Oxidation-Reduction Titrations of Copper Ions in Rhus-Laccase Properties of Two Types of Copper Ions in the Molecule

(1) The molecular weight of laccase [EC 1.10. 3. 2, ^-diphenol: oxygen oxidoreductase] from the latex of Rhus venicifera was re-estimated by the method of sedimentation equilibrium and found to be 1.04xl05, taking the partial specific volume as 0.734 ml/mg. It was confirmed that as reported previously (1), laccase contains 4.0g-atom of copper per mole of protein. (2) The copper ions in laccase were titrated with hydrogen peroxide as an oxidant and with ascorbate as a reductant, measuring the absorbance changes at 615 m/u and 330 m ^ under anaerobic conditions. The profiles of the titration curves measured at these two wave-lengths differed. It was concluded that these two absorption bands were due to different species of copper ions in laccase. It was estimated that there was one copper ion, Cu2+(615), with an absorption band at 615 m/J and two or three copper ions, Cu2+(330), with an absorption band at 330 rn.fi in the molecule. (3) Cu(615) and Cu(330) seemed to be in oxidation-reduction equilibrium, the oxidation-reduction potential of Cu(615) being lower than that of Cu(330). The redox potential difference, E0n0-E06l \ was estimated to be+45mV at pH 7.0 and 25°C. (4) On oxidative titration of the reduced enzyme with molecular oxygen, the amounts of the cupric forms of Cu(615) and Cu(330) increased almost linearly with increase in the concentration of molecular oxygen added. This suggests that the fully reduced enzyme is spontaneously oxidized with molecular oxygen by a four-electron transfer reaction. Laccase [EC 1.10.3.2], which is blue in the versicolor (3). Rhus-\accase contains four oxidized state and catalyzes the oxidation of copper atoms per molecule as the essential p- and o-diphenol derivatives, phenylene prosthetic group and the oxidation of the diamines and ascorbate by molecular oxygen, substrate is catalyzed by the valence change has been purified from the latex of lacquer of these bound copper ions {4—6). Recently, tree (1,2), and from a fungus, Polyporus Nakamura and Ogura (6,7) found that blu