Models for Copper Dynamic Behavior in Doped Cadmium dl-Histidine

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Models for Copper Dynamic Behavior in Doped Cadmium DL-Histidine Crystals: Electron Paramagnetic Resonance and Crystallographic Analysis Michael J Colaneri, Simon J. Teat, and Jacqueline Vitali J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.5b07864 • Publication Date (Web): 26 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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The Journal of Physical Chemistry

Models for Copper Dynamic Behavior in Doped Cadmium DLHistidine Crystals: Electron Paramagnetic Resonance and Crystallographic Analysis

Michael J. Colaneri†*, Simon Teat§ and Jacqueline Vitali‡* Department of Chemistry and Physics, State University of New York at Old Westbury, Old Westbury, New York 11568, Advanced Light Source, Lawrence Berkeley National Lab, 1 Cyclotron Road MS 15RO317, Berkeley, CA 94720, Department of Physics and Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio 44115

*Authors to whom correspondence should be addressed. E-mail: [email protected] Mailing Address: Department of Chemistry and Physics, SUNY at Old Westbury, Old Westbury, NY 11568, USA, Tel: (516) 876-2756. E-mail: [email protected] Mailing Address: Department of Physics and Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA, Tel: (216) 687-2431.

†SUNY at Old Westbury. §Lawrence Berkeley National Lab. ‡Cleveland State University.

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Abstract

Electron Paramagnetic Resonance and crystallographic studies of copper-doped cadmium DLhistidine, abbreviated as CdDLHis, were undertaken to gain further understanding on the relationship between site structure and dynamic behavior in biological model complexes. X-ray diffraction measurements determined the crystal structure of CdDLHis at100 K and 298 K. CdDLHis crystallizes in the monoclinic space group P21/c with two cadmium complexes per asymmetric unit. In each complex, the Cd is hexacoordinated to two histidine molecules. Both histidines are L in one complex and D in the other. Additionally, each complex contains multiple waters of varying disorder. Single crystal EPR spectroscopic splitting (g) and copper hyperfine (ACu) tensors at room temperature (principal values: g; 2.249, 2.089, 2.050, ACu; -453, -30.5, -0.08 MHz) were determined from rotational experiments. Alignments of the tensor directions with the host structure were used to position the copper unpaired dx2-y2 orbital in an approximate plane made by four proposed ligand atoms; the N-imidazole and N-amino of one histidine, and the N-amino and O-carboxyl of the other. Each complex has two such planes related by non-crystallographic symmetry, which make an angle of 65° and have a 1.56 Å distance between their midpoints. These findings are consistent with three interpretations that can adequately explain previous temperature-dependent EPR powder spectra of this system: (1) a local structural distortion (static strain) at the copper site has a temperature dependence significant enough to affect the EPR pattern, (2) the copper can hop between the two sites in each complex at high temperature and (3) there exists a dynamic Jahn-Teller effect involving the copper ligands. Keywords: EPR, Cu2+, dynamics, g-tensor, hyperfine, cadmium histidine, crystal structure


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Introduction Single crystal Electron Paramagnetic Resonance (EPR) is used to investigate a biological model complex; Cd2+-DL-histidine (CdDLHis), where doped Cu(II) is believed to undergo motional conversion between states. Previous work on this system in powdered samples showed a small but measureable temperature dependency of the EPR spectra, which was thought to arise from copper site dynamic behavior1. By comparing similar results from other histidine-copper complexes2-4, it was proposed that metal site dynamics is mediated by nearby disordered waters. The occurrence of dynamics in these systems depends on vibrational coupling of the lattice, specifically involving copper and ligand atoms, as well as on the presence of thermally accessible states. The flexibility afforded by motional disorder of waters hydrogen bonded to coordinating ligands affects copper site stability and therefore would have direct bearing on vibrational processes. Studies of simple molecule crystals are therefore of special interest because they can reveal the effect that metal site geometry and stability have on dynamics. Investigating such phenomena also provides fundamental understanding on correlations between transition energies, and different states which can co-exist, interconvert or otherwise affect each other and may help in interpreting the role of copper active site dynamics and plasticity in some important metalloproteins5-9. Two different metal-histidine model crystals have been previously studied where bound water molecules was proposed to influence the dynamics of doped copper; Zn2+-(DL-histidine)2 pentahydrate2 (ZnDLHis) and bis(L-histidinato)cadmium dihydrate3 (CdLHis). The observed temperature-dependent changes in the EPR pattern of Cu2+-doped ZnDLHis was explained as a dynamic Jahn-Teller averaging of its two symmetry-related low temperature (80 K) EPR patterns. The movement of a water hydrogen bonded to the coordinating histidine amino group


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nitrogen as a possible trigger for subsequent copper switching between conformational states was postulated to account for the continuous temperature-dependent changes and relatively sharp transition temperature (Tc ≈ 268 K) in the EPR spectra. This proposition was based in part on Xray diffraction results which found disorder of one of the waters upon comparing the 150 K and 293 K crystal structures2. Single crystal EPR studies performed on Cu2+-doped CdLHis also revealed dynamic averaging of low temperature (77 K) states in the room temperature spectra3. The 298 K crystal structure3,10,11 shows cadmium bound equally to two histidines similar to zinc in ZnDLHis. Results from EPR at 77 K and Electron Spin Echo Envelope Modulation at 4.2 K showed that the doped copper ion lies closer to one of the two symmetry-related histidines12. Later a more detailed 77 K EPR analysis supported this view that the copper binds strongly and closer to one histidine and weakly to the other3. The temperature-dependent EPR study showed that at temperatures lower than 150 K, the spectra exhibited two copper patterns, each related by the crystallographic two-fold axis that runs through the host cadmium ion and relating its two coordinating histidines3. At temperatures approaching room, the EPR consists of only a single copper species displaying spectral parameters close to the averaged values of the two symmetryrelated 77 K species. Experiments also displayed a very interesting 1:1 conversion from the low to high temperature copper species within a relatively narrow temperature range having Tc ≈ 165 K. The observations in CdLHis were explained by the copper rapidly converting between the two low temperature symmetry-related states by switching between its histidine partners at room temperature. This model is similar to that proposed for ZnDLHis but with important differences. Although the CdLHis 298 K crystal structure also displays a water molecule bound to the


