ATTACHMENT OF CUZN-SUPEROXIDE DISMUTASE TO THYLAKOID MEMBRANES AT THESITE OF SUPEROXIDE GENERATION (PSI) IN SPINACH-CHLOROPLASTS - DETECTION BY IMMONUGOLD LABELING AFTER RAPID FREEZING AND SUBSTITUTION METHOD

In chloroplasts O-2(-) is photoproduced via the univalent reduction of O-2 in PSI even under conditions that are favorable for photosynthesi s. The photogenerated O-2(-) is disproportionated to H2O2 and O-2 in a reaction that is catalyzed by superoxide dismutase (SOD). The H2O2-sc avenging ascorbate peroxidase is bound to the thylakoid membranes at o r near the PSI reaction center [Miyake and Asada (1992) Plant Cell Phy siol. 33: 541], and the primary product of oxidation in the peroxidase -catalyzed reaction, the monodehydroascorbate radical, is photoreduced to ascorbate in PSI in a reaction mediated by ferredoxin [Miyake and Asada (1994) Plant Cell Physiol. 35: 539]. Therefore, SOD should be lo calized at or near the PSI complex. We report here the microcompartmen talization of the chloroplastic CuZn-SOD on the stromal-faces of thyla koid membranes where the PSI-complex is located. Spinach leaves were f ixed and substituted by a rapid freezing and substitution method that allows visualization of intact chloroplasts. The embedded sections wer e immune labeled with the antibody against CuZn-SOD by the immunogold method. About 70% of the immunogold particles were found within 5 nm f rom the surface of the stromal-faces of thylakoid membranes. Of these particles, about 40% were found at the ends and margins of the grana t hylakoids and 60% were found on the stromal side of the stromal thylak oids. From these results, the local concentration of CuZn-SOD on the s troma-facing surfaces of the thylakoid membranes was estimated to be a bout 1 mM. The effect of the microcompartmentalization of CuZn-SOD on the scavenging of superoxide radicals is discussed.