Light-dependent reduction of dehydroascorbate by ruptured pea chloroplasts

Abstract

Glutathione dehydrogenase (EC 1.8.5.1) was partially purified from pea shoots. The pH optimum was 7.6. The K(m) values for GSH and dehydroascorbate were 4.4 and 0.44 millimolar, respectively. The enzyme was inhibited by iodoacetate and CuSO(4) but not significantly by ZnCl(2) or NaN(3). Part of the total enzyme activity was associated with isolated chloroplasts.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H) and substrate concentrations of GSH or GSSG, catalyzed (dehydroascorbate plus glutathione)-dependent O(2) evolution with the concomitant reduction of dehydroascorbate to ascorbate. Oxidation of ascorbate by ascorbate oxidase activity associated with the chloroplasts was relatively insignificant. ZnCl(2) inhibited (dehydroascorbate plus glutathione)-dependent O(2) evolution but not ascorbate formation. The reaction was attributed to light-dependent reduction of GSSG (involving glutathione reductase) coupled to the reduction of dehydroascorbate (involving glutathione dehydrogenase). Light-dependent reduction of GSSG appears to be the rate-limiting step in this reaction sequence at physiological concentrations of GSH.