The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants: A SPECIFIC REQUIREMENT FOR CAROTENOIDS WITH A 3-HYDROXY-β-END GROUP

Denise Phillip,Stephan Hobe,Harald Paulsen,Peter Molnar,Hideki Hashimoto,Andrew J Young

doi:10.1074/jbc.m202002200

Abstract

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.

Highlights

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved
Binding of Native Xanthophylls—A comparison of the procedures for the in vitro reconstitution of the 28-kDa polypeptide from pea overexpressed in E. coli and the high stringency procedure utilizing solubilized spinach thylakoid polypeptides was initially performed
The data obtained in the current study reveal that for all three procedures available for reconstitution of LHCIIb from higher plants, a maximum of three molecules of xanthophyll are bound per monomer of reassembled LHCIIb

Summary

The abbreviations used are

Chlorophyll; HPLC, high performance liquid chromatography; LHC, light-harvesting complex; LHCIIb, major light-harvesting complex of photosystem II of higher plants. The pigments show affinity to the native (trimeric) complex in the order Chl b Ͼ neoxanthin Ͼ Chl a Ͼ lutein Ͼ zeaxanthin Ͼ violaxanthin This affinity can alter upon monomerization of the complex (e.g. neoxanthin is tightly bound to trimeric LHCIIb but not to monomers of the complex, suggesting that it occupies a site in the interior of the trimeric array). Yields of these complexes were poor, and the complexes were less stable than the complex assembled using the full native complement of pigments These studies indicated that lutein was an essential component for the assembly of LHCIIb. More recent evidence suggests that, in vivo, there is some plasticity with regard to the specificity of xanthophyll binding and that some plants deficient in lutein are viable [16]. The ability of a range of xanthophylls with different structural features including chain length, end group type and substituents, and in-chain substitutions together with a number of geometric and optical isomers to facilitate reassembly of LHCIIb was determined

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION