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Copper Dynamics in Doped Metal Bis-Histidine Complexes Michael J Colaneri, and Jacqueline Vitali J. Phys. Chem. A, Just Accepted Manuscript • Publication Date (Web): 12 Jun 2014 Downloaded from http://pubs.acs.org on June 16, 2014

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The Journal of Physical Chemistry

Copper Dynamics in Doped Metal Bis-Histidine Complexes

Michael J. Colaneri†* and Jacqueline Vitali‡* Department of Chemistry and Physics, State University of New York at Old Westbury, Old Westbury, New York 11568, 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. ‡Cleveland State University.

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Abstract Electron Paramagnetic Resonance (EPR) temperature dependent measurements were undertaken on three Cu(II)-doped metal-histidine complexes to assess copper-site dynamic behavior. Previous single crystal EPR analysis on two of these; zinc D,L-histidine pentahydrate (ZnDLH) and bis(L-histidinato)cadmium dihydrate (CdLH) found that doped Cu2+ can be modeled as hopping between two neighboring conformational states, with a temperature-dependent rate becoming large enough at room temperature to produce an “averaged” spectrum. By comparing spectra from their powdered form, we show that Cu2+ doped into a third system: Cd2+-D,Lhistidine (CdDLH) also exhibits temperature dependent EPR with features indicating a similar motional-averaging process. In addition, the change of g and copper hyperfine parameters from low to high temperature for CdDLH resemble that in ZnDLH, whereas the change in these parameters for CdLH are like that found in a fourth copper-doped system; zinc L-histidine dihydrate (ZnLH). Taken together, these results suggest that averaging motion between neighboring copper sites is common in metal bis-histidine compounds. More detailed studies on biological models are thus warranted, especially since they reveal unique relationships between structure, dynamic processes and stability, and can lead to a better understanding of the role played by site flexibility in copper proteins.

Keywords: EPR, Copper, Dynamics, g-tensor, hyperfine, histidine, zinc histidine, cadmium histidine


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Introduction

An Electron Paramagnetic Resonance (EPR) temperature-dependent study was performed on crushed crystals of copper doped Cd2+ -DL-histidine (CdDLH) in order to complement our recent investigations of copper dynamic behavior in biological models. Previous single crystal EPR investigations on two host systems; zinc D,L-histidine pentahydrate1 (ZnDLH) and bis(Lhistidinato)cadmium dihydrate2,3 (CdLH) postulated that the doped copper can be modeled as hopping between neighboring conformational states. Figure 1 depicts the proposed copper binding sites in the crystal structures along with the orientations of the g-tensors. Their EPR spectral tensor parameters were used to correlate the hopping states with symmetry-related histidine binding configurations, and their temperature dependences were explained by the copper moving between these sites. Employing Anderson’s theory7, this motion became rapid enough to cause an averaging of the spectra associated with two symmetry-related, low temperature sites1,3. In the zinc system, the movement of a nearby water molecule was hypothesized to mediate the copper dynamics. DSC and X-ray diffraction results were consistent with an order-disorder transition of this water, which could act as the trigger for the copper-site motion. This accounted for the continuous temperature dependent changes in the EPR and the sharp transition at Tc = 268 K that distinguished the two low temperature patterns from the “averaged” high temperature pattern. In doped CdLH, an equally sharp EPR spectral transition temperature was found but at a much lower temperature, Tc ≈ 165 K. However, no evidence for water disorder was found in the CdLH crystal structure3, and the explanation thus offered for the EPR temperature dependence was a jumping of the copper between two symmetry-related, histidine binding positions. So in CdLH, the doped copper itself can be viewed as undergoing a local thermally induced order-disorder transition. The larger ionic radius8 of cadmium versus 3 ACS Paragon Plus Environment


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zinc in the two systems allowed this dynamic, and this is believed to be one of the reasons for the significant differences in transition temperatures, potential energy barrier between states and room temperature hop rate for the two systems.3 A study of the present system is therefore of particular interest because here also, the doped copper can substitute for the larger cadmium ion in the structure, and this disposition should lead to motional behavior. In this work we present evidence that CdDLH does indeed exhibit temperature dependent EPR spectra. However, its dependence was found to have characteristics similar to both CdLH and ZnDLH.