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coordinated amino nitrogen, X-ray diffraction experiments at 130 K and 200 K (on the low and high side of the 165 K transition temperature) displayed identical structural water3. This refuted the contention that water motion was necessary for the copper dynamic behavior. Second, the distinct 1:1 conversion between low and high temperature copper states suggested a more complicated mechanism. The explanation proposed was a hopping of the copper ion between the two different histidines which is facilitated by the larger ionic radius of the replaced cadmium versus zinc in the two systems3. The present CdDLHis crystal study provides an additional opportunity to investigate how structural and geometric features may support copper site dynamic behavior. Earlier EPR measurements on powders of CdDLHis displayed temperature-dependent spectra which were consistent with previous observations of motional averaging1. In this work specific interest is to correlate the crystal structure with the EPR measured g and copper hyperfine coupling tensors as a means to deduce the copper binding site and to examine its possible role in this process. Experimental Rod shaped single crystals of cadmium DL-histidine were grown by slow evaporation of aqueous solutions containing DL-histidine and cadmium carbonate. For copper-doped hosts, a small ~1-2% amount of copper carbonate was also added to the solution. The doped crystals were pale blue. All compounds were of highest purity obtained from Sigma. The crystals readily cracked upon brief exposure to air and quickly dehydrated into a white powder. However, it was found that they could be stabilized by their immediate coating with Duco cement. Crystallography X-ray diffraction data were collected on a single crystal coated with Duco cement using a Bruker PHOTON100 CMOS area-detector diffractometer at 298(2) K and 100(2) K with


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synchrotron radiation (0.77490 Å) at beamline 11.3.1 of the Advanced Light Source at Lawrence Berkeley National Laboratory. The Bruker Apex2 program was used for the data collection13. Intensity data integrations, cell refinement and data reduction were performed using the Bruker SAINT software package13. Absorption correction was made with SADABS13,14. Crystal data are given in Table 1. The 298 K structure was determined with intrinsic phasing using SHELXT 201415 and refined using SHELXL 201415. The amino nitrogens in each complex are connected by a bracelet of disordered waters in five sites which were refined in pairs using different PART numbers and occupancies 0.9, 0.1, 0.9, 0.2, 0.8 in sequence from N1 to N4 and 0.9, 0.1, 0.9, 0.1, 0.9 from N1’ to N4’. Most water molecules, especially the high occupancy sites of the bracelets, had high temperature factors (Figure 1A, Tables S2 and S4). Hydrogen atoms bonded to the amino nitrogens N1, N4, N1’ and N4’ were obtained from difference syntheses and refined with their N-H distances restrained to be equal. The remaining hydrogens were placed in calculated positions using a riding model with their Uiso = 1.2 Ueq(N,C). No hydrogen atoms were placed in the water molecules. The non-hydrogen atoms were refined anisotropically except for the minor sites of disordered waters which were refined with an isotropic thermal factor. The 298 K structure (excluding the disordered water molecules) was the starting model for the refinement of the 100 K structure with SHELXL 201415. The bracelet of disordered waters between the amino nitrogens was re-determined in the 100 K structure and a sixth site near N1 and N1’ was also obtained owing to the better quality of the data at 100 K. The occupancy factors were 0.7, 0.35, 0.65, 0.5, 0.5 in sequence from N1 to N4 and 0.3 for the sixth site near N1, and 0.75, 0.25, 0.7, 0.3, 0.7 from N1’ to N4’ and 0.25 for the additional site near N1’. The treatment of the hydrogen atoms was identical with the 298 K structure. All the non-


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hydrogen atoms in the 100 K structure were refined anisotropically. The water molecules had low temperature factors at 100 K (Figure 1B, Tables S9 and S11). Data collection and refinement details for both structures are given in Table 1. Further details, including atomic parameters, complete distances and angles and hydrogen bonds are found in the supporting information. Superpositions were carried out with the lsq superpose command in coot16 and automol fit command in PLATON17,18. Figures 1 and 3 were prepared with ORTEP-319, Figure 2 was prepared with PYMOL (http://pymol.sourceforge.net), and Figures 4, 7 and 8 with ORTEP-3 and POVRAY20. EPR Spectroscopy EPR measurements were made on fresh copper-doped crystals which, when harvested, were also quickly immersed into Duco cement. The samples were immediately aligned and glued to the end of Pasteur pipettes under a Leica StereoZoom microscope, and subsequently placed in 3x4 mm quartz sample tubes. The crystallographic a' b and c axes (a' = b x c) were selected as reference axes for the external magnetic field directed EPR measurements. The tubes were held in a Bruker goniometer and placed inside the EPR microwave cavity. Rotational spectra were collected using a Varian E-109 X-band spectrometer, whose operating microwave frequency and magnetic field strength setting were monitored with an Agilent 53150A frequency counter and F.W. Bell 7010 gaussmeter, respectively. The spectrometer was interfaced to a Dell Dimension 2400 PC using a National Instruments 6024E PCI card for analog/digital signal conversion, and the gaussmeter digital output was connected through the PC’s serial port. Home written code based on the LabView language, including commercial code supplied with the Bell gaussmeter, was used to acquire and record the EPR signals as a function of magnetic field strength. Single crystal g-tensor and copper hyperfine tensor (ACu) parameters in the spin Hamiltonian operator:


