The dinuclear copper site structure of Agaricus bisporus tyrosinase in solution probed by X-ray absorption spectroscopy.

We have measured the x-ray absorption near edge structure (XANES) spectra of the enzyme tyrosinase from the mushroom Agaricus bisporus in solution in the oxy and deoxy forms. The spectra, obtained under the same conditions as the analogous forms of mollusc hemocyanin (Hc), show that the oxidation state of copper changes from Cu(II) (oxy form) to Cu(I) (deoxy form), and the copper active site(s) of A. bisporus tyrosinase in solution undergoes the same main conformational changes as Hc. We have applied the multiple scattering theory to simulate the XANES spectra of various alternative geometries of the copper site, accounting for the residual differences between Hc and tyrosinase. While oxy-Hc is reasonably fitted only by the pseudo-square-pyramidal geometry reported by its crystallographic data, oxytyrosinase can be fitted, starting from the Hc coordinates, either by distortions toward a pseudo-tetrahedral geometry, with inequivalent copper sites, or by an apically distorted square-pyramidal geometry (with an elongation of the apical distance of no more than 0.2 A).