De-epoxidation of Violaxanthin in Light-harvesting Complex I Proteins

Abstract

The conversion of violaxanthin (Vx) to zeaxanthin (Zx) in the de-epoxidation reaction of the xanthophyll cycle plays an important role in the protection of chloroplasts against photooxidative damage. Vx is bound to the antenna proteins of both photosystems. In photosystem II, the formation of Zx is essential for the pH-dependent dissipation of excess light energy as heat. The function of Zx in photosystem I is still unclear. In this work we investigated the de-epoxidation characteristics of light-harvesting complex proteins of photosystem I (LHCI) under in vivo and in vitro conditions. Recombinant LHCI (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids. All four LHCI proteins exhibited unique de-epoxidation characteristics. An almost complete Vx conversion to Zx was observed only in Lhca3, whereas Zx formation in the other LHCI proteins decreased in the order Lhca4 > Lhca1 > Lhca2. Most likely, these differences in Vx de-epoxidation were related to the different accessibility of the respective carotenoid binding sites in the distinct antenna proteins. The results indicate that Vx bound to site V1 and N1 is easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially and/or with the slower kinetics convertible to Zx. The de-epoxidation properties determined for the monomeric recombinant proteins were consistent with those obtained for isolated native LHCI-730 and LHCI-680 in the same in vitro assay and the de-epoxidation state found under in vivo conditions in native LHCIs.

Pigment Binding, Fluorescence Properties, and Oligomerization Behavior of Lhca5, a Novel Light-harvesting Protein

“…To find out whether this is also true for Lhca5, we compared the pigment stoichiometry of r-Lhca5 with those published for r-Lhca1-r-Lhca4 (21). From Table I it is obvious that r-Lhca5 binds pigments in a unique stoichiometry, although there are similarities to other r-Lhcs.…”

“…Recombinant Lhca2 and Lhca4 bind only two carotenoids per monomer (5,7,19,21), in contrast to three in Lhca3 (19,21,24). Whether Lhca1 binds two or three carotenoids is still under debate (5,15,19).…”

“…Pigment analyses of native LHCIs (e.g. 5,19,[21][22][23] and reconstituted Lhca1-Lhca4 (r-Lhca1-r-Lhca4) (5,7,19,21,24) revealed that all four Lhca proteins coordinate Chl a, Chl b, lutein (lut), violaxanthin (vio), and ␤-carotene (␤-car) at different ratios. In addition, the Chl a/b ratio of LHCs seems to correlate with the chlorophyll species required for LHC formation (17-19, 24 -27).…”

Expression of LHC Genes and their Relation to Photo-Oxidative Stress Tolerance Tolerance in Solanum lycopersicum L. and Solanum chilense (Dunal) Reiche

“…high-light) there is a differential expression of LHCI genes (Bailey et al, 2001;Ensminger et al, 2004;Gáspár et al, 2006). On the other hand, Wehner et al (2004) suggested that the zeaxanthin formed in LHCI proteins is involved in an antioxidative mechanism implicated in suppression of ROS-triggered lipid peroxidation rather than heat energy dissipation (NPQ), which could be consistent with our observations on the accumulation of zeaxanthin ( Figure 3B) which can function as antioxidant, as evidenced by the amount of peroxidized lipids (Figure 4) and the invariable value of NPQ ( Figure 2C). …”

“…On the other hand, zeaxanthin also appears to have a role in tolerance to photo-oxidative stress protecting membranes against lipid peroxidation by a different mechanism from NPQ (Lichtenthaler and Schnidler, 1992). Carotenoids are essential components of light-harvesting chlorophyll (chl) a/b binding (LHC) protein (Wehner et al, 2004) and therefore the chemical transformation of the xanthophyll cycle induces a reorganization in light-harvesting complex (LHC) proteins (i.e. from a state of efficient light harvesting to a state of high thermal energy dissipation; Nelson and Yocum, 2006).…”