Thermal denaturation of a blue-copper laccase: Formation of a compact denatured state with residual structure linked to pH changes in the region of histidine protonation

Abstract

The partial (absolute) heat capacity of a laccase enzyme from Myceliophthora thermophila (MtL) was determined from calorimetric scans in the 4.5–10.0 pH range. Above pH 7.5, the heat capacity of the thermally denatured state (CpD) of this blue-copper glycoprotein is consistent with that for an unfolded, fully solvated polypeptide chain, if its carbohydrate content is taken into account. Below pH 7.5, CpD decreases and eventually levels off within the 5.5–4.5 pH region, where a compact, partially solvated denatured state is formed. In the compact state, denatured MtL is an oligomer, and exhibits considerable native-like secondary structure and a perturbed environment of its copper atoms. Analysis of the pH dependence of CpD and the content of secondary structure gives results implying that His residues play an important role in the stability of the compact denatured state.