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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 33; p. e2205619119
• Main Authors: Kipouros, Ioannis, Stańczak, Agnieszka, Ginsbach, Jake W, Andrikopoulos, Prokopis C, Rulíšek, Lubomír, Solomon, Edward I
• Format: Journal Article
• Language: English
• Published: United States 16.08.2022
• Subjects: Bacterial Proteins; Binding Sites; Catalysis; Copper - chemistry; Melanins - biosynthesis; Monophenol Monooxygenase - chemistry; Oxygen - metabolism; Peroxides - chemistry; Phenols - chemistry; Streptomyces - enzymology; melanin biosynthesis; binuclear copper; tyrosinase; oxygen activation; monooxygenase
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2205619119

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 -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 to form a μ:η :η -peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of -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 /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 :η -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 :η -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 :η -peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic -hydroxylation by the nonprotonated μ-oxo. This study provides insights into O activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.