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 (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] + the EPR spectrum of which is remarkably similar to that of Tyr272 in Cu-free, oxidized, apo-GO. Density Functional Theory calculations, have shown that the proton-transferred (R–O ···H–N+) form of [MeSLH] + 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 Tyr272 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 Tyr272 in Cu-free, oxidized, apo-GO is involved in a O ···H–O/N hydrogen bond.