Kinetics and Thermodynamics of Copper(II) Binding to Apoazurin

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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.