Evidence for H-bonding interactions to the μ-η²:η²-peroxide of oxy-tyrosinase that activate its coupled binuclear copper site

Abstract

The factors that control the diverse reactivity of the μ-η2:η2-peroxide dicopper(II) oxy-intermediates in the coupled binuclear copper proteins remain elusive. Here, spectroscopic and computational methods reveal H-bonding interactions between active-site...

Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

“…2 B ). Since the geometric structure of the oxy-Ty active site is not perturbed upon solvent deuteration ( 27 ), the observed solvent KIE likely reflects the participation of one or more exchangeable protons in the transition state (TS) of the RLS, clearly implicating the phenolic substrate proton in the monooxygenation reaction mechanism. This is fully consistent with our pH dependence results ( SI Appendix , Fig.…”

“…Our experimental data, coupled with quantum-mechanics/molecular-mechanics (QM/MM) calculations, indicate that the monophenolic substrate binds to the active-site pocket of oxy-Ty fully protonated, without cleavage of the μ-η 2 :η 2 -peroxide O-O bond and without direct coordination to a Cu center. Formation of this ternary intermediate involves the displacement of the recently reported active-site water molecules ( 27 ), and replacement of their H-bonding interactions with the μ-η 2 :η 2 -peroxide by a single H bond from the hydroxyl group of the docked full-protonated monophenol substrate. This unprecedented substrate binding mode to a μ-η 2 :η 2 -peroxide dicopper(II) active site has direct mechanistic implications.…”

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

“…Since previous studies were mostly focused on oxy‐Ty either in its pro‐form (i.e., the latent enzyme form that is activated by post‐translational proteolytic cleavage of its extended terminal) or its complex with the caddie protein, in all of which the surface‐exposed active site pocket is fully occupied by terminal or caddie residues, we proceeded to obtain the rR spectrum of the catalytic and monomeric oxy‐Ty. Interestingly, upon solvent isotopic perturbations (H 2 O→D 2 O), the rR of oxy‐Ty exhibited frequency upshifts in its fundamental (Δ ν Cu–O = +4 cm −1 ), and first‐overtone Cu–O modes (Δ ν Cu–O(overtone) = +7 cm −1 ), suggesting solvent interactions with the μ‐η 2 :η 2 ‐peroxide dicopper(II) active site [71]. These H 2 O→D 2 O frequency upshifts were not observed in rR studies for oxy‐Hc [72], consistent with the fact that its μ‐η 2 :η 2 ‐peroxide dicopper(II) active site is buried within the protein matrix, precluding solvent access.…”

“…Molecular dynamics (MD) simulations predicted the presence of active site solvent water molecules in close proximity to the μ‐η 2 :η 2 ‐peroxide of oxy‐Ty, with subsequent QM frequency calculations indicating that H‐bonding interaction between active site water molecules (W1, W2) and the μ‐η 2 :η 2 ‐peroxide (Fig. 3E) is required to reproduce the spectroscopically observed frequency upshift of the Cu–O mode [71]. This frequency upshift of the Cu–O mode upon solvent deuteration is due to its mode coupling with close‐in‐energy W1 and W2 modes since in H 2 O the water modes are higher in energy than the Cu–O mode, and thus mixing decreases the Cu–O frequency, while in D 2 O the energy of the water modes are below the energy of the Cu–O mode and mixing increases the Cu–O frequency [71].…”

“…3E) is required to reproduce the spectroscopically observed frequency upshift of the Cu–O mode [71]. This frequency upshift of the Cu–O mode upon solvent deuteration is due to its mode coupling with close‐in‐energy W1 and W2 modes since in H 2 O the water modes are higher in energy than the Cu–O mode, and thus mixing decreases the Cu–O frequency, while in D 2 O the energy of the water modes are below the energy of the Cu–O mode and mixing increases the Cu–O frequency [71]. Having defined the reference rR spectra and active site structure for the monomeric oxy‐Ty, we proceed to trap the ternary intermediate via 100‐ms rapid freeze quench (RFQ) for the reaction of deoxy‐Ty with an O 2 ‐saturated solution of 4‐COOCH 3 (10 m m ).…”