On the nature of copper in ascorbate oxidase: I. The valence state of copper in the denatured and native enzyme

Abstract

1. 1. The valency state of Cu in the enzyme ascorbate oxidase ( l-ascorbate: O 2 oxidoreductase, EC 1.10.3.3) has been determined by liberating the copper from the enzyme in the presence of valence-specific Cu-chelating agents. The procedures were designed to prevent electronic changes in the copper valency as a result of enzyme denaturation during the assay process. 2. 2. By means of the two Cu(I)-specific reagents, cuproine and bathocuproine, it has been found that the prosthetic copper in ascorbate oxidase exists in a mixed valency state, corresponding to 25% Cu(I) and 75% Cu(II). This ratio of 1:3 corresponds to 2 atoms of Cu(I) and 6 atoms of Cu(II) per enzyme molecule. 3. 3. This same ratio was found for the mixed valency state in the denatured enzyme when the Cu(I) reagent, bathocuproine, and the Cu(II) reagent, cuprizone, were used simultaneously. 4. 4. Neither bathocuproine nor cuprizone reacted directly with the native enzyme at physiological pH 7.2.