Molecular Basis of Light Harvesting and Photoprotection in CP24

Abstract

CP24 is a minor antenna complex of Photosystem II, which is specific for land plants. It has been proposed that this complex is involved in the process of excess energy dissipation, which protects plants from photodamage in high light conditions. Here, we have investigated the functional architecture of the complex, integrating mutation analysis with time-resolved spectroscopy. A comprehensive picture is obtained about the nature, the spectroscopic properties, and the role in the quenching in solution of the pigments in the individual binding sites. The lowest energy absorption band in the chlorophyll a region corresponds to chlorophylls 611/612, and it is not the site of quenching in CP24. Chlorophylls 613 and 614, which are present in the major lightharvesting complex of Photosystem appear to be absent in CP24. In contrast to all other light-harvesting complexes, CP24 is stable when the L1 carotenoid binding site is empty and upon mutations in the third helix, whereas mutations in the first helix strongly affect the folding/stability of the pigment-protein complex. The absence of lutein in L1 site does not have any effect on the quenching, whereas substitution of violaxanthin in the L2 site with lutein or zeaxanthin results in a complex with enhanced quenched fluorescence. Triplet-minus-singlet measurements indicate that zeaxanthin and lutein in site L2 are located closer to chlorophylls than violaxanthin, thus suggesting that they can act as direct quenchers via a strong interaction with a neighboring chlorophyll. The results provide the molecular basis for the zeaxanthin-dependent quenching in isolated 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.…”

“…For example, the four Chl-binding sites located in the center of the molecule (602, 603, 610 and 612, nomenclature according to [78]) accommodate Chl a in all antenna complexes, with Chls 602-603 absorbing around 675 nm and Chl 610-612, absorbing around 680 nm, thereby representing the lowest energy state of the system [92,94]. On the other hand, the domain including helix C has a higher tendency to coordinate Chl b [92,97], although the occupancy of some of the sites by either Chl a or Chl b varies for the different complexes and it has been suggested that this difference is mainly related to be possibility/impossibility for the formyl group of Chl b to form H bonds [96], thus stabilizing the Chl b binding. While the L1 site of all complexes coordinates lutein, the L2 site accommodates lutein in LHCII and CP26, and violaxanthin in CP29 and CP24.…”

“…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.…”

<em>In Vitro</em> Reconstitution of Light-harvesting Complexes of Plants and Green Algae

“…For most analyses this is not a problem, as the apoprotein does not contain pigments and thus does not interfere with the functional measurements. However, in case it is necessary to fully remove the apoprotein from the fraction containing the reconstituted complex (for example, to calculate the pigment to protein stoichiometry), an anionic exchange column can be used (see Passarini et al 2009 29 for details).…”

“…The importance of different protein domains on the stability and folding of the complexes, or their involvement in the protein-protein interactions, have been determined by truncating the apoprotein or performing random mutagenesis 8,[41][42][43][44] . Single amino acid residues important for the coordination of different pigments can be altered through site-directed mutagenesis in order to analyze the properties of individual pigments or assess their contribution to the function and stability of the complex 10,28,29,[45][46][47][48][49][50][51][52] . Figure 6 shows reconstituted Lhcb4 (CP29) with a mutation of the histidine at position 216 53 .…”