Elucidation of the tyrosinase/O(2)/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho-hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O(2) to form a μ:η(2):η(2)-peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para-substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O(2)/monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantum-mechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the μ:η(2):η(2)-peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the μ:η(2):η(2)-peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the μ-η(2):η(2)-peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho-hydroxylation by the nonprotonated μ-oxo. This study provides insights into O(2) activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.