Insights into the nature of the hydrogen bonding of *Tyr272 in apo-galactose oxidase.

The synthesis and structure of an o-methylthio-phenol-imidazole, 2-(2'-(4'-tert-butyl-6'-methylsulfanyl)-hydroxyphenyl))-4,5-diphenyl-imidazole ((MeS)LH), is reported; X-ray crystallographic studies have shown that (MeS)LH involves an O-H...N(+) hydrogen bond between the phenol and an imidazole nitrogen. (MeS)LH undergoes a reversible, one-electron, oxidation to form the radical cation (MeS)LH(+) the EPR spectrum of which is remarkably similar to that of ()Tyr(272) in Cu-free, oxidized, apo-GO. Density Functional Theory calculations, have shown that the proton-transferred (R-O()...H-N(+)) form of (MeS)LH(+) has a spin density distribution--with a substantial delocalization of the unpaired electron spin density onto the ortho sulfur atom--and EPR properties that are in good agreement with those of ()Tyr(272) in Cu-free, oxidized, apo-GO whereas the non-proton-transferred (R-O()(+)-H...N) form does not. The results reported herein are a further demonstration of the influence of hydrogen bonding on the nature and properties of phenoxyl radicals and strongly suggest that the phenoxyl oxygen of ()Tyr(272) in Cu-free, oxidized, apo-GO is involved in a O()...H-O/N hydrogen bond.