Role of Copper Ion in Bacterial Copper Amine Oxidase:  Spectroscopic and Crystallographic Studies of Metal-Substituted Enzymes

The role of the active site Cu2+ of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu2+ from the active site. The enzymes reconstituted with Co2+ and Ni2+ (Co- and Ni-AGAO) exhibited 2.2 and 0.9% activities, respectively, of the original Cu2+-enzyme (Cu-AGAO), but their K m values for amine substrate and dioxygen were comparable. X-ray crystal structures of the Co- and Ni-AGAO were solved at 2.0−1.8 Å resolution. These structures revealed changes in the metal coordination environment when compared to that of Cu-AGAO. However, the hydrogen-bonding network around the active site involving metal-coordinating and noncoordinating water molecules was preserved. Upon anaerobic mixing of the Cu-, Co-, and Ni-AGAO with amine substrate, the 480 nm absorption band characteristic of the oxidized form of the topaquinone cofactor (TPQox) disappeared rapidly (