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Eq. 1

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H = βS•g•H - I•ACu•S - gnβnH•I,

with S=1/2, I=3/2 and remaining terms defined in the usual way21, were least-square fit to measured resonant field rotational data using local methods as described in earlier reports3,12. Results Description of the Structures The asymmetric unit consists of two complexes. In each complex Cd2+ is hexacoordinated to two histidine molecules as seen in Figure 1A for the 298 K structure and Figure 1B for the 100 K structure. The coordination of Cd2+ is similar to that seen in the structure of Cd2+-L-histidine10,11. In one complex both histidines are in the L configuration and in the other both histidines are in the D configuration. Each complex contains two ordered water molecules that appear to be hydrogen bonded to the carboxyl oxygen that is not coordinated to Cd2+ (Figures 1A and 1B; supporting information, Tables S6 and S12). In addition, the amino nitrogens of each complex form hydrogen bonds to the water sites of the bracelet of disordered waters closest to them (Figures 1A and 1B; Tables S5 and S11). These two amino nitrogens are of particular interest because of their involvement in the putative copper binding site as discussed below. The two temperature structures are closely similar. There is a decrease in the axial lengths in all three directions (Table 1) at 100 K and, as seen in Figure 2, the LL complex is shifted away from the DD complex by (0.29940 Å, -0.07427 Å, -0..02135 Å) along the axes of the standard cartesian system while the DD complex is essentially in the same position [a shift by (-0.05984 Å, -0.00250 Å, -0.04780 Å) along the axes of the standard coordinate system]. The root-mean-square (rms) deviations between corresponding atoms are 0.034 Å for both DD and LL complexes. These superpositions were carried out with the lsq superpose feature in coot16. In


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addition, the 298 K structure has much higher temperature factors (Figures 1A and 1B, Tables S1, S3, S4, S7, S9 and S10).

Figure 1 The two histidines in each complex are related by a non-crystallographic two-fold axis parallel to b through the cadmium ions and the two complexes are related by a noncrystallographic center of inversion. For the 298 K structure, the (x, z) coordinates of the noncrystallographic two-fold axes are (0.5267, 0.7478) for the LL complex and (-0.0021, 0.7505) for the DD complex. These non-crystallographic dyads combined with the crystallographic 21 axis generate a non-crystallographic translation of b/2 in the structure. The non-crystallographic center of inversion is located at (0.2623, 0.1250, 0.7492) for the 298 K structure. These approximate symmetry elements combined with the space group symmetry generate a “pseudo unit cell” with unit vectors a, b/2, c and symmetry I2/a. The rms fit between the DD and LL


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complexes computed with automol fit in PLATON is 0.042 Å for the 23 non-hydrogen and nonwater atoms. The situation is analogous in the 100 K structure. The complexes form a three-dimensional network in the crystal with hydrogen bonds between them (Tables S5 and S11) and slipped imidazole-imidazole π stacking interactions between the D and L complexes across the non-crystallographic inversion center (Figure 3). The disordered water bracelets interact with this network only with hydrogen bonds to the amino nitrogens (Figures 1A and 1B; Tables S5 and S11) and hydrogen bonds to the ordered water molecules that form potential hydrogen bonds with the carboxylate groups (Figures 1A and 1B; O…O distances in Tables S6 and S12). They form an infinite column of disordered waters parallel to the a-axis weakly held by the network of complexes. A view of the packing looking down the a-axis illustrating this infinite column of disordered water molecules is shown in Figure 4. This is possibly the reason that the crystals readily dehydrate.

Figure 2 Room Temperature EPR Spectra The EPR single crystal patterns show g-tensor and copper nuclear (I=3/2) hyperfine tensor anisotropy similar to that observed in many copper amino acid complexes22. The spectra however lack any resolved super hyperfine splitting from nearby coupled nitrogen nuclei or


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protons. In addition, only one copper resonance pattern is visible when the external field (H) is directed in the a'c plane. This pattern site-splits into two when H is placed in the a'b and bc planes, which is consistent with a single magnetic copper species that follows the point group symmetry of the P21 cell. Because the asymmetric unit contains two different complexes (Figure 1), each with two structurally distinct histidine molecules bound to cadmium, the EPR was initially expected to display four distinct patterns (or two if the copper exactly substitutes for the cadmium ion). The lone copper pattern can be explained however by presence of the two noncrystallographic symmetries found in the structure (see discussion above), i.e., the approximate two-fold rotation axis along the b-axis through each cadmium ion, and the approximate inversion center at the midpoint of the two complexes. These cause the four expected patterns to coalesce into one magnetically unique copper per asymmetric unit. Figure 5 displays EPR spectra observed at three different orientations of the crystal. The upper spectrum (A) was obtained with the magnetic field H directed along the b-axis, the middle (B) is with H aligned at c+20° in the bc plane and the lower spectrum (C) is when H is placed in the a'c plane at 135° from the c-axis. The Figure 5A spectrum is centered at g = 2.132 and displays a copper nuclear hyperfine splitting of 85 G, which is found to collapse to a small value in Figure 5C (centered at g=2.059). The hyperfine tensor interaction has both an isotopic component due to Fermi contact of the unpaired electron spin with the magnetic nucleus and an anisotropic component which is commonly modeled by a magnetic dipole-dipole interaction between the spin moments of the unpaired electron and nucleus.21 These components can have opposing algebraic signs which can then lead to a near zero hyperfine spectral splitting at certain orientations of the copper complex in the external magnetic field, as displayed in Figure 5C. The doubling of patterns (site-splitting) shown in Figure 5B is consistent with the presence of two copper sites related by the point group


