QUANTITATIVE Cu X-RAY ABSORPTION EDGE STUDIES : OXIDATION STATE AND SITE STRUCTURE DETERMINATION
ABSTRACT X-ray absorption edge studies of Cu(I) complexes with different coordination number and covalency reveal that the 8983-8984 eV feature (assigned as the 1s→4p transition) can be correlated with ligation and site geometry. These Cu(I) features have been qualitatively interpreted using a Ligand Field model, and this has been applied to analyze the polarized single crystal, pH dependent edge spectra of reduced plastocyanin. In addition, normalized difference X-ray absorption edge analysis has been used to quantitatively determine the percent of Cu(I) in several derivatives of the multicopper oxidase, laccase.
JOURNAL DE PHYSIQUE
Colloque C8, supplement au no 12, Tome 47, dbcembre 1986
QUANTITATIVE Cu X-RAY ABSORPTION EDGE STUDIES : OXIDATION STATE AND
SITE STRUCTURE DETERMINATION
LUNG-SHAN KAU, J.E. PENNER-HAHN, E.I. SOL OM ON(^) and
Department of Chemistry, Stanford University, Stanford,
CA 94305, U.S.A.
X-ray absorption edge studies of C~(I) complexes with different
coordination number and covalency reveal that the 8983-8984 eV feature
(assigned as the Is-tQp transition) can be correlated with ligation and site
geometry. These Cu(1) features have been qualitatively interpreted using a
Ligand Field model, and this has been applied to analyze the polarized single
crystal, pH dependent edge spectra of reduced plastocyanin. In addition,
normalized difference X-ray absorption edge analysis has been used to
quantitatively determine the percent of Cu(I) in several derivatives of the
multicopper oxidase, laccase.
Determination of oxidation state and g e o r n e . t r y is essential for the
interpretation of metal ion active site chemistry but can be difficult for many
copper centers. This problem derives from the fact that the
spectroscopic methods, in particular EPR, cannot probe the 3dY6U$iprous
configuration nor distinguish reduced copper from an antiferromagnetically
coupled EPR nondetectable cupric pair. However, Cuprous complexes exhibit a
strong X-ray absorption edge feature at -8984 ev which is absent in Cu(I1)
complexes [I]. We have found X-ray absorption edge spectroscopy to be a most
useful qualitative probe of Cu(1) geometry, and and can be further used to
quantitate the amount of reduced copper present in multicopper enzymes [21.
All X-ray absorption edge data were measured at Stanford Synchrotron
Radiation Laboratory utilizing several different beam lines. ~ l l
edges were measured using a Si double crystal monochromator and were
recorded as fluorescence excitation spectra with an array of NaI(T1)
scintillation detectors. The copper model compound data were collected in
transmission mode. To insure a consistent energy reference, the internal
calibration method with Cu foil was used . To allow proper normalization,
the absorption was measured for at least 300 eV below and 200 eV above the Cu K
edge. The data presented are pre-edge background-subtrated and normalized to
give an edge jump of 1.0 at 9000 eV.
We have systematically studied a number of copper model compounds (20
m(I) and 43 Cu(11)) to correlate copper X-ray absorption features with
oxidation state and geometry. The Cu(1) compounds studied represent different
Result and discussion
TO whom all correspondence should be addressed
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868230
JOURNAL DE PHYSIQUE
coordination numbers, geometries and degrees of covalency. Most Cu(I1)
complexes studied have geometries close to tetragonal but with different ligand
sets a $
Using the energy calibration
and normalization procedure
described above, we find that, in
all cases, Cu(I1) complexes show
no low energy pre-edge peak below
8985.0 eV and that their
absorption intensities in this
region are significantly lower
than those of Cu(1) complexesr
which exhibit a pre-edge maximum
in the 8983-8984 eV region. As
summarized in figure 1, we find
that the shape, energy and
intensity of the pre-edge maximum
varies significantly over the
different Cu( I ) complexes studied.
These Cu(1) pre-edge spectral
changes can be correlated
systematically with coordination
9000 number and geometry of the metal
ion and interpreted using a simple
8970 8980 8990
Figure 1. Absorption edge spectra of repre- ligand field model which predicts
sentative 2-, 3- and 4-coordinate that the 4p
Cu(1) complexes with those of
normal and covalent Cu(I1)
complexes (below 8985.0 eV).
in the free Cu( I)
ion will bex6$lft differently by
the ligand field associated with
The Cu(1) model compounds with a linear CuNZ ligation environment have the
m s t intense pre-edge peak. Polarized single crystal studies on linear
2-coordinate Cu(1) complexes have shown that the 8983-8984 eV feature is a x,y
polarized, ls+4p electric dipole'allowed transition (ligands are along the z
axis) . The transition from the 1s to the doubly degenerate 4p
state then results in an intense pre-edge peak at lower energy thaff'yhat of
ls+4p and the covalent overlap along z axis will reduce the intensity of
1s+4~' transition. For a 3-coordinate T-shaped complex (the third ligand along
the yzaxis), the 4p
pair would be further split with the 4p level shifted
to higher energy refdyive to 4p . In the limit of a trigonal gomplex (-D
with C3 along the x axis), 4p ghould be lower in energy than the degener%
4p . p;ll 3-coordinate Cu(1q complexes have an -8984 eV transition with
r o & f i l y half the intensity of those with linear ligation and have a second
feature on the higher energy side of the 8984 eV peak which is reasonably
assigned as the ls+4p transition. Finally, for the four coordinate
tetrahedral Cu(1) com$lexes, a broad pre-edge maxim shifted to higher energy
(-8985.5 eV) is observed in figure 1.
