Light-Dependent Reduction of Hydrogen Peroxide by Ruptured Pea Chloroplasts

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Abstract
Ruptured pea (P. sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H2O2-dependent oxidation of ascorbate and ascorbate-dependent reduction of H2O2. The ratio of ascorbate peroxidase to NADP-glyceraldehyde-3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. Evidently, the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the Km value for ascorbate was 0.6 mM. Pyrogallol, glutathione and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN3, KCN and 8-hydroxyquinoline but not ZnCl2 or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate. Illuminated ruptured chloroplasts, in the presence of 50 .mu.M NADP(H), 2 mM L-ascorbate and substrate concentrations of oxidized or reduced glutathione, catalyzed O2 evolution when H2O2 was added. Since the reaction was not inhibited by 0.1 mM NaN3 and did not occur in the dark, catalase was not involved. Light-plus-H2-O2-dependent O2 evolution consisted of 2 distinct phases. The 1st phase was ascorbate-dependent and typically represented 10% of the total amount of O2 evoked. The 2nd phase was dependent on ascorbate and glutathione. The properties of the 2nd phase were consistent with the operation of light-coupled glutathione reductase sequentially coupled to glutathione dehydrogenase and ascorbate peroxidase.