X-ray absorption spectra are reported for the multi-Cu oxidase Rhus vernicifera laccase in oxidized and fully reduced forms and for laccase from which the type 2 Cu has been depleted (T2D). The structure of the Cu K edge for both preparations shows the presence of CuII and CuI in the oxidized and reduced 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 first and outer shell scattering peaks. Analysis of the first 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, it is shown that all of the first shell peaks contain 2.7-3.5 near neighbors per Cu, at an average distance of 1.97-1.98 Å. For T2D laccase, the fit is improved by inclusion of one-third of a sulfur atom at 2.19 Å, corresponding to the presumptive cysteine ligand of the type 1 Cu, which remains in the preparation containing three Cu atoms per molecule. The outer shell region shows two 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, however, imidazole alone cannot satisfactorily model the outer shell contribution. Scattering is required from an additional half-atom, at a distance 3.4 Å, 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.
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