Substrate specificity and functional aspects of violaxanthin-de-epoxidase, an enzyme of the xanthophyll cycle

The fast light-dependent xanthophyll transformations (<<xanthophyll-cycle>>) in thylakoids of green plants are catalysed by two enzymes: the strictly pH controlled and ascorbate-dependent (violaxanthin)de-epoxidase (VDE) and the NADPH(2)- and O-2-dependent (zeaxanthin)-epoxidase. The substrate specificity of the VDE was studied by using structurally different epoxy-xanthophylls. For this purpose the enzyme was isolated by a freeze-thaw treatment of thylakoid-vesicles of spinach at pH 7.5 and incubated with various epoxy-substrates in the presence of the cosubstrate ascorbate and a lipid factor (phosphatidylcholine) at pH 5.2. Under these conditions the K-m-value for the substrate violaxanthin (Vio) was 11.1 mu mol/L and for antheraxanthin (Ant) 5.3 mu mol/L. Only the epoxy-oxygen at the 5,6 (5',6') position of xanthophylls was cleaved by the VDE, whereas ring-spanning epoxides at position 3,6 (3',6') were not accessible to the enzyme. Moreover, the structure and chemical ligands of the second jonon ring were insignificant for the de-epoxidation of the 5,6-epoxy-groups of the first ring. Therefore, the epoxy-free (or also epoxy-containing) second jonon ring is not involved in the binding of the xanthophyll to the catalytic center and does not affect the enzyme reaction. However, due to a steric hindrance, any tested cis-configuration in the polyene chain of the xanthophylls, as well as the 8-oxy group in fucoxanthin, prevent the deepoxidation. The epoxy-xanthophylls available for the VDE are suggested to occur as rod-like, trans-configurated pigments within the lipid bilayer of thylakoids. When the mobile VDE is bound to the lumenal side of the thylakoid at pH less than or equal to 6.5 (Hager and Holocher, 1994), the epoxy-xanthophylls, guided by lipids, invade a fold, channel or tube-like structure of the enzyme, which functions as the catalytic center for the de-epoxidation. Functional aspects of the Vio-de-epoxidation and of the xanthophyll-cycle are discussed.