Activation Mechanism of the <i>Streptomyces</i> Tyrosinase Assisted by the Caddie Protein

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding <i>ortho</i>-quinone. The enzyme from the genus <i>Streptomyces</i> is generated as a complex with a “caddie” protein that assists the transport of two copper ions into the active center. In this complex, the Tyr⁹⁸ residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr⁹⁸ residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of μ-η²:η²-peroxo-dicopper­(II) and Cu­(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr⁹⁸ residue to dopaquinone, may ensure the generation of fully activated tyrosinase