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symmetry of the unit cell. The blue lines beneath indicate the resonant field positions for these two patterns that were calculated using tensors in Table 2. Other broad lines of low signal strength were visible at certain orientations but because of extensive overlap with the major pattern they could not be followed. The linewidth of the individual copper lines are approximately 40 G and display only a small variation over the copper mI lines and orientation of the crystal over the three planes. This situation is different than found in the room temperature EPR study of ZnDLHis where a large linewidth dependence on orientation (ranging from 40 up to 150 G!) was observed and subsequently analyzed in terms of a dynamic averaging of conformational states2. In CdDLHis the structural differences in the

Figure 3 complexes (Figure 2) lead to a distribution of copper EPR spectral parameters and this static strain23 contributes to the inhomogeneous broadening of the lines (40 G). A small but finite g and ACu strain would effectively smear out resolution of any ligand hyperfine splittings without creating an abnormally large linewidth. The copper resonance fields as a function of crystal orientation are traced in Figure 6. The solid curves represent simulations using the g and copper hyperfine tensors in Table 2. The


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least-square refinement of ACu was slightly ill-conditioned due to small oscillations about those elements contributing to the collapsed region (Figures 5C and 6) i.e., where the hyperfine value approaches zero. Therefore the procedure was considered complete when the goodness-of-fit for the data fell below 15 MHz (~5 G). The refined hyperfine principal values were assumed to have a similar uncertainty.

Figure 4 The tensor parameters obtained from the rotational data have an eight-fold ambiguity (four pairs) due partly to the symmetry site-splitting. Only one pair is correct for a specific copper site. Each solution in a pair is related by the two-fold b-axis of the unit cell. Two of the possible pairs can be quickly discounted because their principal values do not match the powder EPR. That leaves two pairs which are experimentally indistinguishable because the a-axis (that is, the beta angle) could not be unequivocally identified on the crystals used in the EPR measurements. The tensors reported in Table 2 correspond to one of these two possible angular assignments of the a'c plane data (Figure 6). The alternate tensor solution (or assignment) was rejected on the basis that no possible binding site in the host crystal was found that provided a


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reasonable correspondence between g-tensor orientation and possible copper-ligand directions. The g and ACu tensors in Table 2 (principal values: g; 2.249, 2.089, 2.050, ACu; -453, -30.5, 0.08 MHz) are collinear within experimental uncertainty, and together with gmax > gmid≈gmin > gfs=2.0023, predicts a distorted tetragonal site geometry with a predominantly dx2-y2 copper unpaired orbital22,24.

Figure 5 The proposed copper binding site (Figure 7) is located at the midpoint of the best-plane25 made up of four ligand atoms; the amino group and imidazole nitrogens of one histidine, and the amino group nitrogen and carboxyl oxygen of the other. There are four such sites in the asymmetric unit made of planes N4-N1-N2-O3 and N1-N4-N5-O1 for the DD complex, and N4’-N1’-N2’-O3’ and N1’-N4’-N5’-O1’ for the LL complex. This leads to four slightly different copper sites possible in the asymmetric unit. The gmax (and ACu maximum) direction was found to deviate by only 2.9° from the ligand plane normal (average over the four sites), and this tight


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correlation orients the half-filled copper dx2-y2 orbital approximately in this plane. Figure 7A shows the proposed copper site in one of the complexes (in the N4-N1-N2-O3 plane), excluding the cadmium. The fractional coordinates for the site are −0.0206, 0.0124, 0.681, and is displaced 0.8 Å from the host cadmium position. Both ions cannot be simultaneously present in the complex due to the size of their respective radii26 and therefore the doped copper removes the cadmium from the complex. Figure 7B shows the copper at the middle of this distorted quadrilateral with distances: Cu-N4 1.98 Å, Cu-N1 2.50 Å, Cu-N2 2.03 Å and Cu-O3 2.22 Å, and inter-ligand angles: N4-Cu-N1 101°, N1-Cu-N2 88.3°, N2-Cu-O3 94.5° and O3-Cu-N4 78.0° (average values over the four sites). The angle between the imidazole plane and the copper ligand plane is ~25° and the Cα-N-Cu angles range from ~111-114°, both of which are reasonable values found for copper-histidine27. The view normal (z) to the ligand plane (in Figure 7B) displays the correspondences between the gmin and gmid principal directions and the copper-ligand contacts. The small angular deviations found between gmin: Cu-N1 (11.6°) and CuO3 (12°), gmid: Cu-N4 (13.6°) and Cu-N2 (14.6°) strongly support the suggested copper location. The two similar planes that exist in each complex have a common hinge of the amino nitrogens forming an inter-plane angle of 65° and a midpoint (copper site) separation of 1.56 Å. As discussed above, the slight structural difference in the four histidine molecules (Figure 1) leads to the four slightly different copper dope sites. The resulting EPR parameter strain is believed to be mainly responsible for the lack of any resolved ligand hyperfine splitting. The in-plane g and ACu tensor principal directions are slightly twisted (~13°) away from the directed copper ground-state orbital lobes (Figure 7B). This could be due to deformation of the site geometry from a square-plane28 (the atom rms deviation is ~0.12 Å and angular deviation from square is ~12°) or from “apical” ligands which can introduce excited dxz and dyz states into


