Elucidation of the tyrosinase/O2/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 mu:eta(2):eta(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 mu:eta(2):eta(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 mu:eta(2):eta(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 mu-eta(2):eta(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 non-protonated mu-oxo. This study provides insights into O-2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.

Automated summary

Melanins are important biopolymer pigments that provide photoprotection, and their biosynthesis involves the catalytic role of tyrosinase (Ty). This study combines spectroscopic, kinetic, and computational methods to investigate the mechanism of the rate-limiting step in melanin biosynthesis. The results provide insights into the O-2 activation and reactivity by the active sites of coupled binuclear copper, with implications in biocatalysis.