Experimental The variable temperature EPR measurements were carried out on a computer-interfaced Varian E-109 X-band (9.2058 GHz) spectrometer equipped with an Oxford Cryostat system. Rod shaped single crystals of CdDLH were grown by slow evaporation of aqueous solutions containing D,L-histidine and cadmium carbonate, and for doped hosts, a small ~1% amount of copper carbonate. Energy Dispersive X-ray Spectroscopic analysis carried out by the Instrumentation Center of the University of Toledo using a JEOL JSM-7500F scanning electron microscope confirmed the presence of Cd, N, C and O in the host crystal. 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. Measurements were made on fresh copper-doped crystals which were harvested, crushed into a powder and placed in 3 by 4 mm quartz EPR sample tubes. It was observed that dry fresh powder samples when placed in boiling water for 8 min produced water droplets in the sealed tubes, indicating the presence of water in the crystal. Crystals of copper-doped ZnDLH and CdLH were grown according to the published procedures1,6 and were also crushed into powders for EPR measurements. DPPH (g=2.0036) was used as magnetic field calibration (3283 G). EasySpin9 was used to fit and simulate the EPR 4 ACS Paragon Plus Environment

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powder spectra measured at 80 K and 298 K. The EasySpin utilities “pepper” and “chili” were used for the rigid-limit (static) and motional-regime (dynamic) EPR powder simulations, respectively. Fit spectral parameters include the principal values of the g-tensor and copper hyperfine tensor (ACu) in the Spin Hamiltonian (Eq. 1):

Eq. 1

H = βS•g•H - I•ACu•S - gnβnH•I,

with S=1/2, I=3/2 and remaining terms in Eq. 1 defined in the usual way10. In all EasySpin calculations, spectral linewidth parameters were set to 40 G to be consistent with those found in previous single crystal EPR studies of ZnDLH1 and CdLH3 at low temperature. Dynamic fittings of the room temperature EPR spectra were accomplished employing the Stochastic Liouville equation9 using a rotational isotropic correlation time (τ). Results and Discussion The temperature dependence of the EPR spectra of CdDLH shows a gradual but significant change in the copper pattern as the sample temperature ranges between 80 K and 298 K (Figure 2A). The spectrum remains mostly unchanged from 80 K up to 165 K. At temperatures higher than this, alterations occur, namely: (a) the parallel region (low field) copper hyperfine lines broaden and shift in resonance to higher magnetic field and (b) the pattern symmetry first becomes more axial, then more rhombic. This change is slight at first but becomes more pronounced around 235 K and continues up to room temperature (298 K). In addition, a second, copper pattern (species II) becomes apparent above 245 K, seen slightly to the high field side of each of the resolved low field copper hyperfine-split (mI) lines of the major species (I). This secondary pattern grows in relative amount to about 30% of the total pattern as the temperature reaches 298 K. 5 ACS Paragon Plus Environment


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These features are similar to the EPR changes for copper complexes in ZnDLH and in CdLH. As shown in Figure 2B, the low temperature patterns (80 K) for both initially appear to become more axially symmetric, then more rhombic, as the temperature rises to room temperature. The transition temperature is apparent in ZnDLH (Tc = 268 K)1 by the setting just higher than where the maximum broadening of the copper hyperfine lowest field line occurs (~265 K), and in CdLH (Tc ≈ 165 K)3, by the setting where the low temperature pattern has diminished with the first appearance of the high temperature pattern (~165 K). These features are consistent with the critical temperatures obtained from EPR single crystal measurements1,3, and also locate the temperature where the powder spectrum exhibits the most accelerated change. The transition in CdDLH (Fig. 2A) is not nearly as distinguished as in ZnDLH or CdLH, owing possibly to its low signal strength, increasing line broadening and relative shrinkage of the lower temperature species (I) with the overlap of a growing secondary species (II) at higher temperatures. An analysis of the maximum rate of change for species I places the transition temperature for CdDLH at roughly 265 K. This is about the same as the transition temperature found for ZnDLH (268 K). The growth of species II at the expense of the low temperature species continues up to, and possibly extends beyond, room temperature. The discrete conversion between a low and high copper species is like that observed in CdLH near its transition temperature. The EPR temperature dependence in CdDLH therefore has features found in both ZnDLH and CdLH. This will be discussed further below. Figure 3 compares EPR powder spectra of Cu2+ doped in ZnDLH, CdLH (Fig. 3A) and CdDLH (Fig. 3B) measured at 80 K and at 298 K with simulations calculated from EasySpin9 fits. The rigid-limit simulations closely matched the low temperature experiments. However, the room temperature spectra, especially measured from CdLH, were poorly modeled by static 6 ACS Paragon Plus Environment