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the spin wavefunction29. The postulated site has two such weakly interacting “apical” ligands, a carboxyl oxygen and an imidazole nitrogen that lie above the equatorial plane at 3.28 Å (3.25 Å at 100 K) and 2.84 Å (2.86 Å at 100 K) from the copper (Figure 7A) which then could theoretically account for the slight misalignment of the tensor. However, there are undoubtedly

Figure 6 local changes in the host structure upon doping that can also affect these correspondences. Two of the copper-ligand distances Cu-N4, 2.50 Å and Cu-O3, 2.22 Å are far from the normally found value of 2.0 Å30. In addition, the flexibility afforded by the disordered waters, especially the “bracelet” linking the two amino group nitrogens (Figure 1), can introduce adjustments that stabilize the copper. The copper could also be bound closer to one or the other of the two histidines in the complex, as was proposed earlier in CdLHis3,12. Discussion One aim of this study is to try to understand the structural basis behind the temperaturedependent results of CdDLHis powders1. EPR of the powder CdDLHis showed a continuous shifting of g (and ACu) principal values from 2.265, 2.083, 2.041 (488, 52, 10 MHz) at 80 K to


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2.258, 2.070, 2.069 (487, 3, 9 MHz) at room temperature1,31. Based on the crystallographic and single crystal EPR findings reported above, three interpretations can be offered. Two involve dynamic processes and one doesn’t, though they are not mutually exclusive. The static model considers the static structural distortions at the bonding site which change with temperature. In Figure 7B the Cu-ligand distances and angles using the room temperature structure are compared with the 100 K structure (parentheses). Small differences (~0.02 Å and ~1°) are found in the proposed copper-ligand distances and geometry. And although it appears unlikely that such small structural variation would have any significant impact, the changes in the powder EPR spectra were also slight (∆g’s ~0.01)1,31. Such small changes in host structure with temperature have been previously shown to produce a similar level of change in the g-tensor values of copper-doped Tutton salts32. In the present case, the question on whether these slight differences can emulate the powder EPR results1 must be considered in the context of the structural heterogeneity of the dope site in the asymmetric unit. The magnitude of the atomic positional differences in the structures at the two temperatures (Figure 2 and Figure 7B) is roughly the same as the structural heterogeneity of the four copper sites at 298 K. And since the single crystal EPR spectra exhibit no abnormal line broadening beyond the blurring of ligand hyperfine splitting, it seems unlikely that the slight changes in structure can account for the previous EPR powder observations1,31. As discussed above, the temperature dependence of the single crystal EPR of CdLHis3 was explained by a hopping model, where copper jumps or hops between neighboring histidine binding sites. At temperatures greater than Tc ≈ 165 K, the hopping motion becomes rapid enough to cause a transformation in the EPR from individual copper site patterns into an “averaged” room temperature pattern. A possible hopping model in CdDLHis and that proposed


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in CdLHis3 are shown in Figure 8. In CdLHis (Figure 8B), the doped copper is slightly displaced from the approximate ligand plane of an amino and imidazole nitrogen of one histidine, and the amino nitrogen and carboxyl oxygen of the other, but lies closer to the histidine contributing both nitrogens. Two such planes exist per complex and intersect with an angle of 85°. This places the two binding sites to within ~0.8 Å of each other. In CdDLHis (Figure 8A), the planes containing the copper sites are hinged by cis amino group ligands, forming a smaller angle (~65°) than in

Figure 7 CdLHis but having a larger distance between sites (1.56 Å). Nevertheless, the structural similarities in the two systems (Figure 8) make it appealing to speculate that the copper in CdDLHis also hops between binding sites, when given a large enough thermal activation. This would account for the EPR temperature dependence seen in the powder, where a partial averaging of the 80 K pattern occurs at 298 K1. It is notable that the average taken of the single crystal tensors (Table 2) over the two sites in each complex predicts an “averaged pattern” at high temperature with principal values; g: 2.20, 2.13, 2.06 and |ACu|: 332, 145, 5 MHz. These are very close to those measured for a minor species found in the room temperature CdDLHis powders1,31 (g: 2.22, 2.11, 2.08 and ACu: 375, 130, 20 MHz). Unfortunately, no such secondary


