Kinetics of copper(II) uptake by apoazurin in complexing media

We have studied the kinetics of copper uptake by apoazurin in imidazole and 1-methylimidazole buffers in the pH range of 7-9 with mu = 0.5 M and copper(II) in large excess. The reaction has been monitored by measuring the visible absorbance and circular dichroism as a function of time. The uptake occurs in a stepwise fashion, and at least two intermediates are implicated. Overall, the rate of uptake varies inversely with the concentration of the proton and the (complexing) buffer, but depends directly on the copper concentration. A model involving a weakly absorbing intermediate is proposed to rationalize the data taken at the lower end of the pH range. According to the model the intermediate forms with a second order rate constant of about 30 M-1 S-1 and is probably described as a ternary complex of copper, buffer, and one or more of the histidine ligands of the binding site. This then decays by a pH-dependent process to give product. At higher pH values there is evidence that relaxation to product occurs via a second intermediate form in which the cysteine ligand is bound to copper. The relevance of these results to the question of how copper is selectively incorporated into the protein is considered. Finally, a milder, more reliable route to the preparation of apoazurin is described.