Oxidation-reduction potentials of the electron acceptors in laccases and stellacyanin

Abstract

1. Laccase ( p-diphenol: O 2 oxidoreductase, EC 1.10.3.2) obtained from the fungus Polyporus versicolor and laccase and stellacyanin from the lacquer tree Rhus vernicifera were titrated with reducing and oxidizing agents in an anaerobic optical cell equipped with a combined metal electrode for simultaneous optical and potentiometric titrations of the chromophores absorbing at about 610 nm in all three proteins and the 330-nm chromophores in the laccases. 2. The oxidation-reduction potential of the “blue” copper atom in stellacyanin was found to be 184 mV when the protein was dissolved in a sodium phosphate buffer, pH 7.1 and ionic strength 0.3. 3. The oxidation-reduction potential of Type 1 copper and the two-electron acceptor, connected with the 330-nm chromophore, in Rhus laccase was found to be dependent on the concentration of hexacyanoferrate which was used as an electron mediator in the titrations. With about three times excess of this substance, compared to the enzyme concentration, the oxidation-reduction potentials of Type 1 copper and the two-electron acceptor were 434 mV and 483 mV, respectively, when the enzyme was dissolved in sodium phosphate buffer, pH 7.5 and ionic strength 0.2. If the concentration of hexacyanoferrate was only about one third the concentration of enzyme, under otherwise similar conditions as above, the oxidation-reduction potentials of Type 1 copper and the two-electron acceptor decreased to 394 mV and 434 mV, respectively. The increase in oxidation-reduction potential of the Type 1 copper and the two-electron acceptor in the presence of excess hexacyanoferrate seems to be due to a specific reduction of Type 2 copper by the hexacyanoferrate (II). 4. For Polyporus laccase the oxidation-reduction potential of Type 1 copper was extremely high. A value of 785 mV was obtained for an enzyme solution in sodium phosphate buffer, pH 5.5 and ionic strength 0.2, which also contained small amounts of some metal cyanides as electron mediators. The 330-nm chromophore was found to be a two-electron acceptor with an oxidation-reduction potential of 782 mV under these experimental conditions. In the presence of excess of the electron-mediating metal cyanides or with about 100 times excess of the inhibitor NaF the oxidation-reduction potential of Type 1 copper was about the same as above. The potential of the two-electron acceptor decreased to 570 mV, however, when fluoride was present.