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pattern could be distinguished in the single crystal spectra to support this contention. In addition, as mentioned above, the large water disorder in CdDLHis should allow sufficient structural flexibility at the proposed site to stabilize the doped copper coordination. The probability of hopping between sites over any significant distance would be diminished as the copper binding becomes more energetically stable. The alternative to the hopping model is a dynamic Jahn-Teller effect. Like the hopping model, it also produces a spectral averaging over accessible copper states. This effect has been proposed to explain the temperature-dependent EPR in ZnDLHis crystals, where vibrational motions of the amino and imidazole nitrogen ligands were responsible for the observed averaging2. For CdDLHis, the arrows directing the ligand atoms in Figure 7B provide hypothetical movements that could contribute to a dynamic Jahn-Teller effect. This process would be consistent with the partial averaging of the 80 K in-plane g-values (gmin and gmid) observed in the room temperature powder spectra1,31. The similarities in the ZnDLHis and CdDLHis systems also extend to their transition temperature Tc ≈ 268 K1,2. Conclusion The origin of changes in the EPR spectra at different temperatures in the CdDLHis powder was presumed dynamic in origin by a comparison with similar investigations on ZnLHis4, ZnDLHis1,2 and CdLHis1,3. This study was undertaken to shed light on structural factors that may affect such processes in this system. This alignment of EPR tensor directions with atomic positions determined by the crystal structure reveal a putative copper binding site between the two histidine molecules in the complex. The copper is located at the midpoint of an approximate ligand plane made up of the amino and carboxyl groups of one histidine and the amino and imidazole nitrogen of the other. Each complex has two such planes related by a non-


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crystallographic two-fold rotation axis about the b-axis. The temperature dependence of the EPR powder pattern of CdDLHis reported in a previous study1 can therefore be explained by three

Figure 8 models; a static structural distortion at the copper site that depends on temperature, copper hopping between the two sites in a given complex, or a local dynamic Jahn-Teller effect from the vibrational motions of the copper ligand atoms. All three models are equally applicable to the observations and are not totally exclusive to each other. More specifically, results from this study shed further light on the possible role of disordered water in stabilizing the copper dope site and regulating its dynamic behavior. The aim of the present work was to investigate how structural and geometric features support copper site dynamic behavior. By comparing with previous results on similar systems, this study favors the contention that disordered waters bound near the copper complex promote a pseudo-dynamic Jahn-Teller mechanism through the mediation of ligand-copper motions. The ligand vibrational couplings with the copper electron spin then leads to the previously reported temperature dependence of the EPR powder spectra. However, the two other models, a


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temperature-dependent static strain and a copper hopping model, cannot be fully discounted for this system. More advanced-EPR spectroscopic approaches may be able to distinguish among these three mechanisms. Acknowledgements The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. We thank SUNY/Old Westbury undergraduate student Ms. Yanli Li for her help in the early stages of growing crystals and the EPR analysis. Supporting information CIF files for the 298 K (rt_a.cif) and 100 K (100k_a.cif) structures. Tables S1-S6 for the 298 K structure, Tables S7-S12 for the 100K structure, and Table S13 for correspondence of naming of water sites in the bracelets at the two temperatures. This material is available free of charge via the Internet at http://pubs.acs.org.


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References (1) Colaneri, M. J.; Vitali, J. Copper Dynamics in Doped Metal Bis(histidine) Complexes. J. Phys. Chem. A 2014, 118, 4688-4694. (2) Dalosto, S. D.; Calvo, R.; Pizarro, J .L.; Arriortua, M .I. Structure, Disorder, and Molecular Dynamics in Zn(DL-histidine)2: EPR of Copper Ion Dopants, X-ray Diffraction, and Calorimetric Studies. J. Phys. Chem. A 2001, 105, 1074-1085. (3) Colaneri, M. J., Vitali, J.; Kirschbaum, K. Electron Paramagnetic Resonance Spectroscopic Study of Copper Hopping in Doped Bis(L-Histidinato)Cadmium Dihydrate. J. Phys. Chem. A 2013, 117, 3414-3427. (4) Rockenbauer, A.; Gydr, M.; Szabo-Planka, T. Electron Spin Resonance Detection of JahnTeller Effect Induced Phase Transition with Thermal Hysteresis in the Copper(II) Doped Zinc(II)-Bis-Histidine Systems: Free and Hindered Rotation of Histidine Molecules in Solid Lattice. J. Chem. Phys. 1987, 86, 976-979. (5) Bacci, M.; Cannistraro, S. Temperature Dependence of the g Values in Blue Copper Protein EPR Spectra. Chem. Phys. Lett. 1987, 133, 109-112. (6) Bacci, M.; Cannistraro, S. Role of Vibronic Coupling and of Conformation Substrate Distribution in Determining the Features of Copper-Protein EPR Spectra. Applied Magn. Reson. 1990, 1, 369-378. (7) Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. Crystallographic Evidence that the Dinuclear Copper Center of Tyrosinase Is Flexible during Catalysis. J. Biol. Chem. 2006, 281, 8981-8990. (8) Sendovski, M.; Kanteev, M.; Ben-Yosef, V. S.; Adir, N.; Fishman, A. First Structures of an Active Bacterial Tyrosinase Reveal Copper Plasticity. J. Mol. Biol. 2011, 405, 227-237. (9) Matoba, Y.; Bando, N.; Oda, K.; Noda, M.; Higashikawa, F.; Kumagai, T.; Sugiyama, M. A Molecular Mechanism for Copper Transportation to Tyrosinase that is Assisted by a Metallochaperone, Caddie Protein. J. Biol. Chem. 2011, 286, 30219-30231. (10) Candlin, R.; Harding, M. M. The Crystal Structure of Bis(Histidino)Cadmium Dihydrate. J. Chem. Soc. (A) 1967, 421-423. (11) Fuess, H.; Bartunik, H. Neutron Diffraction Structure Determination of Bis-(Lhistidinato)Cadmium Dihydrate. Acta Cryst. 1976, B32, 2803-2806. (12) Colaneri, M. J.; Peisach, J. A Single Crystal EPR and ESEEM Analysis of Cu(II)-Doped Bis(L-Histidinato)Cadmium Dihydrate. J. Amer. Chem. Soc. 1995, 117, 6308-6315. (13) APEX2, SAINT and SADABS. Bruker AXS Inc., Madison Wisconsin, 2014.


