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.

Department of Chemistry Stanford University Stanford CA 94305

Faculty of Science Charles University 128 00 Prague 2 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences 166 10 Prague 6 Czech Republic

Stanford Synchrotron Radiation Lightsource SLAC National Accelerator Laboratory Stanford University Menlo Park CA 94025

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Kirti K., Amita S., Priti S., Mukesh Kumar A., Jyoti S., Colorful world of microbes: Carotenoids and their applications. Adv. Biol. 2014, 1–13 (2014).

Simon J. D., Peles D., Wakamatsu K., Ito S., Current challenges in understanding melanogenesis: Bridging chemistry, biological control, morphology, and function. Pigment Cell Melanoma Res. 22, 563–579 (2009). PubMed

Solomon E. I., et al. , Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014). PubMed PMC

Nosanchuk J. D., Casadevall A., The contribution of melanin to microbial pathogenesis. Cell. Microbiol. 5, 203–223 (2003). PubMed

Ciui B., et al. , Wearable wireless tyrosinase bandage and microneedle sensors: Toward melanoma screening. Adv. Healthc. Mater. 7, e1701264 (2018). PubMed

Buitrago E., et al. , Are human tyrosinase and related proteins suitable targets for melanoma therapy? Curr. Top. Med. Chem. 16, 3033–3047 (2016). PubMed

Carballo-Carbajal I., et al. , Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis. Nat. Commun. 10, 973 (2019). PubMed PMC

Ananya N., Kumar J. S., Ram S. H., L-DOPA, a promising pro-drug against Parkinson’s disease: Present and future perspective. Res. J. Biotechnol. 14, 10 (2019).

Tinello F., Lante A., Recent advances in controlling polyphenol oxidase activity of fruit and vegetable products. Innov. Food Sci. Emerg. Technol. 50, 73–83 (2018).

Lobba M. J., et al. , Site-specific bioconjugation through enzyme-catalyzed tyrosine-cysteine bond formation. ACS Cent. Sci. 6, 1564–1571 (2020). PubMed PMC

Struck A.-W., et al. , An enzyme cascade for selective modification of tyrosine residues in structurally diverse peptides and proteins. J. Am. Chem. Soc. 138, 3038–3045 (2016). PubMed

Frangu A., Pravcová K., Šilarová P., Arbneshi T., Sýs M., Flow injection tyrosinase biosensor for direct determination of acetaminophen in human urine. Anal. Bioanal. Chem. 411, 2415–2424 (2019). PubMed

Florescu M., David M., Tyrosinase-based biosensors for selective dopamine detection. Sensors (Basel) 17, 1314 (2017). PubMed PMC

Min K., Park G. W., Yoo Y. J., Lee J.-S., A perspective on the biotechnological applications of the versatile tyrosinase. Bioresour. Technol. 289, 121730 (2019). PubMed

Matoba Y., Kumagai T., Yamamoto A., Yoshitsu H., Sugiyama M., Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. J. Biol. Chem. 281, 8981–8990 (2006). PubMed

Baldwin M. J., et al. , Spectroscopic studies of side-on peroxide-bridged binuclear copper(II) model complexes of relevance to oxyhemocyanin and oxytyrosinase. J. Am. Chem. Soc. 114, 10421–10431 (1992).

Baldwin M. J., et al. , Spectroscopic and theoretical studies of an end-on peroxide-bridged coupled binuclear copper(II) model complex of relevance to the active sites in hemocyanin and tyrosinase. J. Am. Chem. Soc. 113, 8671–8679 (1991).

Halfen J. A., et al. , Reversible cleavage and formation of the dioxygen O-O bond within a dicopper complex. Science 271, 1397–1400 (1996). PubMed

Pidcock E., Obias H. V., Zhang C. X., Karlin K. D., Solomon E. I., Investigation of the reactive oxygen intermediate in an arene hydroxylation reaction performed by Xylyl-bridged binuclear copper complexes. J. Am. Chem. Soc. 120, 7841–7847 (1998).

Mirica L. M., et al. , Tyrosinase reactivity in a model complex: An alternative hydroxylation mechanism. Science 308, 1890–1892 (2005). PubMed

Spada A., Palavicini S., Monzani E., Bubacco L., Casella L., Trapping tyrosinase key active intermediate under turnover. Dalton Trans. (33):6468–6471 (2009). PubMed

Goldfeder M., Kanteev M., Isaschar-Ovdat S., Adir N., Fishman A., Determination of tyrosinase substrate-binding modes reveals mechanistic differences between type-3 copper proteins. Nat. Commun. 5, 4505 (2014). PubMed

Decker H., Solem E., Tuczek F., Are glutamate and asparagine necessary for tyrosinase activity of type-3 copper proteins? Inorg. Chim. Acta 481, 32–37 (2018).

