HIGH-FREQUENCY ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY OF THE APOGALACTOSE OXIDASE RADICAL

The activated form of galactose oxidase from the fungus Dactylium dend roides contains a single divalent copper ion which is antiferromagneti cally coupled to a protein-based free radical. Chemical oxidation of t he apoenzyme generates the free radical which is localized on a covale ntly cross-linked tyrosine-cysteine residue. This species, together wi th model radicals generated by UV irradiation of protonated and select ively deuterated o-(methylthio)cresol (MTC), has been studied by high- frequency EPR spectroscopy (139.5 GHz/5 T) in conjunction with molecul ar orbital calculations employing self-consistent local density functi onal (LDF) methods. The Zeeman interactions (g values) determined from the high-frequency spectra of the apogalactose oxidase and the MTC mo del radicals are remarkably similar and support the assignment of the protein radical to a sulfur-substituted tyrosyl moiety. Molecular orbi tal calculations accurately reflect the experimental data, including a n increase in the axial symmetry of the Zeeman interaction for the MTC radical compared with the unsubstituted tyrosyl radical species. An e xplanation of this effect based on an analysis of individual atomic co ntributions to the molecular g values is presented. High-frequency ech o-detected EPR spectroscopy of the apogalactose oxidase radical resolv es hyperfine splittings. Based on the molecular orbital calculations a nd the EPR spectroscopic results presented here, the hyperfine splitti ngs are assigned to two methylene protons-one derived from tyrosine an d one from cysteine. These findings are consistent with the radical sp in density being localized on the tyrosine-cysteine moiety, rather tha n delocalized throughout an extended pi-network involving a nearby try ptophan as had been previously suggested as a possible explanation of the stability of the radical species.