De-epoxidation of violaxanthin after reconstitution into different carotenoid binding sites of light-harvesting complex II

In higher plants, the de-epoxidation of violaxanthin (Vx) to antheraxanthin and zeaxanthin is required for the pH-dependent dissipation of excess ligh t energy as heat and by that process plays an important role in the protect ion against photo-oxidative damage. The de epoxidation reaction was investi gated in an in vitro system using reconstituted light-harvesting complex II (LHCII) and a thylakoid raw extract enriched in the enzyme Vx de-epoxidase . Reconstitution of LHCII with varying carotenoids was performed to replace lutein and/or neoxanthin, which are bound to the native complex, by Vx. Re combinant LHCII containing either 2 lutein and 1 Vx or 1.6 Vx and 1.1 neoxa nthin or 2.8 Vx per monomer were studied. Vx de epoxidation was inducible f or all complexes after the addition of Vx de epoxidase but to different ext ents and with different kinetics in each complex. Analysis of the kinetics indicated that the three possible Vx binding sites have at least two, and p erhaps three, specific rate constants for de-epoxidation. In particular, Vx bound to one of the two lutein binding sites of the native complex, most l ikely L1, was not at all or only at a slow rate convertible to Zx. In reiso lated LHCII, newly formed Zx almost stoichiometrically replaced the transfo rmed Vx, indicating that LHCII and Vx de-epoxidase stayed in close contact during the de-epoxidation reactions and that no release of carotenoids occu rred.