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 tr ee laccase under anaerobic reducing conditions and developed a mechani stic model. We identify two key steps in the reaction: (i) dissociatio n 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,l0-phenanthroline. We maintain a stea dy-state concentration of cyanide in a pH 5.5 acetate buffer under a c onstantly exchanging nitrogen atmosphere by introducing a cyanometalat e ion as a cofactor or by continuously injecting the ion into the prot ein solution. The type 2-depleted product is identical to previous pre parations as regards its spectral properties, activity level and abili ty 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. (C) 1995 A cademic Press, Inc.