Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a “caddie” protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr98 residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the μ-η2:η2-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now—despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations—the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16–1.70 Å). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of CuA, which induces the syn-to-anti rearrangement of the copper ligands after the formation of μ-η2:η2-peroxo-dicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu2O2 oxidant.

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 ortho-quinone. The enzyme from the genus Streptomyces 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 Tyr 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 Tyr residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of μ-η:η-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 Tyr residue to dopaquinone, may ensure the generation of fully activated tyrosinase.

Human indoleamine 2,3-dioxygenase 1 (IDO) is a heme enzyme that catalyzes the first reaction of the main metabolic pathway of L-tryptophan (Trp) to produce N-formylkynurenin. The reaction involves cleavage of the C 2 ¼C 3 bond in the Trp indole ring and insertion of two atomic oxygens from the iron-bound O 2 into the indole 2 and 3 position. For establishment of the chemical mechanism of this unique enzymatic reaction, it is necessary to determine the conformation and electronic state of the substrate Trp bound to IDO. In this study, we measured the ultraviolet resonance Raman spectra of IDO in the presence of Trp to detect the vibrational modes of the substrate Trp. We compared the ultraviolet resonace Raman spectra of Trp in a ternary complex (Trp-bound cyanide enzyme) and a binary complex (Trp-bound reduced enzyme) of IDO with that of free Trp in solution and found that binding to IDO influences the conformation of Trp, resulting in similar changes in the two complexes, especially around the C 3 -C b bond. However, the presence of the diatomic ligand at the heme sixth coordination site in the ternary complex significantly alters the mobility and electronic structure of Trp, most likely resulting in the C 2 ¼C 3 bond cleavage in the enzymatic reaction.

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