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calculations. This is because the dynamic averaging in these systems can lead to large copper mI dependent line broadenings.1,3 EPR room temperature powder patterns were subsequently treated with the motion-regime, giving isotropic rotational correlation times of 3.82 ns and 1.51 ns for ZnDLH and CdLH, respectively. These values are effective estimates, owing to the approximate nature of the Stochastic Liouville analysis for copper9 and the simplified hopping model (isotropic) employed. They are significantly larger but nonetheless relatively comparable to those from previous single crystal EPR temperature dependent analyses of hop rates: νh-1 = 1.25 ns reported for ZnDLH1, and νh2-1 = 0.384 ns and νh4-1 = 0.439 ns, extrapolated from Arrhenius plots (Figure 17) of CdLH3. Similar to ZnDLH, correlation times for CdDLH were found to be 3.62 ns and 2.92 ns for species I and II, respectively. As mentioned above, satisfactory simulations of the room temperature spectra of CdDLH required the presence of two mostly overlapped patterns: a major species I which provides 70% of the spectrum, and an underlying secondary species II supplying 30%. Figure 3B shows the separate contributions of the two; species I (green curve) and species II (red curve), to the total simulation (blue curve) of the experimental spectra (black curve). The best fit principal g-tensor, copper hyperfine tensor (ACu) and correlation time (τ) quantities from the ZnDLH, CdLH and CdDLH are given in Table 1 as well as parameters reported in an earlier EPR study11 of ZnLH at 269 K and 285 K. Also included are the tensor components obtained from the previous single crystal EPR measurements on ZnDLH and CdLH. The powder quantities for ZnDLH are in better agreement with the available single crystal g-tensors than CdLH, whose g-values are systematically smaller in the crystal. It may be noted that the single crystal measurements for CdLH were particularly difficult because of the extensive spectral overlap of the high field resonances3 and this might account for the differences in the tensors. The more easily assessed of 7 ACS Paragon Plus Environment


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the powder parameters is giso, since it can be directly tied to the experimental centroid of the pattern12, followed by the resolved maximum g-value (gmax) and copper hyperfine (Amax) components. A more tentative approach is required when comparing the other quantities at different temperatures and systems due to higher uncertainties resulting from spectral overlap and ambiguity in modeling unresolved broadenings. Even so, the lower temperature gmax and Amax tensor parameters for all four systems are found to be in the normal range for tetragonally distorted, axial type copper-nitrogen or mixed copper-nitrogen/oxygen ligand model complexes13. On the other hand, the EPR parameters found for the systems above their respective transition temperatures, with the exception of species I of CdDLH, are far different from the observed ranges for normal complexes. The higher temperature (room) tensors in Table 1 for CdLH and ZnDLH are also, as reported earlier1,3, close to but not exactly the averaged values of tensors corresponding to the two symmetry-related copper-histidine low temperature sites. The giso values for the four copper-histidine complexes in Table 1 were determined from the trace of the principal g values determined from the powders and from the single crystal gtensors. While powders of ZnDLH give approximately the same giso; 2.131 at 80 K and 2.133 at 298 K, it decreases in CdLH from 2.152 to 2.148. In general the giso values at the two temperatures for each system have differences slightly more than the estimated uncertainty, i.e., ± 0.00312. As mentioned above, giso is not expected to be the same at 80 K and 298 K in either ZnDLH and CdLH, as previous single crystal1,3 and powder measurements11 show a small but continuous shift of the dynamically averaging resonances to higher magnetic field (or lower gvalue) as the temperature rises above Tc. This shift can be seen in Table 1 by comparing the giso from the single crystal measurements; 2.136 for ZnDLH, and 2.146 for CdLH at 80(77) K with ~2.132 at 298 K for both ZnDLH and CdLH. This is consistent with the differences between the 8 ACS Paragon Plus Environment

