REVERSIBLE REMOVAL OF ONE SPECIFIC COPPER(2) FROM FUNGAL LACCASE

One of the four copper atoms of fungal laccase forms a Cu1+‐chelate with bathocuproine disulfonate at pH 4.0 in 1 M guanidine hydrochloride and in the presence of excess ascorbate. Electron paramagnetic resonance (EPR) analysis of the native and copper‐depleted proteins has shown that one of the two Cu2+ in the protein has been removed under these conditions. The Cu2+ removed has been identified as the Type 2 or “non‐blue” Cu2+. The spectral characteristics of the other Cu2+ present in the molecule (the Type 1 Cu2+) appear unchanged after removal of the second Cu2+. The copper‐depleted protein is almost completely devoid of enzymic activity after removal of the Type 2 Cu2+ but both the activity and original copper content can be restored after incubation of the copper‐depleted protein with Cu2+ and ascorbate under anaerobic conditions. No activity is restored by the addition of Cu2+ alone. The rate of removal of the Type 2 Cu2+ closely parallels the rate of decrease of enzymic activity of the protein during the reaction with the chelating agent. These results show that the Type 2 Cu2+ is an integral part of the laccase molecule and has a functional role in the catalytic reaction. Copyright © 1969, Wiley Blackwell. All rights reserved