Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites

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Abstract
1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.