Abstract
The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important.
Highlights
Carotenoids are involved in many aspects of higher plants photosynthesis
We report the results of a detailed analysis of the chlorophyll a/b/xanthophyll protein CP29 (Lhcb4) with respect to the xanthophyll stoichiometry, location, and function as well as of the sequence determinants involved in their interaction with the polypeptide chain
The first step of this work consisted into the determination of the number of carotenoids bound to each CP29 polypeptide
Summary
Chlorophyll; Car, carotenoid; Lhc, light-harvesting complex; L, lutein; LD, linear dichroism; N, neoxanthin; V, violaxanthin; WT, wild type; PSII, Photosystem II; HPLC, high performance liquid chromatography; CTL, control. The reason why Lhc proteins require a number of xanthophyll species while reaction center proteins only require -carotene is not completely understood; these carotenoid species have very similar physico-chemical properties, enabling, in each case, efficient light harvesting, triplet quenching, and singlet oxygen scavenging (11). Despite the high homology in the transmembrane regions, which suggests a similar folding, the number of xanthophyll binding sites, their selectivity, and their occupancy in the structure seem to be different in each Lhc gene product. Theless, four short sequences, located in the hydrophilic domains and consisting into four hydrophobic amino acids and a charged residue, could be involved in the binding by forming a hydrophobic pocket hosting xanthophylls end-rings, whereas the charged side chain was supposed to interact with oxygenated ring substituents (20). We report the results of a detailed analysis of the chlorophyll a/b/xanthophyll protein CP29 (Lhcb4) with respect to the xanthophyll stoichiometry, location, and function as well as of the sequence determinants involved in their interaction with the polypeptide chain
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