High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical

The activated form of galactose oxidase from the fungus Dactylium dendroides contains a single divalent copper ion which is antiferromagnetically coupled to a protein-based free radical. Chemical oxidation of the apoenzyme generates the free radical which is localized on a covalently cross-linked tyrosine - cysteine residue. This species, together with model radicals generated by UV irradiation of protonated and selectively deuterated o-(methylthio)cresol (MTC), has been studied by high-frequency EPR spectroscopy (139.5 GHz/5 T) in conjunction with molecular orbital calculations employing self-consistent local density functional (LDF) methods. The Zeeman interactions (g values) determined from the high-frequency spectra of the apogalactose oxidase and the MTC model radicals are remarkably similar and support the assignment of the protein radical to a sulfur-substituted tyrosyl moiety. Molecular orbital calculations accurately reflect the experimental data, including an increase in the axial symmetry of the Zeeman interaction for the MTC radical compared with the unsubstituted tyrosyl radical species. An explanation of this effect based on an analysis of individual atomic contributions to the molecular g values is presented. High-frequency echo-detected EPR spectroscopy of the apogalactose oxidase radical resolves hyperfine splittings. Based on the molecular orbital calculations and the EPR spectroscopic results presented here, the hyperfine splittings are assigned to two methylene protons - one derived from tyrosine and one from cysteine. These findings are consistent with the radical spin density being localized on the tyrosine - cysteine moiety, rather than delocalized throughout an extended π-network involving a nearby tryptophan as had been previously suggested as a possible explanation of the stability of the radical species.