SPECTROSCOPIC STUDIES OF RHUS-VERNICIFERA AND POLYPORUS-VERSICOLOR LACCASE - ELECTRONIC-STRUCTURES OF THE COPPER SITES

Low-temperature absorption and room temperature circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic measurements were made on the multicopper oxidases R. vernicifera laccase and P. versicolor laccase. Near-IR bands at 6000, 9500 and 11,700/cm for Rhus (tree) laccase and 7250 and 11,000/cm for Polyporus (fungal) laccase are assigned to d-d transitions of the type 1 (blue) Cu(II). The low energies of these bands, when considered with the EPR g values, are consistent only with a tetragonally (D2d) distorted tetrahedral geometry for type 1 Cu(II). Lignad field stabilization energy contributions to the redox potential of the type 1 copper are 269 (tree) and 203 mV (fungal). Analysis of all spectroscopic results suggests that the characteristic bands near 13,000, 16,000 and 23,000/cm in the native tree and fungal enzymes be assigned to the charge-transfer transitions .pi.S(thiolate) .fwdarw. dx2-y2, .sigma.S(thiolate) .fwdarw. dx2-y2, and .pi.N(imidazole) .fwdarw. dx2-y2 in a 2B2(dx2-y2) ground-state blue center of probable ligand composition CuN2SX (N = His, S = Cys, X = Met or substitute). Binding of fluoride at the type 2 site in both tree and fungal enzymes perturbs the type 1 circular dichroism spectrum. Bands attributable to the Cu types 2 and 3 are observed in visible region absorption and CD spectra of NO-treated tree laccase and a high pH (> 8.7) form of fungal laccase. Both NO-treated tree and high pH fungal proteins display intense absorption at 30,000/cm and a weak shoulder near 16,700/cm. Prominent activity in the CD spectra is centered at 18,000 and 24,000/cm in NO-treated tree laccase, and 15,000, 19,500 and 22,500/cm in high pH fungal laccase. The 16,700/cm absorption shoulder and 24,000/cm CD activity are absent in type 2 depleted, NO-treated tree laccase. The energies of the observed bands rule out near tetrahedral geometry for types 2 and 3 Cu, but are consistent with structures based on tetragonal 6, 5 or square-planar 4 coordination.