Reaction System for Violaxanthin De-Epoxidase with PSII Membranes

PSII membranes were used as a substrate for violaxanthin de-epoxidase (VDE) that had been solubilized from spinach thylakoids by sonication. Inclusion of Tween 20 in the assay mixture was essential, although the detergent apparently inhibited the activity in the conventional assay with purified violaxanthin and lipid as substrate. The maximum enhancing effect of the detergent was observed near its critical micellar concentration. It is likely that the monomer of the detergent helped VDE react with the substrate in the membranes. Dependence of the activity on the substrate concentration suggested that VDE functions at least at two sites in the membranes, probably on both their lumenal and stromal surfaces. The ability of the enzyme to function on the stromal surface in in vitro assays was demonstrated by using intact thylakoids as the substrate. Under such conditions where the endogenous VDE was functioning in the lumen, the exogenously added VDE converted antheraxanthin to zeaxanthin in the absence of Tween 20. This result suggests that, in the reaction with PSII membranes, the detergent was required for VDE to react with violaxanthin but not with antheraxanthin. Otherwise, the detergent was necessary for the reaction on the lumenal surface.