Chlorophyll triplet quenching and photoprotection in the higher plant monomeric antenna protein Lhcb5.

In oxygenic photosynthetic organisms, chlorophyll triplets are harmful excited states readily reacting with molecular oxygen to yield the reactive oxygen species (ROS) singlet oxygen. Carotenoids have a photoprotective role in photosynthetic membranes by preventing photoxidative damage through quenching of chlorophyll singlets and triplets. In this work we used mutation analysis to investigate the architecture of chlorophyll triplet quenching sites within Lhcb5, a monomeric antenna protein of Photosystem II. The carotenoid and chlorophyll triplet formation as well as the production of ROS molecules were studied in a family of recombinant Lhcb5 proteins either with WT sequence, mutated into individual chlorophyll binding residues or refolded in vitro to bind different xanthophyll complements. We observed a site-specific effect in the efficiency of chlorophyll-carotenoid triplet-triplet energy transfer. Thus chlorophyll (Chl) 602 and 603 appear to be particularly important for triplet-triplet energy transfer to the xanthophyll bound into site L2. Surprisingly, mutation on Chl 612, the chlorophyll with the lower energy associated and in close contact with lutein in site L1, had no effect on quenching chlorophyll triplet excited states. Finally, we present evidence for an indirect role of neoxanthin in chlorophyll triplet quenching and show that quenching of both singlet and triplet states is necessary for minimizing singlet oxygen formation

Photoprotection in the antenna complexes of Photosystem II: role of individual xanthophylls in chlorophyll triplet quenching

In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins

Singlet and triplet state transitions of carotenoids in the antenna complexes of higher-plant photosystem I.

In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T 12S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while \u3b2-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The \u201cred\u201d lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T 12S maxima:\u2009 one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The \u201cblue\u201d-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T 12S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2 12Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale

Dynamics of zeaxanthin binding to the photosystem II monomeric antenna protein Lhcb6 (CP24) and modulation of its photoprotection properties.

Lhcb6 (CP24) is a monomeric antenna protein of photosystem II, which has been shown to play special roles in photoprotective mechanisms, such as the Non-Photochemical Quenching and reorganization of grana membranes in excess light conditions. In this work we analyzed Lhcb6 in vivo and in vitro: we show this protein, upon activation of the xanthophyll cycle, accumulates zeaxanthin into inner binding sites faster and to a larger extent than any other pigment-protein complex. By comparative analysis of Lhcb6 complexes violaxanthin or zeaxanthin binding, we demonstrate that zeaxanthin not only down-regulates chlorophyll singlet excited states, but also increases the efficiency of chlorophyll triplet quenching, with consequent reduction of singlet oxygen production and significant enhancement of photo-stability. On these bases we propose that Lhcb6, the most recent addition to the Lhcb protein family which evolved concomitantly to the adaptation of photosynthesis to land environment, has a crucial role in zeaxanthin-dependent photoprotection

FTIR Study of the Non-heme Iron Binding Site in Photosystem II: Bicarbonate 13C-Labeling and Exchange by Glycolate

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