A spectroscopic and kinetic investigation of anion binding to ascorbate oxidase

The binding of azide, fluoride, and cyanide to ascorbate oxidase has been investigated in detail. Both azide and fluorid inhibit the enzyme competitively with respect to ascorbic acid and noncompetitively with respect to oxygen. Cyanide inhibition is much more complex and also results in inactivation of the enzyme. The binding of azide and fluoride to the resting enzyme is partially competitive. Fluoride binds more strongly to the resting enzyme, while azide binds more strongly to the functioning enzyme. It is proposed that both azide and fluoride bind to type 2 copper and that this copper is also part of an ascorbate binding site. It seems likely that type 2 copper is a reductant binding site in all of the "blue" oxidases. This proposal is used to explain the effect of fluoride on the enzymes and also to suggest a mechanism for the internal electron transfer which is necessary for the reduction of oxygen to water.