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gmax values averaged over the two low temperature (80/77 K) single crystal site tensors (2.214 for ZnDLH, and 2.186 for CdLH) with their 298 K single crystal parameters (2.204 for ZnDLH, and 2.168 for CdLH). These deviations from the fully averaged tensor values were explained in terms of a temperature dependent vibrational mixing of two slightly-higher-in-energy states into the low temperature states (ZnDLH)1, and/or by a dependence of the copper state energy profile with temperature (CdLH)3. For ZnLH, the low to high pattern transition occurs over a narrow temperature range by considering the great change in the EPR spectra between 269 K and 285 K (compare Figs. 1 and 2 in reference 11). The midpoint, 277 K, is therefore estimated as the transition temperature for this system. Keeping with the trend found in ZnDLH and CdLH, the giso for ZnLH at 285 K (2.134) decreases slightly from its value at 269 K (2.139). For CdDLH, the gsio value for species II at 298 K (2.147) opposes this trend, being greater than at 80 K (2.130). However, one expects a high uncertainty in the parameters for species II given its lower contribution to the overall EPR pattern. Even with this caveat, the close correspondences of the low and high temperature giso values in all four systems are consistent with the presence of temperature induced dynamic averaging over two, near-energy and neighboring, copper-histidine binding sites. As stated, the higher temperature EPR powder pattern for CdDLH is complicated by the growth of the minor species (II) to about 30% of the total intensity at room temperature. This is reminiscent of CdLH where the low temperature copper pattern signal decreased with temperature as it converted into the high temperature pattern3. At its Tc (~165 K) equal populations of these patterns are observed. This is not the case for CdDLH where only ~15% of the minor pattern exists at its estimated Tc (265 K). Here the conversion between patterns appears to continue up to, and possibly extend beyond, room temperature. This would argue that the Tc 9 ACS Paragon Plus Environment


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for this complex should be greater than 265 K, and possibly higher than 298K, and that species II represents the dynamically “averaged” state of the low temperature sites. The rhombic-like form and values of the powder EPR spectral g and ACu tensors at 298 K for the copper species II in CdDLH (g; ACu: 2.238, 2.137, 2.065; 398, 111, 78 MHz) resemble those in ZnDLH, (g; ACu 2.226, 2.117, 2.049; 359, 116, 28 MHz), and the parameters in CdLH (g; ACu 2.204, 2.178, 2.061, 281, 154, 34 MHz) are more similar to the axial tensors in ZnLH (g; ACu 2.162, 2.162, 2.078; 196, 196, 28 MHz). The features of the high temperature “averaged” spectra are largely a consequence of how far the two averaging low temperature sites’ tensor principal axes depart from each other. Figure 1 shows the relative orientations of the low temperature g-tensors for the two hopping sites (primed and unprimed) in each system as determined from previous single crystal measurements1,2,3. In CdLH, the gmax (and Amax) principal direction for one site is 85º from the second symmetry-related site, whereas in ZnDLH the comparable angle is 65º. The more different the principal axes alignments are produces a bigger change from the low temperature to the high temperature powder spectra when averaging occurs, and vice versa. One can therefore infer by comparing the change in gmax and Amax from low temperature to room temperature in Table 1, (gmax; Amax 2.265; 488 MHz changing to 2.238; 398 MHz, species II) for CdDLH and (gmax; Amax 2.278; 390 MHz changing to 2.162; 196 MHz) for ZnLH, that the angle between the copper sites’ gmax directions in CdDLH should be less than or near 65º, whereas in ZnLH, this angle should be closer to 85º. Verification of this awaits the full single crystal EPR analyses. CdDLH exhibits a transition temperature (Tc ≥ 265 K) near those for ZnDLH (268 K) and ZnLH (277 K), but far from CdLH (Tc ≈ 165 K). Another difference is found in the available crystal structures. Both ZnDLH1 and ZnLH14 crystals have disordered water molecules, whereas 10 ACS Paragon Plus Environment

