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-peroxo dicopper(II) oxy-intermediates in the coupled binuclear copper proteins remain elusive. Here, spectroscopic and computational methods reveal H-bonding interactions between active-site waters and the μ-η²:η²-peroxide of oxy-tyrosinase, and define their effects on the Cu(II)₂O₂ electronic structure and O₂ activation.

Fig. 1.

Resonance Raman (351nm laser excitation) spectral regions for the key [Cu(II)₂O₂]²⁺ vibrational modes of oxy-Ty from S.Glaucescens (0.75mM, 0.1M CHES, pH/D 9.0) in H₂O (red) and D₂O (blue) corrected for background contributions, with gaussian fits (in regions with multiple peaks, fits without the baseline components are also shown), and each region scaled for clarity (reference normalized peak intensity ratios are ~16:1:2:6 for Cu-Cu : Cu-O fundamental : O-O : Cu-O overtone), (A) the Cu-Cu (indicated with solid line) and Cu-N stretches (dashed lines), the negative feature (asterisk) at the solvent ice peak region results from background correction; the insert expands the region of the two higher-energy isotope-sensitive Cu-N stretches, (B) the Cu-O (B_3u) fundamental stretch (the black gaussian indicates a non-isotope sensitive peak present in both solvents), (C) the O-O stretch, and (D) the Cu-O (B_3u) overtone stretch. For entire rR spectra before and after background corrections and for band-shape discussion see Fig. S5; for additional experimental details see SI methods 1.1.5–1.1.6.

Fig. 2.

(A) The full-length QM/MM-optimized structure for the experimentally-calibrated oxy-Ty active site from Streptomyces castaneoglobisporous (oxy_48, blue), (B) its active site, and (C) its alignment with the met-CaOx from Ipomoea batatas (PDB:1BT1, red). Key second-sphere residues and the μ-η²:η²-peroxide ligand and waters (W1–3) in oxy-Ty are shown as solid sticks, the water (W3) and μ-hydroxide ligand in CaOx as red spheres, the first-sphere histidines as transparent sticks, and the copper centers as brown spheres. H-bonds are indicated as dashed yellow lines.

Fig. 3.

Vibrational coupling of the Cu-O(B_3u) stretch of the Cu(II)₂O₂ active site with two key water modes for W1 (twisting in H₂O and rocking in D₂O).