THERMODYNAMICS OF BINDING OF THE DISTAL CALCIUM TO MANGANESE PEROXIDASE

We previously demonstrated that manganese peroxidase from Phanerochaet e chrysosporium was susceptible to thermal inactivation due to release of the distal calcium, which maintained the distal heme environment o f the enzyme [Sutherland, G. R. J., Zapanta, L. S., Tien, M., & Aust, S. D. (1997) Biochemistry 36, 3654-3662]. In this investigation the bi nding of calcium to the distal calcium binding site of manganese perox idase was studied by optical absorption spectroscopy and isothermal ti tration calorimetry. The dissociation constant for the distal calcium binding site was 11 +/- 1 mu M and the Hill coefficient was 1.1 +/- 0. 1. The binding of calcium was accompanied by decreases in enthalpy and entropy that were large compared to those of other calcium binding pr oteins. The decreases were consistent with the large conformational ch anges proposed to occur in manganese peroxidase as a result of the bin ding and release of the distal calcium. Studies involving binding of t he hydrophobic fluorescent probe, 4,4'-dianilino-1,1' -binaphthyl-5,5' -disulfonic acid, dipotassium salt (bis-ANS), to manganese peroxidase indicated that the active, calcium-containing form of the enzyme had l ess exposed hydrophobic surface area, which would contribute to an inc rease in enthalpy and entropy upon calcium binding. Therefore, the neg ative changes in enthalpy and entropy associated with calcium binding were attributed to a large increase in the structural rigidity and com pactness of the enzyme. The dissociation constant for calcium decrease d and the rate of thermal inactivation decreased with decreasing pH. H owever, both the ability of calcium to prevent thermal inactivation of manganese peroxidase and the rate of calcium binding decreased as the pH decreased. Therefore it was proposed that, at lower pH, calcium bi nding to manganese peroxidase was more thermodynamically favorable, bu t the rate of calcium binding decreased because the flexibility of the calcium binding site, and in turn exposure of the ligands to the inco ming ion, decreased.