Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts and Photoreduction of Its Primary Oxidation Product Monodehydroascorbate Radicals in Thylakoids

Ascorbate peroxidase, a key enzyme for the scavenging of hydrogen peroxide in chloroplasts, was found in a thylakoid-bound form in spinach chloroplasts at comparable activity to that in the stroma. The activity of peroxidase was detectable in the thylakoids only when prepared by an ascorbate-containing medium, and enriched in the stroma thylakoids. The thylakoid enzyme was not released from the membranes by either 2 mM EDTA, 1 M KCl, 2 M NaBr or 2 M NaSCN, but was solubilized by detergents. Enzymatic properties of the thylakoid-bound ascorbate peroxidase were very similar to those of the stromal ascorbate peroxidase. Thylakoid-bound ascorbate peroxidase could scavenge the hydrogen peroxide either added or photoproduced by the thylakoids. No photoreduction of hydrogen peroxide was observed, however, in the thylakoids whose ascorbate peroxidase was inhibited by KCN and thiol reagents or inactivated by the treatment with ascorbate-depletion. The primary oxidation product of ascorbate in a reaction of ascorbate peroxidase, monodehydroascorbate (MDA) radical, was photoreduced in the thylakoids, as detected by the quenching of chlorophyll fluorescence, disappearance of EPR signals of the MDA radicals and the MDA radical-induced oxygen evolution. Thus, ascorbate is photoregenerated in the thylakoids from the MDA radicals produced in a reaction of ascorbate peroxidase for the scavenging of hydrogen peroxide.