Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies of the Radical Site in Galactose Oxidase and of Thioether-Substituted Phenol Model Compounds

The organic free radical in galactose oxidase is exchange coupled to the mononuclear copper atom in the active site of the holoenzyme. By removing the copper, the radical can be generated in high concentrations in its electron paramagnetic resonance (EPR)-detectable form. We have carried out a detailed study of the magnetic resonance properties of the radical in the apoenzyme and of a series of phenol model compounds. In the protein, the radical EPR spectrum has partially resolved fine structure with a center crossing at g = 2.0055 and an overall peak-to-trough line width of 33 G. The electron nuclear double resonance (ENDOR) spectrum of the radical reveals strong hyperfine couplings to two classes of protons. The first has A_iso = 14.6 G and tensor components characteristic of a β-proton; the second has A_iso ≈ 8 G and exhibits hyperfine anisotropy typical of an α-proton. Simulation of the radical EPR spectrum with the ENDOR parameters shows that there is only a single proton in each of the two classes of strongly coupled protons. In the weakly coupled region of the ENDOR spectrum, H₂O/D₂O exchange measurements indicate the occurrence of a proton hydrogen-bonded to the phenol oxygen of the radical. In conjunction with earlier data indicating a tyrosine origin for the radical, these observations provide strong experimental support for suggestions that the radical site is the o-cysteine-substituted tyrosine residue (Y₂₇₂) in the immediate coordination sphere of the copper. EPR and ENDOR spectra have been obtained for a series of substituted phenol radicals that support this conclusion. Introduction of a thioether at the ortho position perturbs the g tensor and spin density distribution of the phenol moiety only slightly; our ENDOR results indicate, for example, that the spin density at the ring para position is decreased by ~25% upon thioether substitution. The ring thioether substituent is expected to perturb the redox potential of the tyrosine, however, and contribute to the relative ease with which the modified tyrosine in galactose oxidase can be oxidized in comparison with redox-active tyrosines that have been observed in other protein systems.