X-ray absorption study of Rhus laccase: evidence for a copper-copper interaction, which disappears on type 2 copper removal

Publisher Website
Google Scholar
Abstract
X-ray absorption spectra are reported for the multi-Cu oxidase R. vernicifera laccase in oxidized and fully reduced forms and for laccase from which the type 2 Cu was depleted (T2D). The structure of the Cu K edge for both preparations shows the presence of CuII and CuI in the oxidized and reduce states, respectively. As previously reported by LuBien et al. (1981), removal of the type 2 Cu leads to reduction of the type 3 center, which can be reoxidized with H2O2. Fourier transforms of the extended X-ray absorption fine structure (EXAFS) give well-defined 1st and outer shell scattering peaks. Analysis of the 1st shell peak is complicated by the heterogeneity of the Cu sites. When (imidazole)4CuIISO4 is used as a model of the average Cu-ligand interactions, all of the 1st shell peaks contain 2.7-3.5 near neighbors/Cu, at an average distance of 1.97-1.98 .ANG.. For T2D laccase, the fit is improved by inclusion of 1/3 of a S atom at 2.19 .ANG., corresponding to the presumptive cysteine ligand of the type 1 Cu, which remains in the preparation containing 3 Cu atoms/molecule. The outer shell region shows 2 peaks characteristic of scattering from distant imidazole atoms. For T2D laccase the filtered outer shell contribution can be satisfactorily fit by scattering from an average of 2.1-2.4 imidazole groups. For native laccase, imidazole alone cannot satisfactorily model the outer shell contribution. Scattering is required from an additional half-atom, at a distance 3.4 .ANG., presumably the Cu at the type 3 binuclear site. Thus EXAFS gives evidence for a binuclear site in native laccase that is significantly perturbed by type 2 Cu removal. This perturbation might involve a substantial increase in the Cu-Cu distance of the type 3 site or, alternatively, a substantial decrease in rigidity of the site (increased Debye-Waller factor), perhaps by disruption of a bridging group.
Keywords