mode).
The case of the C. aestuarii derivative is more complex.
There is some correspondence of shape and position at high energy ( and derivative features) and at low energy ( and features in the L-6,6 case only) with the azide models of the 2-BB and L-6,6 ligands. Both ligands provide 6-T chelate rings and similarities of the met-Hc derivative of both phyla with the L-6,6 family have been
observed. However, with respect to the 2-BB-N3
model (end-on binding mode), the coordination number for the
Carcinus met-azido-Hc derivative is expected to be five, and
the symmetry of the Cu cluster should be different. These differences
appear clearly when comparing the shape and the position of the other
peaks. The similarities with the L-6,6-N3 model
(µ-1,3 bridging mode) propose analogous constraints and the
presence of a µ-bridge for the binding of azide, but the differences
noted suggest a different coordination fashion of the bridge. By
comparing the XANES features of the L-5,5-N3
model (µ-1,1 bridging mode, 5-T chelate rings) and the
met-N3 derivative from C. aestuarii,
we see differences, which could be attributed to the different
constrains imposed by the L-5,5 ligand and by the protein matrix. The
mode of coordination proposed by the L-5,5 model complex cannot be
excluded. The spectroscopic model (Alzuet et al., 1997) suggested an
end-on geometry, and, in this case, two different symmetries can be
present at the copper site. The results obtained for the met-azido-Hc
derivative of C. aestuarii require a very careful MS XANES
calculation because the XANES spectrum is expected to contain
information on each copper site.
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CONCLUDING REMARKS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
|
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The EXAFS analysis of the first shell, in combination with a
qualitative XANES analysis and the preliminary results of the MS
analysis, is allowing us to establish some conclusions on our general
study of the oxidized-Hc derivatives and on their related models. The
results obtained for the mononuclear compounds and for the binuclear
L-5,5-bis(hydroxo) complex, which are in agreement with the data of the
crystallographic structures, validate the approach followed for the
analysis of data. The results obtained for the remaining
poly(benzimidazole)-complexes (with unknown structure) are compatible
with previous spectroscopic studies (Casella et al., 1993). The
L-6,6-bis(hydroxo) complex is characterized by a shorter apical
distance, but the binuclear copper(II) centers with the two ligands
present similar bis(hydroxo)-bridged structure. The results obtained on
the aquo complexes seem to confirm the presence of a bis(aquo)-bridge
structure as suggested by previous NMR studies. The XANES features are
suggesting an overall symmetry of the Cu clusters similar to the
correspondent bis(hydroxo) centers, but with some local differences.
This is in agreement with the EXAFS results for which one of the two
water molecules is at a longer distance with respect to the hydroxo
complexes, thus bridging the
Cu2(H2O)2
units to a less rigid conformation. The hints that we have from the
first analyses for the azide compounds suggest a similarity with the
aquo compounds. The values of the L-5,5-azido are in agreement with a
µ-1,1 coordination and the presence of a second bridge. The
L-6,6-N3, clearly, shows a coordination mode of
the anion different with respect to the L-5,5 case. The results obtained seem to be compatible with a µ-1,3 binding mode for the azide and with the presence of a second bridge.
In the case of the proteins, the work presented here extends our
knowledge of the met-Hc compounds by favoring the hypothesis of a
bis(hydroxo) structure for both species and thus discerning the origin
of the magnetic coupling of the site. The EXAFS results that are
similar, at this pH, for the O. vulgaris and C. aestuarii Hc met forms do not explain the catalase activity
present in the met forms of molluscs with the coordination number, type
of ligands, and bond distances. The results obtained for the
met-azido-Hcs are in agreement with previous spectroscopic
characterizations (Octopus derivative), but are still
ambiguous and require a very careful MS calculation for the C. aestuarii derivative. By comparing the XAS features of the
met-azido-Hcs derivatives to those of the model compounds, it seems
possible to confirm the µ-bridging mode for O. vulgaris
previously proposed (Beltramini et al., 1995). The µ-1,3 appears more
probable. The XAS features for a terminal binding mode and for a
µ-1,1 bridging mode present some structural similarity. Therefore,
the result of C. aestuarii derivative, for which the
reaction models for the binding of azide propose a terminal mode
(Alzuet et al., 1997), is not yet fully clarified.
This investigation allowed the determination of a correct value of the Cu-Cu distance. The features of the XANES edge region and the EXAFS analysis in the MS approach validate an apical distortion at the copper site, as a consequence of the movement of the apical ligand involved in the ligand association.
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ACKNOWLEDGMENTS |
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The authors are grateful to Prof. L. Casella (University of Pavia, Italy) for the kind gift of all model compounds and for the private communication on x-ray data of one model compound.
This work was supported by the grant 9803184222 from the Ministero dell'Università e della Ricerca Scientifica, Italy (to E.B. and P.L.S.) and by a French Government fellowship to P.L.S. Our research project was partially supported, under the decision of the European Committee, by the European Synchrotron Radiation Facility in Grenoble, France.
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FOOTNOTES |
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.
Address reprint requests to Elena Borghi, Dipartimento di Chimica, Università "La Sapienza", P. le A. Moro 5, I-00185 Roma, Italy. Tel.: +39-06-4991-3678; Fax: +39-06-490324; E-mail: e.borghi{at}caspur.it.
Submitted October 6, 2001 and accepted for publication November 23, 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
|
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2:2-peroxo complex.
J. Am. Chem. Soc.
115:7874-7875.
