FAD IS A FURTHER ESSENTIAL COFACTOR OF THE NAD(P)H AND O-2-DEPENDENT ZEAXANTHIN-EPOXIDASE

In chloroplasts of plants the xanthophyll cycle is suggested to functi on as a protection mechanism against photodamage, Two enzymes catalyze this cycle, One of them, violaxanthin de-epoxidase, transforms violax anthin (Vio) to zeaxanthin (Zea) via antheraxanthin (Anth) and is boun d to the lumenal surface of the thylakoid vesicles, when being in its active state, The other enzyme, Zea-epoxidase, is responsible for the backward reaction (Zea --> Anth --> Vio) and is active at the stromal side of the thylakoid, For the epoxidation of Zea this enzyme requires NAD(P)H and O-2 as cosubstrates. Using isolated thylakoid membranes w e found that FAD enhances the epoxidase activity (decrease of apparent K-m for NAD(P)H and two-fold increase of V-max). The flavin functions as a third cofactor which is partially lost during the isolation proc edure of thylakoids. Other flavins, such as FMN or riboflavin are with out effect, The involvement of FAD in the enzymatic reaction is also d emonstrated by the inhibitory action of diphenyleneiodoniumchloride (D PI) (IC50 = 2.3 mu M), a compound that blocks the reoxidation of reduc ed flavins within enzymes, The Zea-epoxidase is a multicomponent enzym e system which can be classified as FAD-containing, NAD(P)H- and O-2-d ependent monooxygenase that is able to epoxidize 3-hydroxy beta-ionone rings of xanthophylls in the 5,6 position.