The role of cyanide in the removal of type 2 copper from laccase.

We have improved a method for the removal of the type 2 copper from tree laccase under anaerobic reducing conditions and developed a mechanistic model. We identify two key steps in the reaction: (i) dissociation of copper(I) catalyzed by trace levels of cyanide in a weakly acidic medium and (ii) sequestration of the released metal by an appropriate chelator such as 2,9-dimethyl-1,10-phenanthroline. We maintain a steady-state concentration of cyanide in a pH 5.5 acetate buffer under a constantly exchanging nitrogen atmosphere by introducing a cyanometalate ion as a cofactor or by continuously injecting the ion into the protein solution. The type 2-depleted product is identical to previous preparations as regards its spectral properties, activity level and ability to recombine with copper(I). The mechanistic insights appear to be quite general and should form the basis for the development of methods for removing the type 2 copper from other related systems.