Fujieda N., et al. , Copper-oxygen dynamics in the tyrosinase mechanism. Angew. Chem. Int. Ed. Engl. 59, 13385–13390 (2020). PubMed

Conrad J. S., Dawso S. R., Hubbard E. R., Meyers T. E., Strothkamp K. G., Inhibitor binding to the binuclear active site of tyrosinase: Temperature, pH, and solvent deuterium isotope effects. Biochemistry 33, 5739–5744 (1994). PubMed

Matoba Y., Oda K., Muraki Y., Masuda T., The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity. Int. J. Biol. Macromol. 183, 1861–1870 (2021). PubMed

Kipouros I., et al. , Evidence for H-bonding interactions to the μ-η2:η2-peroxide of oxy-tyrosinase that activate its coupled binuclear copper site. Chem. Commun. (Camb.) 58, 3913–3916 (2022). PubMed PMC

Yamazaki S., Itoh S., Kinetic evaluation of phenolase activity of tyrosinase using simplified catalytic reaction system. J. Am. Chem. Soc. 125, 13034–13035 (2003). PubMed

Rodríguez-López J. N., Tudela J., Varón R., García-Carmona F., García-Cánovas F., Analysis of a kinetic model for melanin biosynthesis pathway. J. Biol. Chem. 267, 3801–3810 (1992). PubMed

Op’t Holt B. T., et al. , Reaction coordinate of a functional model of tyrosinase: Spectroscopic and computational characterization. J. Am. Chem. Soc. 131, 6421–6438 (2009). PubMed PMC

Eickman N. C., Solomon E. I., Larrabee J. A., Spiro T. G., Lerch K., Ultraviolet Resonance Raman Study of Oxytyrosinase. Comparison with Oxyhemocyanins. J. Am. Chem. Soc. 100, 6429–6431 (1978).

Solem E., Tuczek F., Decker H., Tyrosinase versus catechol oxidase: One asparagine makes the difference. Angew. Chem. Int. Ed. Engl. 55, 2884–2888 (2016). PubMed

Decker H., Dillinger R., Tuczek F., How Does Tyrosinase Work? Recent Insights from Model Chemistry and Structural Biology. Angew. Chem. Int. Ed. Engl. 39, 1591–1595 (2000). PubMed

Matoba Y., et al. , Activation mechanism of the Streptomyces tyrosinase assisted by the caddie protein. Biochemistry 56, 5593–5603 (2017). PubMed

Wilcox D. E., et al. , Substrate analog binding to the coupled binuclear copper active site in tyrosinase. J. Am. Chem. Soc. 107, 4015–4027 (1985).

Jiang H., Lai W., Monophenolase and catecholase activity of Aspergillus oryzae catechol oxidase: Insights from hybrid QM/MM calculations. Org. Biomol. Chem. 18, 5192–5202 (2020). PubMed

Lind T., Siegbahn P. E. M., Crabtree R. H., A quantum chemical study of the mechanism of tyrosinase. J. Phys. Chem. B 103, 1193–1202 (1999).

Inoue T., Shiota Y., Yoshizawa K., Quantum chemical approach to the mechanism for the biological conversion of tyrosine to dopaquinone. J. Am. Chem. Soc. 130, 16890–16897 (2008). PubMed

Matoba Y., et al. , Catalytic mechanism of the tyrosinase reaction toward the Tyr98 residue in the caddie protein. PLoS Biol. 16, e3000077 (2018). PubMed PMC

Osako T., et al. , Oxidation Mechanism of Phenols by Dicopper-Dioxygen (Cu2/O2) Complexes. J. Am. Chem. Soc. 125, 11027–11033 (2003). PubMed

Snyder B. E. R., Bols M. L., Schoonheydt R. A., Sels B. F., Solomon E. I., Iron and copper active sites in zeolites and their correlation to metalloenzymes. Chem. Rev. 118, 2718–2768 (2018). PubMed

Feng X., et al. , Rational construction of an artificial binuclear copper monooxygenase in a metal-organic framework. J. Am. Chem. Soc. 143, 1107–1118 (2021). PubMed

Chang T.-S., An updated review of tyrosinase inhibitors. Int. J. Mol. Sci. 10, 2440–2475 (2009). PubMed PMC

Gabrielle M., et al. , Targeted prodrug design for the treatment of malignant melanoma. J. Dermatol. Res. Ther. 2, 1–8 (2016).

Ginsbach J. W., et al. , Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF. Proc. Natl. Acad. Sci. U.S.A. 109, 10793–10797 (2012). PubMed PMC

Noguchi A., Kitamura T., Onaka H., Horinouchi S., Ohnishi Y., A copper-containing oxidase catalyzes C-nitrosation in nitrosobenzamide biosynthesis. Nat. Chem. Biol. 6, 641–643 (2010). PubMed

Stańczak A., Chalupský J., Rulíšek L., Straka M., Comprehensive theoretical view of the [Cu2 O2] side-on-peroxo-/bis-μ-oxo equilibria. ChemPhysChem, e202200076 (2022). PubMed

Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase

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