Radical copper oxidases, one electron at a time

Radical copper oxidases (including the fungal enzymes galactose oxidase and glyoxal oxidase) are emerging as an important family of metalloenzymes based on the free radical-coupled copper catalytic motif. The active sites of these enzymes combine a redox active copper ion with a stable protein free radical, forming a two-electron redox unit capable of oxidizing a variety of alcohols and aldehydes with reduction of dioxygen to hydrogen peroxide. This active site is remarkable in the extent to which the ligands participate in catalysis. One of the tyrosine residues dissociates from the metal center and abstracts a proton to activate substrate for oxidation, while a second tyrosine (post-translationally modified to form a tyrosine-cysteine dimer) serves as the free radical redox site. Computational studies of the substituted phenoxyl indicates that in the ground state the majority of the unpaired electron is localized on the phenoxyl oxygen and thioether sulfur atoms, accounting for the special properties of this site. Isotope kinetics have been investigated for the substrate oxidation half-reaction indicating a dramatic isotope effect (k_H/k_D∼20) consistent with homolytic cleavage of the methylene C-H bond and hydrogen atom transfer to the phenoxyl in the transition state.