THE CHLOROPHYLL BIOSYNTHETIC ENZYME MG-PROTOPORPHYRIN IX MONOMETHYL ESTER (OXIDATIVE) CYCLASE - CHARACTERIZATION AND PARTIAL-PURIFICATION FROM CHLAMYDOMONAS-REINHARDTII AND SYNECHOCYSTIS SP PCC-6803

A universal structural feature of chlorophyll molecules is the isocycl ic ring. This ring is formed by the action of the enzyme Mg-protoporph yrin IX monomethyl ester (oxidative) cyclase, which catalyzes a comple x reaction in which Mg-protoporphyrin IX monomethyl ester is converted to divinyl protochlorophyllide (also called Mg-2,4-divinylpheoporphyr in a(5)), with the participation of NADPH and O-2. Cyclase activity wa s demonstrated in lysed Chlamydomonas reinhardtii chloroplasts and ext racts of Synechocystis sp. PCC 6803. The yield of the reaction product was increased by the addition of catalase and ascorbate or isoascorba te to the incubation mixture. These compounds may act by preventing de gradation of the tetrapyrroles by reactive oxygen species. Cyclase act ivity from C. reinhardtii was not inhibited by the flavoprotein inhibi tor quinacrine or by the hemoprotein inhibitors CO, KCN, or NaN3. In c ontrast, cyclase activity in extracts of C. reinhardtii and Synechocys tis sp. PCC 6803 was inhibited by chelators of Fe, suggesting that non heme Fe is involved in the reaction. Cyclase in lysed C. reinhardtii c hloroplasts was associated with the membranes, and attempts to further fractionate or solubilize the activity were unsuccessful. In contrast , cyclase in Synechocystis sp. PCC 6803 extracts could be separated in to soluble and membrane components, both of which were required for re constitution of activity. The membrane component retained activity aft er it was solubilized by the detergent n-oetyl-beta-D-glucopyranoside in the presence of glycerol and Mg2+. The solubilized membrane compone nt was purified further by dye-affinity and ion-exchange chromatograph y.