Occupancy and Functional Architecture of the Pigment Binding Sites of Photosystem II Antenna Complex Lhcb5

Abstract

Lhcb5 is an antenna protein that is highly conserved in plants and green algae. It is part of the inner layer of photosystem II antenna system retained in high light acclimated plants. To study the structure-function relation and the role of individual pigments in this complex, we (i) "knocked out" each of the chromophores bound to multiple (nine total) chlorophyll sites and (ii) exchanged the xanthophylls bound to the three xanthophyll sites. The occupancy and associated energy of the pigment binding sites were determined. The role of the individual pigments in protein folding, stability, energy transfer, and dissipation was studied in vitro. The results indicate that lutein has a primary role in the folding and stability of the complex, whereas violaxanthin and zeaxanthin have a negative effect on folding yield and stability, respectively. The data showed a distinct function for the L1 and L2 carotenoid binding sites, the former preferentially involved in gathering the excitation energy to chlorophyll a (Chl a), whereas the latter modulates the concentration of chlorophyll singlet excited states dependent on the xanthophylls bound to it, likely via an interaction with Chl-603. Our results also underscored the role of zeaxanthin and lutein in quenching the excitation energy, whereas violaxanthin was shown to be very effective in energy transfer. The characteristics of the isolated proteins were consistent with the observed role of Lhcb5 in vivo in catalyzing fluorescence quenching upon zeaxanthin binding.Sunlight energy is absorbed and converted into chemical energy by pigments bound to photosystems I and II (PSI 2 and PSII). Photosystems are composed of two moieties: (i) a reaction center devoted to the conversion of light energy into chemical energy and (ii) an antenna system that increases the capacity of light absorption and contributes to photoprotection (1). In photosynthetic eukaryotes, the antenna system is composed of members of a multigenic family called light-harvesting complexes (Lhc). In higher plants, 10 different subunits are associated with the photosystems, Lhca1-4 with PSI and Lhcb1-6 with PSII (2). All of these proteins share the same evolutionary origin and a common structural organization (3). Only the structure of LHCII, a trimer composed mainly of Lhcb1 (4), has been resolved at the atomic level, revealing the presence of three transmembrane and two amphipathic helices named, respectively, A-C and D-E (5). Each Lhcb1 polypeptide has been shown to coordinate four carotenoids and 14 Chl molecules. Two carotenoid binding sites, L1 and L2, 3 are close to helices A and B, respectively (6), and a third site, N1, specific for neoxanthin, is located in proximity to helix C (7). Finally, a peripheral and less stable binding site, V1, has been shown to accommodate violaxanthin and zeaxanthin (5,8,9).Other members of the Lhc protein family coordinate a variable number of pigments (8 -14 Chl and 2-4 carotenoid molecules, depending on the polypeptide (9 -14)) implying that some of the binding sites id...

Molecular Basis of Light Harvesting and Photoprotection in CP24

“…Unfortunately, none of the mutants targeting Chls close to the L2 site was stable enough to allow fluorescence time-resolved measurements. However, in CP26 the absence of Chl 603 induces a large increase of the fluorescence yield of the complex (40). Considering that Chl 603 is located in close contact with the carotenoid in L2, it is likely that it is responsible for the quenching also in CP24.…”

Light-harvesting and structural organization of Photosystem II: From individual complexes to thylakoid membrane

“…The pigment binding of most of the complexes was studied with the use of mutation analysis. Mutations of the putative Chl-binding residues followed by in vitro reconstitution [90] has led to the production of complexes lacking individual chromophores, allowing the characterization of each chromophore in each binding site [91][92][93][94][95][96]. This analysis has revealed that the biochemical and spectroscopic properties of Chls in several of the binding sites are conserved across the Lhc family.…”

“…1) was reconstructed [126] using the crystal structures of PSII core [44] and LHCII [78]. For the minor antenna complexes, the structure of a monomer of LHCII was used while the pigment composition/occupancy was assigned based on the results of mutation analysis experiments on in vitro reconstituted complexes [91,92,95,96]. The model shows that LHCII S is connected directly with CP43 and that Chls 612/611 of one of the monomers are facing Chl 43 of CP43.…”

Lutein Accumulation in the Absence of Zeaxanthin Restores Nonphotochemical Quenching in the Arabidopsis thaliana npq1 Mutant

“…CP24, CP26, and CP29 complexes were refolded in vitro from apoproteins expressed in E. coli and chlorophylls a and b plus lutein only or a total carotenoid mix. The resulting complexes were characterized (Table 2) by having lutein in both carotenoid binding sites L1 and L2 (LL complexes), whereas in the control complexes, the L2 binding site could be occupied by lutein, violaxanthin, or neoxanthin (LNV complexes) (Pagano et al, 1998;Ruban et al, 1999;Ballottari et al, 2009), except for CP24, which cannot bind neoxanthin either in vivo or in vitro (Pagano et al, 1998). LHCII trimers were purified from the wild type or the chy1 chy2 lut5 mutant, which has lutein as the only xanthophyll (Fiore et al, 2006).…”