β-Haloethanol substrates as probes for radical mechanisms for galactose oxidase

Ketyl radical anions with a halogen substitutent on the carbon adjacent to ketyl are known to rapidly rearrange by halide anion ejection. Such a rearrangement is an ideal probe for possible ketyl radical anion intermediates in the catalytic mechanism of the monocopper/tyrosine radical enzyme galactose oxidase (GOase). Turnover of β-fluoro-, β-chloro-, β-bromo-, and β-iodoethanol by GOase leads to mechanism-based inactivation of the enzyme by trapping the enzyme in a catalytically inactive one-electron-reduced form. Presuming that mechanism-based inactivation and turnover proceed through the same reactive intermediates, the data reported here narrow down the possible mechanisms for the substrate oxidation step (the two electron transfer from substrate to enzyme) to two similar possibilities. Either the reaction proceeds through a short-lived ketyl radical anion intermediate or it proceeds through a closely related concerted E₂R mechanism with considerable ketyl radical anion character in the transition state.