Konrad Lerch

Extended X-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from Neurospora crassa

Cu K-edge X-ray absorption spectra have been recorded for the enzyme tyrosinase from Neurospora crassa, in its oxy, resting (met-aquo), and inhibitor-bound (met-mimosine) forms. The K-edges proper resemble those of oxy- and met-hemocyanin, and confirm the presence of CuII. The forbidden 1s----3d transition is noticeably stronger for the 1-mimosine-bound enzyme, implying some distortion of the tetragonal Cu coordination group on inhibitor binding. The extended fine structure (EXAFS) beyond the K-edge has been analyzed. The first shell scattering is consistent with the presence of two N- and two O-ligand atoms, at 2.0 and 1.9 A, for all three forms of the enzyme; there is no evidence for heavy atom (S) scattering in the first shell. As in analogous hemocyanin derivatives, the outer shell scattering contains contributions from distant atoms of imidazole ligands, as well as from an addition scattering atom, at 3.4-3.6 A. For oxy-tyrosinase the additional scatterer is unambiguously a heavy atom (Cu), although a larger Debye-Waller factor suggests a somewhat less rigid binuclear site than in oxy-hemocyanin.

The reaction of CN− with the binuclear copper site of Neurospora tyrosinase: its relevance for a comparison between tyrosinase and hemocyanin active sites

The equilibrium and the kinetics of the reaction between Neurospora crassa tyrosinase and cyanide have been studied. Cyanide reacts with the binuclear copper active site of the protein competitively with respect to dioxygen and displaces the metal ions. This process occurs stepwise and involves transient intermediates containing mononuclear Cu(I) sites. The reaction mechanism proved to be the same as described earlier for molluscan and arthropodan hemocyanins, which share with tyrosinase the same copper active site organization, but perform different physiological functions. A comparison of the kinetic parameters between the different proteins shows that the tyrosinase copper active site has a greater accessibility than that of hemocyanin. The relevance of these data in terms of structure-function relationship and evolution of the binuclear copper proteins is discussed.