Tyrosyl radical in galactose oxidase not strongly perturbed by cysteine cross-link

Several density functional methods are applied to calculate the hyperfine coupling constants and spin population distributions of sulfur-substituted and unsubstituted tyrosyl radical. The cysteine-substituted tyrosyl radical is found at the active site of the radical enzyme of galactose oxidase. The main conclusion is that the sulfur center only possesses a small amount of unpaired spin not sufficient to perturb the overall odd-alternant spin distribution in the tyrosyl radical. The original assignment of two hyperfine couplings, one from a tyrosine β-proton and one from H5, is clearly confirmed by calculated isotropic hyperfine coupling constants. The inclusion of solvent effects does not alter any of the conclusions.