Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu 2 O 2 Active Site of oxy-Tyrosinase.
Ioannis KipourosAgnieszka StańczakEleanor M DunietzJake W GinsbachMartin SrnecLubomír RulíšekEdward I SolomonPublished in: Journal of the American Chemical Society (2023)
Tyrosinase is a ubiquitous coupled binuclear copper enzyme that activates O 2 toward the regioselective monooxygenation of monophenols to catechols via a mechanism that remains only partially defined. Here, we present new mechanistic insights into the initial steps of this monooxygenation reaction by employing a pre-steady-state, stopped-flow kinetics approach that allows for the direct measurement of the monooxygenation rates for a series of para -substituted monophenols by oxy-tyrosinase. The obtained biphasic Hammett plot and the associated solvent kinetic isotope effect values provide direct evidence for an initial H-transfer from the protonated phenolic substrate to the Cu 2 O 2 core of oxy-tyrosinase. The correlation of these experimental results to quantum mechanics/molecular mechanics calculations provides a detailed mechanistic description of this H-transfer step. These new mechanistic insights revise and expand our fundamental understanding of Cu 2 O 2 active sites in biology.