New mechanistic insights into coupled binuclear copper monooxygenases from the recent elucidation of the ternary intermediate of tyrosinase

Abstract

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2/monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the μ‐η2:η2‐peroxide dicopper(II) O–O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non‐protonated μ‐oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure–function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites.

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

“…12 A key experimental finding from our previous study 12 was that the monophenol monooxygenation rate of Ty measured in presteady-state conditions was ∼2−3 orders of magnitude faster than its steady-state turnover rate; this result indicates that previous studies based on steady-state rate Hammett plots likely reflect a different rate-limiting step and are thus not relevant to the monooxygenation mechanism of Ty. 11,19,22 As we previously demonstrated, the stopped-flow mixing of deoxy-Ty with an O 2 -containing solution of monophenol results in the fast formation of an intense 345 nm absorption feature corresponding to the μ-η 2 :η 2 -peroxide dicopper(II) active site that is present in both oxy-Ty and the ternary intermediate, which are in a rapid equilibrium. 12 This 345 nm feature subsequently decays at a first-order rate (k obs , Figure 2A inset) with the concomitant and near stoichiometric formation of the monooxygenation product(s) (i.e., catechol and/or quinone, with their ratio determined by the difference between the rates for catechol dissociation versus oxidation by met-Ty for a given substrate; see Scheme S1).…”

Magneto‐structural correlations of oximato‐bridged dinuclear copper(II) complex: A theoretical perspective

“…This aids in expanding the magnetic structure relationship of the complex molecular magnets, which attracts the attention of synthetic chemists to this field [6,7]. It is worth mentioning that dinuclear metal complexes are also active centers of proteases, such as tyrosinase, and copper oxidase, that are important for some life processes [8,9].…”

“…This aids in expanding the magnetic structure relationship of the complex molecular magnets, which attracts the attention of synthetic chemists to this field [6,7]. It is worth mentioning that dinuclear metal complexes are also active centers of proteases, such as tyrosinase, and copper oxidase, that are important for some life processes [8,9].Currently, the organic ligand containing the oxime group ( C N O) is of interest because of its structural and magnetic characteristics. The oximato-bridged dinuclear metal complex has a delocalized conjugate π bond in the plane of the oxime group because the three atoms, C, N,…”

“…Metal complexes comprising Cu(II) ions are among the most ideal compounds for investigating the magnetic characteristics of complex molecules because their magnetic behavior can be easily described. Moreover, Cu(II) ions have an electronic configuration of 3d 9 , and spin quantum number S = 1/2, which is an isotropic system. Cervera et al [21][22][23][24][25][26] observed that in the oximato-bridged dinuclear copper complex, the magnetic coupling constant between the two Cu(II) ions was linear with the Cu O N bond angle of the bridged ligand.…”

Nobel symposium #168 Visions of bio‐inorganic chemistry: metals and the molecules of life

“…The focus is, however, not on the substrate chemistry per se but rather the mechanisms put in place to protect the enzyme from self-inactivation by runaway oxidizing species when substrate oxidation is not possible. Solomon provides an account on the mechanism of binuclear copper monooxygenases [8]. These enzymes constitute a number of families with varied functions.…”