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CdLH does not2. As stated above, spectroscopic results from ZnDLH supported the hypothesis that the thermal motion of disordered water is cooperatively linked to the copper dynamics1. In CdDLH the observation of water droplets in sealed tubes from heated samples also indicate the presence of disordered waters of crystallization. This finding strengthens the contention that the mechanism of copper site dynamics in CdDLH is similar to that in ZnDLH (and possibly in ZnLH), i.e. mediated by water motions, but not to that in CdLH. Conclusion Comparisons with temperature dependent powder EPR spectra of systems known to exhibit dynamic behavior suggest that copper in Cd2+-D,L-histidine (CdDLH) also undergoes motional averaging of two near copper-histidine binding sites at room temperature. Its transition temperature, which distinguishes the low from the high temperature species, is estimated as Tc ≥ 265 K from the changes in the powder pattern. This is close to that found in single crystals of ZnDLH1 (Tc = 268 K) and powders of ZnLH8 (Tc ≈ 277 K). Most notably, the EPR spectra of CdDLH changes from one pattern at 80 K (gmax;Amax: 2.265; 488 MHz) to become two patterns: species I (gmax;Amax: 2.267; 472 MHz at 70%) and species II (gmax;Amax: 2.238; 398 MHz at 30%) at 298 K. The species II parameters are similar those found for the room temperature pattern of ZnDLH, and therefore is believed to represent the “averaged” pattern of the low temperature sites. Species I may be an intermediate averaging pattern that converts into species II with increasing temperature, similar what was observed in the CdLH single crystal study3 near its transition temperature. The dynamic mechanism in CdDLH appears to be mediated by disordered water motions, like that proposed for ZnDLH1. In all four metal bis-histidine complexes addressed here, the doped copper appears to undergo hopping motion between closely



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related states. Dynamic behavior might therefore be expected in other copper bis-histidine complexes. A recent structural study of an active bacterial tyrosinase has implicated copper site plasticity in enzymatic function and structural maintenance15. Here the CuA in the type 3 active site was found to have structural heterogeneity, where two resolved copper positions, 0.5 Å apart with the same three histidine ligands, are each ~50% occupied. That the CuA was also found to be more stable than its partner CuB in the type 3 site suggested to the authors that copper mobility, caused by this structural heterogeneity, leads to a tighter binding and therefore a greater importance for CuA in enzyme activity. Flexibility in coordination has also been shown in the structure of quercetin 2,3 dioxygenase where the copper active site is comprised of a major (~70%) and a minor (~30%) conformation16. Distinct EPR spectral patterns at 77 K reflected these populations17. The tetrahedral distorted copper coordination with three histidines and a water molecule in the major conformer is further distorted in the minor species by a rotation of near glutamate side chain to bind copper with a slight shifting of the apical water. Although no direct evidence for copper site dynamics was reported, inhibition results suggest the conversion of minor to major conformer upon substrate binding. Temperature dependent changes in the EPR spectra of copper proteins have however been observed for laccase18,19, plastacyanin20,21 and azurin20,21. In laccase, correlated Resonance Raman and EPR measurements16 at room and low temperatures led to the conclusion that changes are due to temperature dependent structural changes at the copper site. For the type I sites in the two blue copper proteins, slight but significant gmax variations were explained as originating from vibrational couplings which alter the EPR parameters by causing modulated conformational changes of the copper ligands.20,21 Similar observations from such complex biomolecules are to be anticipated as more systems are 12 ACS Paragon Plus Environment

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investigated. EPR investigations on copper-histidine models, such as those described here, can provide useful references in assessing these future observations, providing state transition rates, barrier energies and further insight into the role of dynamics in protein function. Acknowledgement We thank Dr. Panne Burckel of the Instrumentation Center of the University of Toledo for carrying out the Energy Dispersive X-ray Spectroscopic analysis. A Faculty Development Grant from the State University of New York at Old Westbury is gratefully acknowledged for partial financial support (to MJC).



