Carotenoid-binding Sites of the Major Light-harvesting Complex II of Higher Plants

Abstract

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in 1 O 2 * scavenging.Light energy for the photosynthesis of green plants is collected by an antenna system composed of many homologous proteins belonging to the Lhc multigene family (1). These pigment-protein complexes are organized around photosynthetic reaction centers to form supramolecular complexes embedded into the thylakoid membrane, accounting for ϳ70% of the pigment involved in plant photosynthesis. LHCII 1 is the most abundant light-harvesting complex in higher plants. The structure of this complex has been resolved at 3.4 Å by electron microscopy (2) and is formed by three hydrophobic transmembrane helices connected by hydrophilic loops and an amphipathic helix exposed to the luminal surface of the membrane. LHCII coordinates 7 Chl a, 5 Chl b, and 3-4 carotenoid molecules (lutein, neoxanthin, and a substoichiometric amount of violaxanthin) depending on the genotype (3) and the physiological state of the plant (4). In the structural model of LHCII (2), 2 xanthophyll molecules have been located in the center of the complex, forming an internal cross-brace interacting with helices A and B. These appear to be crucial for protein stabilization, as suggested by the fact that a stable LHCII complex cannot be obtained without lutein in refolding experiments (5, 6). Although the 2 central molecules were tentatively assigned to lutein (2), the structural resolution is insufficient for their identification and for the location of the xanthophyll molecule with respect to the 2 detected by structural analysis. The nature and location of the binding site for the third xanthophyll molecule are presently unknown. It is also unclear if the individual binding sites have different affinities for the three xanthophyll species.Carotenoids have at least five different roles in photosynthesis: 1) light h...

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“…The photobleaching kinetics of pigment-protein complexes was measured as described (Croce et al, 1999). Samples were cooled at 10°C.…”

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