Ascorbate oxidase peroxide form

Fig. 12. Schematic drawing of the peroxide form of ascorbate oxidase around the trinuclear copper site. The included copper-copper distances are the mean values between both subunits.

The first successful observation and characterization of the ascorbate free radical was carried out with ESR (14,15). A 1.7-G ESR doublet was reported and it was correctly concluded that the observed spectrum represented the anionic form (A ) of the radical. These measurements (14,15) showed that the enzyme-generated radical (horseradish peroxidase-hydrogen peroxide-ascorbate) was present as a free radical and decayed by second-order kinetics (see Figure 2). Recent experiments (16,17) have shown that ascorbate oxidase and dopamine-monooxygenase also generate unbound ascorbate radicals. 

Anaerobic reduction of ascorbate oxidase, AOox, by ascorbate ion is an eight-electron process. Initial reduction of the type 1 centre is reversed as the type 3 centre is reduced and it is suggested that type 1 copper is close to the substrate binding site and that type 3 is involved in the interaction with molecular oxygen. Azide, which can bind to type 2 copper, has no effect on the reduction process. A potentially catalytically active intermediate is formed in the reaction between ascorbate oxidase and hydrogen peroxide at pH 7.6, 0.01 M phosphate, and 25 °C ... 

From extracts of the crook-neck squash and cucumber purified preparations containing 0.15 to 0.25% of copper have been obtained. Since ascorbic acid oxidase does not attack mono- or diphenols, it is distinct from the phenol oxidases. A number of synthetic copper protein complexes have been shown to have ascorbic oxidase activity. This activity, however, is much less than that of the isolated enzyme. These complexes form hydrogen peroxide which is not produced in the oxidation catalyzed by ascorbic acid oxidase. 

Recent studies by Nielsen et al. (23) have shown that simultaneous addition of hypoxantiiine and catalase to raw milk preserves ascorbate considerably. This indicates that the formed radical species induced by hydrogen peroxide and milk peroxidases are inq)ortant for die loss of ascorbate in addition to tiie direct oxidation of ascorbate through reaction widi superoxide. This is supported by the fact that addition of hydrogen peroxide to raw milk initiates a dramatic decrease in tile ascorbate content (23). This enzymatically catalyzed pathway is supported by the fiuit that addition of hydrogen peroxide to UHT-milk only results in a minor oxidation of ascorbate vhich in cates tiiat Fenton reaction chemistry is only of minor in rtance. Overall, tiie present data states that xaiiuiine oxidase-and peroxidase-mediated oxidation are crucial tiictors in ascorbate oxidation as suggested by (33). 

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