Oxygen activation in the Copper Amine Oxidases

Abstract

Oxygen‐utilizing enzymes must activate oxygen in a very controlled manner to avoid oxidative damage to themselves and surrounding molecules. By studying the mechanism of oxygen activation in enzymes, we can understand how these enzymes controllably activate oxygen. The copper amine oxidases (CAOs) are a family of enzymes that utilize oxygen two oxidize primary amines to aldehydes and ammonia. In this reaction, O2 is reduced to H2O2 using to cofactors: topaquinone (TPQ) and Cu(II). Two mechanisms have been proposed for oxygen reduction: an outer‐sphere mechanism, where O2 is directly reduced by TPQ forming superoxide, which is stabilized by Cu(II); and an inner‐sphere mechanism, where TPQ initially reduces Cu(II) to Cu(I) to which oxygen binds and is reduced to superoxide. We have evaluated the mechanism of oxygen activation in CAOs through kinetic analysis and metal substitution. Replacement of Cu(II) with Co(II) blocks the inner‐sphere mechanism, since Co(II) would not be reduced to Co(I) in a protein, and can distinguish between the two mechanisms. We have replaced Cu(II) with Co(II) in several CAOs, including the CAOs from pea seedlings (PSAO) and from E. coli (ECAO) and evaluated the effect on catalysis. These results allow us to determine which mechanism each enzyme uses for catalysis. This work was supported by the University of San Diego.