Mechanistic studies on the single copper tyrosyl-radical containing enzyme galactose oxidase

Studies on the single Cu protein galactose oxidase (63kDa; 639 amino acids) from Fusarium NRRL 2903 are described. The Cu is coordinated in a square based pyramid by Tyr-272, Tyr-495 (axial), His-496, His-581 and H2O (the substrate binding site), and the enzyme functions as a 2-equivalent oxidase, O-2 --> H2O2, with oxidation of primary alcohol substrates RCH2OH to RCHO. The active enzyme has a coordinated tyrosyl (Tyr) free radical at Tyr-272, and along with the Cu-II to Cu-I redox change gives the required two- equivalent redox capacity. The three oxidation states are here written as GOase(ox) (Cu-II, Tyr), GOase(semi) (Cu-II, Tyr), and GOase(red) (Cu-I, Tyr). Protonation of Tyr-495 is an important part of the enzymic reaction. Studies described are consistent with a mechanism involving H-atom transfer from substrate RCH2OH to Tyr at Tyr-272.