Stereoelectronic Properties of Metalloenzymes. II. Effects of Ligand Coordination on the Electron Spin Resonance Spectrum of Galactose Oxidase as a Probe of Structure and Function

The addition of various potential copper ligands to solutions of galactose oxidase produces distinctive changes in its esr spectrum. The most strongly bound ligands appear to be those capable of forming-bonds to Cu²⁺ such as cyanide. A substrate, galactose, competes with this ligand for the Cu²⁺ site. Hydrogen peroxide, a product of the enzymatic reaction, appears to bind at the copper site although its effect on the esr spectrum is small. The experiments indicate that only a single coordination site in the copper-protein complex is readily accessible to exogenous ligands and that this site is normally occupied by a water or hydroxide molecule. The manner in which the esr spectrum changes in the presence of strong Π-bonding ligands suggests that a strong Π-bonding ligand may occupy the nonlabile axial coordination site in the protein-metal complex.