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(14) Blessing, R. H. An Empirical Correction for Absorption Anisotropy. Acta Cryst. 1995, A51, 33-38. (15) Sheldrick, G.M. A Short History of SHELX. Acta Cryst. 2008, A64, 112-122. (16) Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and Development of Coot. Acta Cryst. 2010, D66, 486-501. (17) Speck, A.L. Single Crystal Structure Validation with the Program PLATON. Journal of Applied Crystallography 2003, 36, 7-13. (18) A. L. Speck, A.L. Structure Validation in Chemical Crystallography. Acta Cryst. 2009, D65, 148-155. (19) Farrugia, L.J. Ortep-3 for Windows. J. Appl. Cryst. 1997, 30, 565. (20) Persistence of Vision Raytracer (Version 3.7), http://www.povray.org. (accessed Aug. 1, 2013) (21) Wertz, J. E.; Bolton, J. R. In Electron Spin Resonance: Elementary Theory and Practical Applications; McGraw-Hill, Inc.: New York, 1972. (22) Peisach, J.; Blumberg, W. E. Structural Implications Derived from the Analysis of Electron Paramagnetic Resonance Spectra of Natural and Artificial Copper Proteins. Arch. Biochem. Biophys. 1974, 165, 691-708. (23) Froncisz, W.; Hyde, J. S. Broadening by Strains of Lines in the g-Parallel Region of Cu2+. J. Chem. Phys. 1980, 73, 3123-3131. (24) Blumberg, W. E. Some Aspects of Models of Copper Complexes. In The Biochemistry of Copper (Peisach, J.; Aisen, P.; Blumberg, W. E., eds.) Academic Press: New York, 1966, 49-64. (25) Structural Bioinformatics Group, Imperial College London, http://www.sbg.bio.ic.ac.uk/~islam/plane.html. (accessed June 5, 2015). (26) Cotton, F.A.; Wilkinson, G. Advanced Inorganic Chemistry 3rd ed.; John Wiley & Sons: NY, 1972, p.52. (27) Camerman, N.; Fawcett, J.K.; Kruck, T.P.A.; Sarkar, B.; Camerman, A. Copper(II)Histidine Stereochemistry. Structure of L-Histidinato-D-histidinatodiaquocopper(II) Tetrahydrate. J. Amer. Chem. Soc. 1978, 100, 2690-2693. (28) Debuyst, R.; Dejehet, I.; Ggrller-Walrand, C.; Vanquickenborne, L. G. On the Principal Axes of the g- and A-Tensors of Cu(II)-Ions Substituted in Low-Symmetry Sites. Inorganica Chimica Acta. 1981, 51, 117-122.


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(29) Belford, R. L.; Harrowfield, B.; Pilbrows, J. R. Effects of Excited-State Spin-Orbit Coupling and Ground-State Hybridization upon the Behavior of the In-Plane g Tensor in LowSymmetry d1,9 Systems. J. Mag. Reson. 1977, 28, 433-439. (30) Hathaway, B. J.; Hodgson, P. G. Copper-Ligand Bond-Lengths in Axial Complexes of the Copper(II) Ion. J. Inorg, Nucl. Chem. 1973, 35, 4071-4081. (31) The unpublished g and ACu tensor values for major species I and minor species II obtained from EPR powder analysis of CdDLHis reported here were determined using a static model in the fitting procedure. The corresponding tensor values reported in Table 1 of Ref. 1 for this system are different because they were based on fits that included motional correlation time parameters. (32) Riley, M. J.; Hitchman, M. A.; Mohammed, A. W. Interpretation of the TemperatureDependent g Values of the Cu(H2O)62+ Ion in Several Host Lattices using a Dynamic Vibrational Coupling Model. J. Chem. Phys. 1987, 87, 3766-3778.


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Table 1. Crystal data and refinement of the 100 K and 298 K structures.

100 K

298 K

Empirical formula1

C12 H25.95 Cd N6 O8.98

C12 H25.80 Cd N6 O8.90

Formula weight

510.34

508.98

Wavelength, Å

0.7749

Space group

P21/c

Unit cell dimensions

Volume

a = 12.1161(5) Å

a = 12.2555(5) Å

b = 29.0903(12) Å

b = 29.4689(13) Å

c = 11.0501(5) Å

c = 11.1087(5) Å

β= 92.127(2)°

β= 92.396(2)°

3892.0(3)

Å3

4008.5(3) Å3

Z2 Crystal size,

8 mm3

0.600 x 0.300 x 0.300

θ range for data collection

2.151 to 40.399°.

2.138 to 31.149°

Reflections collected

78514

55386

Independent reflections

18921 [R(int) = 0.0467]

9978 [R(int) = 0.0522]

Completeness to theta = 27.706°

100.0 %

99.9 %

Refinement method

Full-matrix least-squares on F2

Data / restraints / parameters

18921 / 56 / 586

9978 / 56 / 553

Goodness-of-fit on F2

1.036

1.042

Final R indices [I>2sigma(I)]

R1 = 0.0461, wR2 = 0.1035

R1 = 0.0576, wR2 = 0.1320

R indices3 (all data)

R1 = 0.0732, wR2 = 0.1162

R1 = 0.0795, wR2 = 0.1485

1.849 and -2.352

1.905 and -2.461

3

Largest diff. peak and hole ,

e.Å-3

1

The reason for the fractional values of O and H is the bracelet of disordered waters connecting the amino nitrogens. It was not possible to locate the corresponding hydrogen atoms in difference maps but these are included in the empirical formula. 2 The asymmetric unit consists of two complexes – each complex has two histidines, one Cd and waters. The histidines in one complex are D and in the other L.