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References (1) Dalosto, S. D.; Calvo, R.; Pizarro, J. L.; Arriortua, M.I. Structure, Disorder, and Molecular Dynamics in Zn(D,L-histidine)2: EPR of Copper Ion Dopants, X-ray Diffraction, and Calorimetric Studies. J. Phys. Chem. A 2001, 105, 1074-1085. (2) 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. (3) Colaneri, M. J., Vitali, J. and Kirschbaum, K. Electron Paramagnetic Resonance Spectroscopic Study of Copper Hopping in Doped Bis(L-Histidinato)Cadmium Dihydrate. Journal of Physical Chemistry A 2013, 117, 3414-3427. (4) ORTEP: A Fortran Thermal-Ellipsoid Plot Program for Crystal Structure Illustrations. Johnson, C.K. ORNL-3794 (Rev.), Union Carbide Corp., Oak Ridge Natl. Lab., June, 1965. (5) http://www.povray.org; Persistence of Vision Raytracer Pty. Ltd., 2006. (6) Fuess, H.; Bartunik, H. Neutron Diffraction Structure Determination of Bis-(Lhistidinato)cadmium Dihydrate. Acta Cryst. 1976, B32, 2803-2806. (7) Anderson, P. W. A Mathematical Model for the Narrowing of Spectral Lines by Exchange or Motion. J. Phys. Soc. Jpn. 1954, 9, 316-339. (8) Cotton, F.A.; Wilkinson, G. Advanced Inorganic Chemistry 3rd ed.; John Wiley & Sons: NY, 1972, p 52. (9) Stoll, S.; Schweiger, A. EasySpin, A Comprehensive Software Package for Spectral Simulation and Analysis in EPR. J. Magn. Reson. 2006, 178, 42-55. (10) Wertz, J. E.; Bolton, J. R. In Electron Spin Resonance: Elementary Theory and Practical Applications; McGraw-Hill, Inc.: New York, 1972. (11) 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. (12) Hyde, J. S.; Pilbrow, J. R. A Moment Method for Determining Isotropic g Values from Powder EPR Spectra. J. Magn. Reson. 1980, 41, 447-457. (13) 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. (14) Kretsinger, R. H.; Cotton, F. A.; Bryan, R.F. The Crystal and Molecular Structure of Di-(LHistidino)-Zinc(II) Dihydrate. Acta Cryst. 1963, 16, 651-657.


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(15) Sendovski, M.; Kanteev, M.; Shuster Ben-Yosef, V.; Adir, N.; Fishman, A. First Structures of an Active Bacterial Tyrosinase Reveal Copper Plasticity. J. Mol. Biol. 2011, 405, 227-237. (16) Steiner, R. A.; Kooter, I. M.; Dijkstra, B. W. Functional Analysis of the Copper-Dependent Quercetin 2,3-Dioxygenase. 1. Ligand-Induced Coordination Changes Probed by X-ray Crystallography: Inhibition, Ordering Effect, and Mechanistic Insights. Biochemistry 2002, 41, 7955-7962. (17) Kooter, I.M.; Steiner, R.A.; Dijkstra, B.W.; van Noort, P.I.; Egmond, M.R.; Huber, M. EPR Characterization of the Mononuclear Cu-Containing Aspergillus japonicus Quercetin 2,3Dioxygenase Reveals Dramatic Changes upon Anaerobic Binding of Substrates. Eur. J. Biochem. 2002, 269, 2971-2979. (18) Morpurgo, L.; Calabrese, L.; Deisderi, A.; Rotilio, G. Dependence on Freezing of the Geometry and Redox Potential of Type 1 and Type 2 Copper Sites of Japanese-Lacquer-Tree (Rhus vernicifera) Laccase. Biochem. J. 1981, 193, 639-642. (19) Blair, D. F.; Campbell, G. W.; Lum, V.; Martin, C. T.; Gray, H .B.; Malmström, B. G. Chan, S. I. A Resonance Raman Investigation of Perturbed States of Tree and Fungal Laccase. J. Inorg. Biochem. 1983, 19, 65-73. (20) Bacci, M.; Cannistraro, S. Temperature Dependence of the g Values in Blue Copper Protein EPR Spectra. Chem. Phys. Lett. 1987, 133, 109-112, (21) 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.



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Table 1. Listed are transition temperatures (Tc), g-tensor principal values and copper hyperfine principal (ACu) values (MHz) for four copper-doped histidine crystalline complexes obtained from EPR measurements and EasySpin fits of powders at low and high temperature. The reported giso quantity is determined from the g-tensor trace. The powder copper hyperfine quantities listed were assumed positive. Estimated errors in the EasySpin fit parameters are 0.002 and 15 MHz for the resolved gmax and Amax values, respectively. The other g and ACu parameters, which were masked by large overlap of resonances at high field, are therefore less reliable than gmax and Amax, and their errors are estimated as 0.004 and 50 MHz, respectively. For three of these systems, the dynamic contribution to broadening in the room temperature powder spectra beyond that from the copper mI static linewidth (40 G) were modeled with an isotropic correlation time (τ) using the EasySpin “chili” utility. These times have a high relative uncertainty, which are estimated as 20% of their values. Quantities in italics were taken from previous single crystal EPR studies. Averaged principal values (< >) are from the single crystal derived tensors at 77(80) K over the two neighboring histidine hopping sites. System

Tc

ZnDLH

Temp.

