Insights into the nature of the hydrogen bonding of ^{{radical dot}}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 (^MeSLH), is reported; X-ray crystallographic studies have shown that ^MeSLH involves an O-H···N⁺ hydrogen bond between the phenol and an imidazole nitrogen. ^MeSLH undergoes a reversible, one-electron, oxidation to form the radical cation [^MeSLH]^{{radical dot}+} the EPR spectrum of which is remarkably similar to that of ^{{radical dot}}Tyr₂₇₂ in Cu-free, oxidized, apo-GO. Density Functional Theory calculations, have shown that the proton-transferred (R-O^{{radical dot}}···H-N⁺) form of [^MeSLH]^{{radical dot}+} 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 ^{{radical dot}}Tyr₂₇₂ in Cu-free, oxidized, apo-GO whereas the non-proton-transferred (R-O^{{radical dot}+}-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 ^{{radical dot}}Tyr₂₇₂ in Cu-free, oxidized, apo-GO is involved in a O^{{radical dot}}···H-O/N hydrogen bond.