R1 = Σ ||Fo| - |Fc||/Σ|Fo|; wR2 = [Σ[w (Fo2 - Fc2)2] /Σ[w (Fo2)2]]1/2.

3


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Table 2. The g and copper hyperfine coupling (ACu) tensors obtained from Cu(II)-doped cadmium DL-histidine crystals at room temperature. The tensor principal directions (direction cosines) refer to the crystallographic a’bc axes (with a’ = b x c). The largest hyperfine value was assumed negative.

Principal Values

g

2.249 2.089 2.050

Principal Values (MHz)

Cu

A

-453. -30.5 -0.08

Direction Cosines a'

b

c

0.5490 0.6270 -0.5527

0.5328 -0.7720 -0.3466

0.6440 0.1042 0.7579

Direction Cosines a'

b

c

0.542 0.621 -0.566

0.530 -0.775 -0.343

0.652 0.114 0.750


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Figure Captions Figure 1 The asymmetric unit. of the 298 K (A) and 100 K (B) structures. The higher temperature factors of the 298 K structure are noteworthy. There is an additional water site in the bracelet of the 100 K structure owing to the better data set at this temperature. The amino nitrogens form hydrogen bonds to their closest sites of the water bracelet and the ordered waters appear to hydrogen bond with the carboxylate oxygens that are not coordinated to cadmium. These hydrogen bonds are illustrated with dashed lines. The primed nomenclature corresponds to the LL complex. The correspondence of the water names in the bracelets at the two temperatures are listed in Table S16. Ellipsoids are drawn at the 50% probability level. Figure 2 Comparison of the asymmetric unit at 298 K and 100 K. Red is the 298 K structure, blue is the 100 K structure. Waters and hydrogens are not shown. While the DD complexes are essentially superimposable, the LL complex at 100 K shifts away from the DD complex by 0.299 Å along a. When the DD and LL complexes at the two temperatures are superimposed, the rms deviations between corresponding atoms are 0.034 Å for both. The figure was prepared using PYMOL (http://pymol.sourceforge.net). Figure 3 Stacking of the imidazole rings between the DD and LL complexes in the asymmetric unit. View is normal to the imidazole rings. The top histidine is from the LL complex and the bottom histidine from the DD complex. The distance between the imidazole rings is 3.435 Å, the distance between their centroids is 3.827 Å and the rings are parallel (angle 0.166º). The Cd ions, Cd1’ and Cd1, that are coordinated to N5’ and N2, respectively, are not shown for clarity. Figure 4 Packing looking down the a-axis. The network of complexes and the infinite columns of disordered waters are illustrated. Figure 5 Single crystal X-band EPR spectra of Cu(II)-doped cadmium DL-histidine (A) at b//H, (B) when H is aligned along c+20° in the bc plane and (C) aligned at c+135°, when a'c plane//H acquired at room temperature. Figure 6 Room temperature EPR measurements showing the dependence of the copper resonance lines on the crystal orientation in the external field. The solid curves represent simulated values using the g and ACu hyperfine tensors listed in Table 1. The arrow at 20° in the bc plane indicates the crystal orientation where the spectrum in Figure 5B was acquired. The a//H direction and beta angle position indicated on the a'c plane correlates this rotational data set with the tensors in Table 1. Figure 7 (A) A view of the proposed copper binding site within a complex (DD shown) in doped cadmium DL-histidine, without the cadmium ion. The proposed copper position was determined by correlating the g and ACu tensors directions with possible ligands in the host crystal structure. (B) A view of the Cu2+ position looking down the direction of gmax which defines the dx2-y2 spin orbital plane. Shown is the alignment of the gmin and gmid directions with respect to the copperligand contacts (dotted lines). The numbers in parenthesis are the corresponding values computed using the 100 K structure coordinates.


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Figure 8 The two possible copper binding sites per complex in doped (A) cadmium DL-histidine (DD complex) and (B) bis(l-histidinato)cadmium dihydrate showing the structural similarities in the dynamic hopping model. The model consists of the copper hopping between these sites at a high enough temperature, causing the growth of a dynamically “averaged” EPR pattern. In (A), the copper lies at the mid-point of the best plane made of N1-O1-N5-N4 (Cu1) or N4-O3-N2-N1 (Cu2). The two planes have a common N1-N4 hinge of the amino groups and make an angle of 65°. They are related by an approximate two-fold axis running through the replaced cadmium (yellow dot). The sites are displaced from the cadmium position by 0.82 Å, and from each other by 1.56 Å. In (B), the copper site lies slightly out of the plane made of N1’-N2’-N1-O1 (Cu) or N1-N2-N1'-O1' (Cu'). Here again the two amino nitrogens are common to both planes, but in this case the two planes intersect each other, making an angle of about 85°. The sites are related by a crystallographic two-fold axis running through the replaced cadmium ion. Previous EPR studies have shown that the copper is bound closer to one of the histidines in the complex (thin lines drawn in the figure represent their coordinate bonds) forming two possible sites separated by 0.82 Å.


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Models for Copper Dynamic Behavior in Doped Cadmium dl-Histidine

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