Kinetics and Thermodynamics of Copper(II) Binding to Apoazurin

Abstract

The binding of copper(II) to apoazurin has been probed by isothermal titration calorimetry in cholamine buffer at pH 7.0. The standard enthalpy change was determined to be −10.0 ± 1.4 kcal/mol. Each calorimetric trace reveals an initial exothermic phase followed by an endothermic phase. The calorimetric data could be fit to a kinetic model involving a bimolecular combination of copper(II) and apoazurin in an exothermic process (k = 2 ±1 × 10³ M^-1 s^-1, ΔH° = −19 ± 3 kcal/mol) to form an intermediate that spontaneously converts to Cu(II)−azurin in an endothermic process (k = 0.024 ± 0.01 s^-1, ΔH° = +9 ± 3 kcal/mol). These data suggest that copper(II) first combines with apoazurin in an irreversible process to form an intermediate that converts to copper(II)−azurin in a process driven by the release of water. The overall standard free energy of copper(II) binding to apoazurin is estimated to be −18.8 kcal/mol.