Circular dichroism and fluorescence studies to probe the conformational properties of Rhus vernicifera laccase

The conformational properties of native, apo- and type-2 copper depleted laccase from Rhus vernicifera have been investigated by circular dichroism and fluorescence spectroscopies. Circular dichroism experiments reveal a high prevalence of random-coil structure in all laccase derivatives, the content of α-helix and β-sheet not exceeding 8% and 21%, respectively. Nevertheless, the microenvironment of the tryptophan residues is deeply shielded from the external medium and exhibits a marked stability against pH-denaturation. Fluorescence data suggest that a class of tryptophan residues close to type-2 site contributes 50% to the overall fluorescence emitted by apo- and type-2 copper depleted laccase. These residues are masked in the native enzyme, due to quenching effects by copper ions and adjacent amino acid side chains. The interaction of 1-anilino-8-naphthalene sulfonate (ANS) with laccase has been studied by following the fluorescence changes of the dye upon binding. Native laccase d...