Inhibition of Ascorbate Oxidase by Phenolic Compounds. Enzymatic and Spectroscopic Studies

Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed
by ascorbate oxidase was investigated at pH 7.0 and 23.0 °C. Inhibition of p-nitrophenol is pH dependent
over the range 5.0-8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol
nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics
of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented,
and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed
upon prolonged incubation of ascorbate oxidase with excess phenol and azide, but not with phenol alone.
It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (ascorbate)
binds. NMR relaxation measurements of the protons of phenols in the presence of ascorbate oxidase
show paramagnetic effects due to the proximity of the bound inhibitor to a copper center, likely type 1
copper. Copper-proton distance estimates between this paramagnetic center and p-cresol or p-nitrophenol
bound to ascorbate oxidase are between 4.4 and 5.9 Å