THE ENZYMATIC-PROPERTIES OF OCTOPUS-VULGARIS HEMOCYANIN - O-DIPHENOL OXIDASE ACTIVITY

Hemocyanin and tyrosinase are dinuclear copper proteins capable of rev ersibly binding dioxygen. Despite the great similarity of structure an d properties of their active site, the two proteins perform different biological functions (oxygen transport/storage versus monooxygenase an d oxidase activity). In this paper, we show that Octopus vulgaris hemo cyanin exhibits a tyrosinase-like activity; namely, it is capable of u tilizing dioxygen for the oxidation of o-diphenol to quinone. The reac tion is specific for this isomer of diphenol, the meta and para isomer s being unreactive, and is strongly controlled by steric factors. Diox ygen represents a cosubstrate of the reaction, and it is involved in t he catalytic turnover by binding to the dinuclear copper site of the p rotein to form, under steady-state conditions, oxy-Hc, which is the ac tive species. The generation of semiquinone radicals, detected by EPR and by their reaction with N,N,N',N'-tetramethyl-1,4-phenylenediamine strongly supports a reaction mechanism in which such radicals represen t the reaction products of one-electron oxidation of the substrate, qu inone being generated by dismutation of semiquinones. Met-Hc is regene rated by the substrate to the deoxy form. To close the catalytic cycle , the proposed reaction mechanism also involves the participation of t wo transient protein forms with the total oxidation state of the activ e site (V and IV) intermediate between that of oxy-Hey, [(CuO22-)-O-II -Cu-II](VI), and deoxy-Hc, [(CuCuI)-Cu-I](II). A mathematical model ha s been elaborated to describe the reaction kinetics. The differences i n reaction mechanisms between hemocyanin and tyrosinase are discussed in terms of accessibility to exogenous molecules of their active sites .