Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts.

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H(2)O(2)-dependent oxidation of ascorbate and ascorbate-dependent reduction of H(2)O(2). The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K(m) value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN(3), KCN, and 8-hydroxyquinoline but not ZnCl(2) 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 micromolar NADP(H), 2 millimolar l-ascorbate, and substrate concentrations of oxidized or reduced glutathione, catalyzed O(2) evolution when H(2)O(2) was added. Since the reaction was not inhibited by 0.1 millimolar NaN(3) and did not occur in the dark, it was concluded that catalase was not involved. Light-plus-H(2)O(2)-dependent O(2) evolution consisted of two distinct phases. The first phase was ascorbate-dependent and typically represented 10% of the total amount of O(2) evolved. The second phase was dependent on ascorbate and glutathione. The properties of the second phase were consistent with the operation of light-coupled glutathione reductase sequentially coupled to glutathione dehydrogenase and ascorbate peroxidase.