Production of a presumptive chlorophyll catabolite in vitro: requirement for reduced ferredoxin

Senescent chloroplasts (gerontoplasts) isolated from primary leaves of barley (Hordeum vulgare L.) contained a group of fluorescent chlorophyll (Chl) compounds designated as FCC-2, FCC-3 and FCC-4. Compound FCC-2 represents an established catabolite of Chl-porphyrin and was the most abundant constituent of this group. One of the minor constituents, FCC-4, was produced in a reconstituted system composed of thylakoids and stroma. The generation of FCC-4 depended on oxygen and required reduced ferredoxin (Fd) which probably acts as a reductant of the putative oxygenating enzyme responsible for the cleavage of Chl-porphyrin. A typical assay mixture consisted of thylakoids and stroma (equalling 10⁸ gerontoplasts), Fd, NADPH and glucose-6-phosphate for NADPH-regeneration. The oxygenating enzyme appeared to be a stromal protein. However, enzyme activities associated with the thylakoids were also required for the production of FCC-4. The senescence-specific part of the reconstituted system resided in the thylakoids. Thus, FCC-4 was produced in assay mixtures of senescent thylakoids and stroma from presenescent chloroplasts, whereas combinations with presenescent thylakoids failed to yield appreciable amounts of this putative primary Chl-catabolite.