Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

0560284 - ÚOCHB 2023 RIV US eng J - Journal Article
Kipouros, I. - Stanczak, Agnieszka - Ginsbach, J. W. - Andrikopoulos, Prokopis C. - Rulíšek, Lubomír - Solomon, E. I.
Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis.
Proceedings of the National Academy of Sciences of the United States of America. Roč. 119, č. 33 (2022), č. článku e2205619119. ISSN 0027-8424. E-ISSN 1091-6490
R&D Projects: GA MŠMT(CZ) LTAUSA19148
Research Infrastructure: e-INFRA CZ - 90140
Institutional support: RVO:61388963
Keywords : tyrosinase * binuclear copper * monooxygenase * oxygen activation * melanin biosynthesis
OECD category: Physical chemistry
Impact factor: 11.1, year: 2022
Method of publishing: Open access
https://doi.org/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 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 O2 to form a μ:η2:η2-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/O2/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:η2:η2-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:η2:η2-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-η2:η2-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 O2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.
Permanent Link: https://hdl.handle.net/11104/0333270