The binding of copper ions to copper-free bovine superoxide dismutase. Kinetic aspects

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Abstract
The kinetics of reconstitution of bovine superoxide dismutase from Cu2+ and the Cu-free enzyme were studied by activity, UV-absorption, EPR and pulsed-NMR measurements. The process appears to be first-order up to 80% completion in most conditions, and is pH-dependent, with an apparent pK of 6.5. UV-absorption and solvent proton relaxation rate measurements show that fast binding of Cu2+ occurs and the initial ligands are likely to be, at least in part, those of the native active site. The recovery of the native activity and spectroscopic properties is a slow process with activation energies of 92 kJ/mol at pH 5.3 and 8.4 kJ/mol at pH 8.1 and can be described as a rearrangement of the site around the bound metal. The rate of this process is lower in partially recombined protein samples, probably because of intersubunit interactions.