The removal of the type-2 copper from Rhus vernicifera laccase

We have studied the removal of the type-2 copper from tree laccase (Rhus vernicifera) by treatment with EDTA at pH 5.2 in the presence of a redox buffer containing ferri- and ferrocyanide. The efficiency with which the copper is removed depends on the Fe(CN) 6(4-)/Fe(CN) 6(3-) ratio. We have varied this ratio from approx. 2:1 to about 50:1 and the best results were obtained with the highest ratio, i.e., the most cathodic solution potential. Nevertheless, the presence of Fe(CN) 6(3-) is required for the procedure to be effective. Although we cannot exclude the possibility that a mixed-valence form of laccase is the reactive species, we believe the results are better explained by a model which assumes that the removal of the type-2 copper depends upon an ordered sequence of oxidation-reduction reactions. Specifically, we propose that the copper is released as the monovalent ion from previously reduced laccase and then reoxidized in solution and sequestered with EDTA. The reoxidation step drives the reaction because recombination with the protein is inhibited when copper is in the divalent form. In testing this model, we have also shown that the type-2 copper can be removed under strictly reducing conditions when 4,4'-dicarboxy-2,2'-biquinoline (BCA) is present to complex the copper(I) ion. Although the BCA method is effective, the reaction takes longer, perhaps because of the limited solubility of BCA at the pH values of interest. Finally, we have found that the best results are obtained with either method when a cyanometalate ion such as Fe(CN) 6(3-) or Co(CN) 6(3-) is present in the medium. The exact role of this factor has yet to be established, but there is no indication that free cyanide has a role in the process. The most likely interpretation is that some type of binding interaction with the protein facilitates copper release.