Most of the Cu(1I) complexes studied have a very weak ls+3d transition at
8978 eV and, in addition, many show structure on the absorption edge at
energies of 8986-8988 eV. In all complexes studied, this Cu(I1) peak is always
observed at energies greater than 8985.0 eV. Alternatively, most Cu(I1)
complexes do exhibit a pre-edge low energy tail through this 8983-8984 eV
region (figure 1). With six exceptions, the Cu(I1) complexes still have quite
low intensity over this 8983-8984 eV range. These exceptions are highly
covalent Cu(I1) complexes (with sulfur ligation) and the higher intensity in
this low energy tail appears to be related to this increased covalency [51.
To further quantify these features, we have calculated the normalized
difference absorption edge spectra (NDAES) by substtating the normalized edge
of a representative Cu(I1) complex from that of a Cu(1) or from a covalent
Cu(I1) complex . The difference of properly normalized Cu(1) and Cu(I1)
edge spectra has a derivative shape (figure 2) with a positive peak at
8983-8984 eV for 2- and 3-coordinate Cu(1) and at 8986 eV for 4-coordinate
8970 8980 8990 9000 9010 9020
Figure 2. Representative normalized
difference edge spectra
for different Cu(1) and
covalent Cu(I1) minus
Cu(I), and a broad negative feature
for all Cu(1) complexes at
-8990-9000 eV. As can be seen in
figure 2, the difference edge
spectra of covalent Cu(II), although
qualitively similar in shape to
those of 2- and 3-coordinate Cu(I),
have lower intensity and in
particular their maxima are shifted
to significantly higher energy. We
have also simulated NDAES for
binuclear &(I) systems where each
copper can have a different
coordination number (2&3, 2&4 and
3&4). From the energy and shape of
the NDAES maxima, we find that it is
possible to distinguish 2, 3, 2&3
and 2&4 coordinate Cu(1) complexes
from 4 coordinate Cu(1) and covalent
Cu(I1) systems. Alternatively,
there is ambiguity in distinguishing
between 4 coordinate Cu(1) and
covalent Cu(I1). Once the
approximate geometry of a copper
site is known, it is further
possible to use the amplitude of the
peak maxima of the NDAES in figure 2
to quantitate the amount of Cu(1)
This NDAES technique has been applied to determine the amount of Cu(1)
present in several derivatives of the multicopper oxidase, laccase [2,6].
Laccase is the simplest of the multicopper enzymes, containing a total of four
copper ions (one type 1 (TI) Cu, one type 2 (T2) Cu and a coupled binuclear
copper center, type 3 (T3)) which together catalyze the four-electron reduction
of dioxygen to water. Knowledge of the amount of reduced copper is very
important in defining 0 and H 0 reactivities of the Type 2 copper depleted
(T2D) and native enzymeg [71. 2dr X-ray absorption studies of laccase
demonstrated that the T2D derivative contains a significant amount of reduced
copper (-70%) and that this can be oxidized by reaction with excess H20 [61.
EPR and optical data shows that the T1 copper is fully oxidized, thus, &eater
than 90% of the T3 centers are reduced in T2D. In addition, from similar NDAES
results, the reduced T3 site is found to be stable to oxidation by 0 or by
25-fold protein equivalents of ferricyanide. These studies have bee4 further
extended to the native enzyme and show that -25% of the T3 sites are reduced in
the presence of O2 but are reoxidized by peroxide .
This analysis of the Cu(1) edge features has been further applied to the
polarized, pH dependent edge spectra of reduced plastocyanin. The pH dependent
protein single crystal structure of reduced plastocyanin has been solved by
Freeman et. al. [El and, in collaboration, we have collected the polarized
single crystcx-ray absorption edge spectra (figure 3) . A t pH =7.0, the
Cu-S (met) bond is the unique, long axis of the active site with Cu-S (met) -
Alternatively at pH -3.9, the Cu-N
Cu-S (Met) ~2.53 A and Cu-N =3.19 A.
the 2 6 orbitals, spectra takgj! with the Pvector of the synchrotron radiation
along the Cu-S (met) bond in the high pH sample would be expected to show a
strong 8984 ev96eak, which should be shifted to higher energy in the low pH
sample. Analogously, with the % vector perpendicular to the Cu-S (met) bond
(with a projection along the Cu-N
direction), a strong 8984 eV 8gak would be
expected for the low pH but not ti2 high pH sample. These predictions fit
nicely the experimental spectra given in figure 3.
bond becomes the long ax?g with
~a@d on the ligand field splitting of
JOURNAL DE PHYSIQUE
8950 1970 8990
9010 9030 9050
Figure 3. Cu absorption zdge for reduced plastocyanin
oriented with E//Cu-S(Met) (left,-from top, pH
7.0, 6.5, 5.5 and 4.5.) and with EICu-S(Met)
(right, from top, pH 4.5, 5.5, 6.5 and 7.0).
We thank D r . ' s Hans Freeman and Darlene J. Spira-Solomon for their
contributions to this research and useful discussions. The work reported
herein was supported by NIH AM 31450 (EIS) and CHE 85:12129 (KOH). Synchrotron
beam time was provided by the Stanford Synchrotron Radiation Laboratory which
is supported by U.S. Department of Energy and the Division of Research
Resources of the National Institute of Health.
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