Determination of the Cleavage Site of the Large Subunit of Rubisco by Active Oxygen Species and Its Inhibition by CABP in the Lysates of Wheat Chloroplasts

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) catalyzes two competing reactions, photosynthetic CO2 fixation and photorespiratory carbon oxidation, in the stroma of the chloroplasts. This enzyme is the most abundant protein in C3 plants, and the amount of Rubisco can be a limiting factor for light-saturated photosynthesis in air. Therefore, the degradation of Rubisco directly affects photosynthesis and nitrogen economy in leaves. However, the mechanism of Rubisco degradation is still unclear. Rubisco is degraded rapidly under light- or oxidative-stress conditions in isolated chloroplasts (1–4). In illuminated chloroplasts, production of active oxygen species occurs as unavoidable event, especially under light- or oxidative-stress conditions (5, 6). Therefore, active oxygen species can be one of candidates, which act as triggering factors for Rubisco degradation. Recently, we have reported that the large subunit (LSU) of Rubisco is directly fragmented by active oxygen species, which is produced by metal-catalyzed reactions, in the lysates of chloroplasts under illumination (7). In the present study, we determined the definite cleavage site of the Rubisco-LSU into the 37-kDa and the 16-kDa fragments in the chloroplast lysates. In addition, we found that the fragmentation was completely blocked by the binding of 2-carboxyarabinitol 1,5-bisphosphate (CABP), a reaction intermediate analogue of the enzyme’s carboxylation reaction, to the enzyme.