The Mn‐binding site and newly identified C‐terminal metal binding site both play important roles in the thermal stability of Mn‐peroxidase.

Extracellular fungal peroxidases such manganese peroxidase (MnP) offer potential in biomass utilization, biomaterial manufacture and detoxification of recalcitrant pollutants. Use of these enzymes in industrial processing requires a detailed understanding of their thermal tolerance limits. Although the role of calcium in protecting MnP from thermal inactivation has been studied extensively, the roles of the Mn‐binding site and C‐terminus in the thermal stability of MnP remain unexplored. In this work, we examine the effects of exogenous metals on the residual activities, heme Soret absorptions, and secondary structures of MnP. Analysis of the wild‐type MnP indicates that, at low pH, addition of exogenous Mn2+ stabilizes the protein to a greater extent than Ca2+. Analyses of the Mn‐binding site variants, D179N and E39D, indicate that the stabilization is due to interactions at the Mn‐binding site. A new variant, D84N, at the newly identified C‐terminal metal binding site is also examined. The data indicate that this secondary site also contributes to thermal stability, although to a lesser extent than mutations at the Mn‐binding or Ca‐binding sites.