Principal gValues

giso

Principal ACu Values (MHz)

80K

2.266, 2.079, 2.053 2.269, 2.081, 2.057

2.133 2.136

481, 37, 26 500, 111, 0

298K

2.226, 2.117, 2.049 2.204, 2.124, 2.071 ˂2.214, 2.132,2.061˃

2.131 2.133 ˂2.136˃

359, 116, 28 360, 144, 75 ˂360, 144, 15˃

80K

2.306, 2.095, 2.054 2.299, 2.092, 2.048

2.152 2.146

461, 59, 1 -456, 66, -0.6

298K

2.204, 2.178, 2.061 2.168, 2.157, 2.074 ˂2.186, 2.163, 2.089˃

2.148 2.133 ˂2.146˃

269K

2.278, 2.070, 2.070

2.139

390, 70, 70

285K

2.162, 2.162, 2.078

2.134

196, 196, 28

80K

2.265, 2.083, 2.041

2.130

488, 52, 10

298K I II

2.267, 2.096, 2.034 2.238, 2.138, 2.065

2.132 2.147

472, 2, 26 398, 111, 78

τ (ns)

268K

CdLH

3.82 1.25

~165K

ZnLH

281, 154, 34 1.51 -256, -188, -37 0.439, 0.384 ˂-265, -191, 64˃

277K

CdDLH ≥265K


3.62 2.92

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Figure 1 Ortep-III4/PovRay5 renderings showing the proposed copper binding sites1,2 in doped zinc D,L-histidine pentahydrate1 and bis(L-histidinato)cadmium dihydrate3 crystals. At low temperature (80 K) Cu2+ binds differently to the two symmetry-related histidine molecules. This gives rise to two copper sites (primed and unprimed), also related by this symmetry. At high temperature, the copper rapidly hops between the two sites resulting in a dynamic averaging of the g and copper hyperfine tensors. The dotted arcs represent reorientations of the site g-tensors as the copper hops.

Figure 2 (A) The temperature dependence of the EPR spectra of crushed crystals of copperdoped Cd2+-D,L-histidine (CdDLH) between 80 K and 298K. The black vertical lines reference the relative changes in the three visible low field copper mI resonance lines of the major pattern (Species I) with temperature. The greatest incremental spectral shift of I occurs at ~265 K. A secondary pattern (Species II) starts to grow on the high field side of each of these mI lines, becoming visible around 245 K. (B) The temperature dependence of the EPR spectra of crushed crystals

of

copper-doped

zinc

D,L-histidine

pentahydrate

(ZnDLH)

and

bis(L-

histidinato)cadmium dihydrate (CdLH) between 80 K and 298K. The greatest incremental spectral changes are evident in each; at ~265 K for ZnDLH, where the dynamically broadened copper mI lines narrow, and at ~165 K for CdLH, where the low temperature pattern is almost completely diminished with the first appearance of the high temperature pattern.

Figure 3 (A) Experimental (black curve) and EasySpin fit simulated (blue curve) powder spectra of ZnDLH and CdLH at 80 K and 298 K. The spectral linewidth parameter was fixed at 40 G during the fits. The fit g and copper hyperfine tensor (ACu) parameters are listed in Table 1. EasySpin motion regime simulations (chili) employed isotropic rotational correlation times (τ) to



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reproduce the 298 K spectrum. (B) Experimental (black curve) and EasySpin simulated (blue curve) powder spectra of CdDLH at 80 K and 298 K. The spectral linewidth parameter was fixed at 40 G during the fits. The fit g and copper hyperfine tensor (ACu) parameters are listed in Table 1. EasySpin motion regime simulations (chili) fit separate isotropic rotational correlation times (τ) for each species in the 298 K spectrum. The simulation curve at 298 K is a composite of patterns from Species I (green curve) at 70% and Species II (red curve) at 30%.


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