Synthetic models of the inactive Copper(II)-tyrosinate and active Copper(II)-tyrosyl radical forms of galactose and glyoxal oxidases

A series of Cu(II) and Zn(II) complexes with new ligands having either one or two substituted phenolates appended to the 1,4,7-triazacyclononane frame were prepared and characterized by optical absorption, EPR, NMR, and/or resonance Raman spectroscopy, cyclic voltammetry, and, in eight cases, X-ray crystallography. Features of the active site geometries of the Cu(II)-tyrosinate forms of galactose and glyoxal oxidases (GAO and GLO) were modeled by these complexes, including the binding of a redox-active phenolate and an exogenous ligand (Cl^-, CH₃CO₂^-, or CH₃CN) in a cis-equatorial position of a square pyramidal metal ion. The role of the unique ortho S-C covalent bond between a cysteine (C228) and the equatorial tyrosinate (Y272) in the proteins was probed through an examination of the optical absorption and electrochemical properties of sets of similar complexes comprised of phenolate ligands with differing ortho substituents, including thioether groups. The o-alkylthio unit influences the PhO^- → Cu(II) LMCT transition and the M(II)-phenolate/M(II)-phenoxyl radical redox potential, but to a relatively small degree. Electrochemical and chemical one-electron oxidations of the Cu(II) and Zn(II) complexes of ligands having tert-butyl protecting groups on the phenolates yielded new species that were identified as novel M(II)-phenoxyl radical compounds analogous to the active Cu(II)-tyrosyl radical forms of GAO and GLO. The M(II)-phenoxyl radical species were characterized by optical absorption, EPR, and resonance Raman spectroscopy, as well as by their stoichiometry of formation and chemical reduction. Notable features of the Cu(II)-phenoxyl radical compounds that are similar to their protein counterparts include EPR silence indicative of magnetic coupling between the Cu(II) ion and the bound radical, a band with ζ(max) approx 410 nm (ε ≃ 3900 M^-1 cm^-1) in UV-vis spectra diagnostic for the phenoxyl radical, and a feature attributable to the phenoxyl radical C-O vibration (ν(7a)) in resonance Raman spectra. Similar Raman spectra and electrochemical behavior for the Zn(II) analogs, as well as an isotropic signal at g = 2.00 in their X-band EPR spectra, further corroborate the formulations of the M(II)-phenoxyl radical species.