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Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis
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SYSNO ASEP 0560284 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis Author(s) Kipouros, I. (US)
Stanczak, Agnieszka (UOCHB-X) ORCID
Ginsbach, J. W. (US)
Andrikopoulos, Prokopis C. (UOCHB-X)
Rulíšek, Lubomír (UOCHB-X) RID, ORCID
Solomon, E. I. (US)Article number e2205619119 Source Title Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences - ISSN 0027-8424
Roč. 119, č. 33 (2022)Number of pages 10 s. Language eng - English Country US - United States Keywords tyrosinase ; binuclear copper ; monooxygenase ; oxygen activation ; melanin biosynthesis OECD category Physical chemistry R&D Projects LTAUSA19148 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Research Infrastructure e-INFRA CZ - 90140 - CESNET, zájmové sdružení právnických osob Method of publishing Open access Institutional support UOCHB-X - RVO:61388963 UT WOS 000891284800019 EID SCOPUS 85135551531 DOI 10.1073/pnas.2205619119 Annotation 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. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Jana Procházková, Tel.: 220 183 418 Year of Publishing 2023 Electronic address https://doi.org/10.1073/pnas.2205619119
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