Spectroscopic and magnetochemical studies on the active site copper complex in galactose oxidase

Galactose oxidase is a radical copper oxidase, an enzyme making use of a covalently modified tyrosine residue as a free radical redox cofactor in alcohol oxidation catalysis. We report here a combination of spectroscopic and magnetochemical studies developing insight into the interactions between the active site Cu(II) and two distinct tyrosine ligands in the biological complex. One of the tyrosine ligands (Y495) is coordinated to the Cu(II) metal center as a phenolate in the resting enzyme and serves as a general base to abstract a proton from the coordinated substrate, thus activating it for oxidation. The structure of the resting enzyme is temperature-dependent as a consequence of an internal proton equilibrium associated with this tyrosine that mimics this catalytic proton transfer step. The other tyrosine ligand (Y272) is covalently crosslinked to a cysteine residue forming a tyrosine-cysteine dimer free radical redox site that is required for hydrogen atom abstraction from the activated substrate alkoxide. The presence of the free radical in the oxidized active enzyme results in formation of an EPR-silent Cu(II) complex shown by multifield magnetic saturation experiments to be a diamagnetic singlet arising from antiferromagnetic exchange coupling between the metal and radical spins. A paramagnetic contribution observed at higher temperature may be associated with thermal population of the triplet state, thus permitting an estimate of the magnitude of the isotropic exchange coupling (J>200 cm^-1, JS₁.S₂) in this complex. Structural correlations and the possible mechanistic significance of metal-radical coupling in the active enzyme are discussed. Copyright (C) 2000 Elsevier Science B.V.