How do xanthophylls protect lipid membranes from oxidative damage?

Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes

Fluorescence of zeaxanthin and violaxanthin in aggregated forms

The fluorescence emission, the absorption, and the fluorescence excitation spectra of zeaxanthin (Z) and violaxanthin (V) aggregates formed in a water-ethanol solvent system have been measured. Fluorescence quantum yield for zeaxanthin and violaxanthin are: ϕ F(Z) = 4.3 × 10 −4 and ϕ F(V) = 1.7 × 10 −3. The fluorescence lifetimes are τ F(Z) = 1.06 ± 0.07 ns and τ F(V) = 0.90 ± 0.10 ns. The calculated natural radiative lifetimes, τ 0(Z) = 2.6 × 10 −6 s and τ 0(V) = 5.3 × 10 −7 s correspond well to the literature values estimated for 1A * g state of β-carotene by indirect measurements. The energy of the 1A * g state of the carotenoid aggregates has been measured as 17860 cm −1. The fluorescence of these carotenoid aggregates is related to the radiative deactivation of both the 1B u electronic singlet state and the symmetrically forbidden lowest excited electronic singlet state 1A * g

Increased heat emission and its relationship to the xanthophyll cycle in pea leaves exposed to strong light stress

In vivo thermal energy dissipation was photoacoustically monitored in pea leaves before and after strong light treatment. Concomitant with the conversion of the carotenoid violaxanthin into zeaxanthin, a marked increase in the heat emission signal was observed in the light-stressed leaves. However, when the xanthophyll cycle was blocked by dithiothreitol, the photothermal signal still increased, indicating that there was no causal relationship between these two phenomena. Increased heat emission was shown to result from pigment uncoupling, which caused the inhibition of the energy transfer from carotenoids to chlorophyrs. It was also observed that the maintenance of a very low zeaxanthin level by dithiothreitol led to an increase in both the oxygen evolution and the photothermal components of the photoacoustic signal in control leaves and to a strong increase in lipid degradation in light-stressed leaves. These results may suggest that a possible function of the xanthophyll cycle is to provide an accessory pigment (violaxanthin) in weak light and to furnish the hpid matrix of the thylakoid membranes with an efficient photoprotector (zeaxanthin) in strong light

The role of xanthophylls in the supramolecular organization of the photosynthetic complex LHCII in lipid membranes studied by high-resolution imaging and nanospectroscopy

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular beta-structures. Meanwhile, the molecules of violaxanthin act as "molecular spacers" preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle

Photothermal Microscopy: Imaging of Energy Dissipation From Photosynthetic Complexes

An idea of a photothermal imaging microscopy (PTIM) is proposed, along with its realization based on a dependence of fluorescence anisotropy of dye molecules on heat emission in their nearest vicinity. Erythrosine B was selected as a fluorophore convenient to report thermal deactivation of the excited pigment–protein complex isolated from the photosynthetic apparatus of plants (LHCII), owing to the relatively large spectral gap between the fluorescence emission bands of chlorophyll <i>a</i> and a probe. Comparison of the simultaneously recorded images based on fluorescence lifetime of LHCII and fluorescence anisotropy of erythrosine shows a high rate of thermal energy dissipation from the aggregated forms of the complex and, possibly, thermal energy transmission along the protein supramolecular structures. Relatively high resolution of this novel microscopic technique, comparable to the fluorescence lifetime microscopy, enables its application in a nanoscale imaging and in nanothermography

Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product

MAGE (melibiose-derived advanced glycation end-product) is the glycation product generated in the reaction of a model protein with melibiose. The in vivo analog accumulates in several tissues; however, its origin still needs explanation. In vitro MAGE is efficiently generated under dry conditions in contrast to the reaction carried in an aqueous solvent. Using liquid chromatography coupled with mass spectrometry, we analyzed the physicochemical properties and structures of myoglobin glycated with melibiose under different conditions. The targeted peptide analysis identified structurally different AGEs, including crosslinking and non-crosslinking modifications associated with lysine, arginine, and histidine residues. Glycation in a dry state was more efficient in the formation of structures containing an intact melibiose moiety (21.9%) compared to glycation under aqueous conditions (15.6%). The difference was reflected in characteristic fluorescence that results from protein structural changes and impact on a heme group of the model myoglobin protein. Finally, our results suggest that the formation of in vitro MAGE adduct is initiated by coupling melibiose to a model myoglobin protein. It is confirmed by the identification of intact melibiose moieties. The intermediate glycation product can further rearrange towards more advanced structures, including cross-links. This process can contribute to a pool of AGEs accumulating locally in vivo and affecting tissue biology