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 alpha-helix and beta-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 does not bind ANS. The type-2 copper depleted derivative and apo-laccase bind one and three moles of dye per mole of protein, respectively. These findings suggest that the three ANS binding sites correspond with the three different copper binding sites in laccase. These can be distinguished on the basis of their dissociation constants (K(D)) for ANS. The type-2 copper site exhibits the highest affinity for ANS.