A SINGLE-CRYSTAL EPR AND ESEEM ANALYSIS OF CU(II)-DOPED BIS(L-HISTIDINATO)CADMIUM DIHYDRATE

A study was undertaken to investigate the nature of the hyperfine and quadrupole coupling parameters of the distant N-14 in Cu(II)-coordinated imidazole in bis(L-histidinato)cadmium dihydrate (I) to further our understanding of the ESEEM patterns obtained for copper proteins, where Cu(II) is coordinated to either N delta or N epsilon. Single crystal EPR data have been collected at 77 K in isotopically enriched Cu-63-doped crystals grown from D2O in order to obtain g and Cu(II) hyperfine tensors. From the obtained spectra, hyperfine splittings from two directly coordinated N-14 nuclei are evident. Single crystal ESEEM measurements at 4.2 K were used to determine the hyperfine and quadrupole tensors of the remote N-14 epsilon of the imidazole group ligated to the Cu(II). These tensors can be compared to those previously obtained for N-14 delta in Cu(II)-doped L-histidine HCl H2O (II) (Colaneri, M. J.; Peisach, J. J. Am. Chem. Sec. 1992, 114, 5335.). The remote N-14 hyperfine tenser found in I is very similar to that previously obtained in II, displaying predominantly a rhombic anisotropy, 0.36, -0.10, -0.26 MHz, and suggesting that this form should exist for similar histidine ligated copper sites in proteins. The hyperfine isotropy in I, 1.61 MHz, is, however, slightly larger than that found in II, 1.35 MHz. The derived quadrupole coupling parameters e(2)qQ/h and eta have values of -1.57 MHz and 0.64, respectively, with the maximum principal component occurring normal to the imidazole plane. This tenser is different from that previously found in II and may reflect the different hydrogen bonding environments of the remote nitrogen in the host crystal systems. The present ESEEM study shows that a single imidazole coordinates to the Cu(II). From these results, a Cu(II) binding site is postulated whereby the metal ion ligates to two equatorial nitrogens and an axial carboxylate oxygen from a single histidine molecule. A carboxylate oxygen from a neighboring histidine is most likely positioned trans to the imidazole nitrogen Ligand perhaps by a small movement about its C-alpha-C-beta bond. This type of complex is thought to be less stable than the more usually observed, four-equatorial Ligand complex. The temperature dependence of the EPR spectra of I is consistent with. this notion. The EPR parameters observed at room temperature are found to represent the dynamic average of the g and Cu-63 hyperfine tensors measured at 77 K. These sets of low temperature tensors correspond to copper sites bound to neighboring histidine molecules. The averaging is believed to occur by the copper hopping between these sites at higher temperatures.