ROLE OF REDUCTION POTENTIALS IN COPPER ABSTRACTION FROM THE TRINUCLEAR CLUSTER OF BLUE OXIDASES

The crucial point in the selective removal of the type 2 Cu from laccase and ascorbate oxidase by chelating agents was found to be the nature of the reducing agent employed in the reaction. No copper was lost when the reductant was either absent or too strong (ascorbate), namely when the trinuclear Cu cluster was respectively fully oxidized or fully reduced. From both proteins, the type 2 Cu was removed only in the presence of a mild reductant such as [Fe(CN)6]4-, the reduction potential of which could be varied by addition of [Fe(CN)6]3- and set at an intermediate value between that of type 3 and type 2 Cu ions. In this way the type 3 Cu ions were reduced with labilization of the oxidized type 2 Cu. Also, on prolonged incubation some type 3 Cu was removed from ascorbate oxidase. The applicability of the procedure to caeruloplasmin and fungal laccase is discussed.