Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase
- Ioannis KipourosIoannis KipourosDepartment of Chemistry, Stanford University, Stanford, California 94305, United StatesMore by Ioannis Kipouros
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- Agnieszka StańczakAgnieszka StańczakInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 2, 166 10 Praha 6, Czech RepublicFaculty of Science, Charles University, Albertov 2038/6, 128 00 Praha 2, Czech RepublicMore by Agnieszka Stańczak
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- Eleanor M. DunietzEleanor M. DunietzDepartment of Chemistry, Stanford University, Stanford, California 94305, United StatesMore by Eleanor M. Dunietz
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- Jake W. GinsbachJake W. GinsbachDepartment of Chemistry, Stanford University, Stanford, California 94305, United StatesMore by Jake W. Ginsbach
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- Martin SrnecMartin SrnecJ. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, 182 23 Prague, Czech RepublicMore by Martin Srnec
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- Lubomír Rulíšek*Lubomír Rulíšek*Email: [email protected]Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 2, 166 10 Praha 6, Czech RepublicMore by Lubomír Rulíšek
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- Edward I. Solomon*Edward I. Solomon*Email: [email protected]Department of Chemistry, Stanford University, Stanford, California 94305, United StatesStanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California 94025, United StatesMore by Edward I. Solomon
Abstract
Tyrosinase is a ubiquitous coupled binuclear copper enzyme that activates O2 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 Cu2O2 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 Cu2O2 active sites in biology.
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