Atanaska Andreeva

Changes in the energy distribution between chlorophyll-protein complexes of thylakoid membranes from pea mutants with modified pigment content. I. Changes due to the modified pigment content.

The low-temperature (77 K) emission and excitation chlorophyll fluorescence spectra in thylakoid membranes isolated from pea mutants were investigated. The mutants have modified pigment content, structural organization, different surface electric properties and functions [Dobrikova et al., Photosynth. Res. 65 (2000) 165]. The emission spectra of thylakoid membranes were decomposed into bands belonging to the main pigment protein complexes. By an integration of the areas under them, the changes in the energy distribution between the two photosystems as well as within each one of them were estimated. It was shown that the excitation energy flow to the light harvesting, core antenna and RC complexes of photosystem II increases with the total amount of pigments in the mutants, relative to the that to photosystem I complexes. A reduction of the fluorescence ratio between aggregated trimers of LHC II and its trimeric and monomeric forms with the increase of the pigment content (chlorophyll a, chlorophyll b, and lutein) was observed. This implies that the closer packing in the complexes with a higher extent of aggregation regulates the energy distribution to the PS II core antenna and reaction centers complexes. Based on the reduced energy flow to PS II, i.e., the relative increased energy flow to PS I, we hypothesize that aggregation of LHC II switches the energy flow toward LHC I. These results suggest an additive regulatory mechanism, which redistributes the excitation energy between the two photosystems and operates at non-excess light intensities but at reduced pigment content.

Temperature dependence of resonance Raman spectra of carotenoids

To understand the mechanism of the photoprotective and antioxidative functions of carotenoids, it is essential to have a profound knowledge of their excited electronic and vibronic states. In the present study we investigate the most powerful antioxidants: β-carotene and lutein by means of resonance Raman spectroscopy. The aim was to study in detail their Raman spectra in solution at room temperature and their changes as a function of temperature. To measure the spectra in their natural environment pyridine has been used as a solvent. It has been chosen because of its polarizability (n=1.5092) which is close to that of membrane lipids and proteins. The temperature dependence of the most intensive ν(1) band in the range from 77 K to 295 K at 514.5 nm excitation has been obtained. It was found that in pyridine the CC stretching frequency, its intensity, line shape, and line width are very sensitive to the temperature (the sensitivity being different for the two studied carotenoids). The observed linear temperature dependence of the CC stretching frequency is explained by a mechanism involving changes of the vibronic coupling and the extent of π-electron delocalization. The different behavior of the temperature-induced broadening of the ν(1) band and its intensity for the two studied carotenoids can be associated with the different nature of their solid matrices: glassy for β-carotene and crystalline-like for lutein, owing to their different chemical structures.

Selective Photobleaching of Chlorophylls and Carotenoids in Photosystem I Particles under High-Light Treatment

Photosystem I particles (PSI‐200) isolated from spinach leaves were studied by means of absorbance, 77K fluorescence and resonance Raman (RR) spectroscopy. The aim was to obtain better insight into the changes of the pigment spectral properties in those particles during prolonged exposure to high‐light intensities and to reveal the involvement of these pigments in the photoprotection of the PSI. During prolonged exposure to high‐light intensities of spinach PSI particles, a loss of a significant amount of photosynthetic pigments was observed. It was shown that various pigments exhibited different susceptibility to photodamage. In addition to bleaching of chlorophyll a (Chl a), bleaching of carotenoids was also clearly observed. RR technique allowed us to recognize the type and conformation of photobleached carotenoid molecules. Raman data revealed a nearly full photobleaching of the long‐wavelength lutein molecules. The observed similar bleaching rate of the lutein molecules and the most‐red shifted long‐wavelength Chl a, located in the antenna membrane protein Lhca4, suggested that these molecules are located closely. Our results showed that the photobleached antenna pigments and especially luteins and the most long‐wavelength absorbing chlorophylls are involved in photoprotection of PSI core complex.

Effect of modification of light-harvesting complex II on fluorescence properties of thylakoid membranes of Arabidopsis thaliana.

The 77 K chlorophyll fluorescence spectra of Arabidopsis thaliana mutants deficient in lipid fatty acid desaturation have been used in order to further explore the influence of the modification of LHC II after mutation and proteolitic treatment on the energy transfer between the chlorophyll-protein complexes, as well as on the structure-function relationship in the supramolecular complex of Photosystem II. The gaussian decomposition and analysis of the fluorescence bands associated with PS II complex show the controversial action of the trypsin in the investigated thylakoid membranes. This reveals that the organization of PS II complexes is different in the wild type and both mutants indicating altered connection between the LHC II and the RC core complexes of PS II in both mutants. The results obtained demonstrate that different amounts of oligomer and monomer forms of LHC II in the mutants (LK3 and JB67), arising from lipid modification, are responsible for different proteolytic action in their thylakoid membranes.

Carotenoid–Lipid Interactions

Abstract Carotenoids are the most widely spread pigments, both in the plant and animal kingdom, that perform numerous important physiological functions. In photosynthetic organisms, carotenoids are involved in the processes of light harvesting, photoprotection, and electron transfer, serve as scavengers of reactive oxygen species, and perform a structural role in photosynthetic membranes. In animals and humans, carotenoids perform a number of functions, of which the best established are their provitamin A activity and antioxidant properties. A great number of investigations devoted to understanding the basic principles of carotenoid impact on membrane organization, dynamics, physical and mechanical properties, as also their antioxidant activities, have been performed using model membranes. In this review, the structural and spectral characteristics of carotenoids are discussed, as well as the most frequently applied methods of investigation, with special attention to pigment–lipid interactions. The carotenoid–lipid interactions are characterized in several aspects: degree of integration, orientation and conformation of the pigment molecules into lipid membranes, and the impact of carotenoids on the thermotropic phase behavior and dynamics of the lipid bilayer.

Resonance Raman Studies of Carotenoid Molecules Within Photosystem I Particles

ABSTRACT In higher plants, carotenoid molecules play particularly important roles in harvesting light, stabilizing protein structures, regulating energy flow, and dissipating excess energy not required by the organism for photosynthesis. Low-temperature resonance Raman spectroscopy was used to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I particles. Photosystem I is a supercomplex of a reaction centre, core and light-harvesting complexes. Resonance Raman spectra of carotenoid molecules have four main groups of intense bands, which provide information on the conformation and configuration of these molecules. The most sensitive to their molecular configurations relaxed or distorted is v4 band exhibiting an increase in both intensity and structure indicating out-of-plane distortion of the molecule. During prolonged exposure to high-light intensities various pigments in Photosystem I exhibited different susceptibility to photodestruction. Resonance Raman technique allowed us to recognize the type and conformation of photobleached carotenoid molecules and to reveal the involvement of these pigments in the photoprotection. Raman data revealed a nearly full photobleaching of the long-wavelength lutein molecules. The observed similar bleaching rate of the lutein molecules and the most red-shifted long-wavelength chlorophyll a, located in the antenna membrane protein Lcha4, suggested that these molecules are located closely. Our results showed that the photobleached antenna pigments and especially luteins and the most long-wavelength absorbing chlorophylls are involved in photoprotection of PSI core complex. We investigated the effect of histidine on the photobleaching of pigments in isolated particles of photosystem I. Our preliminary results showed that histidine reduces the photobleaching of antenna pigments and especially luteins and the most long-wavelength absorbing chlorophylls located in PSI antenna complex.

Polarized fluorescence spectroscopy of oriented isolated spinach Photosystem I particles.

The fluorescence anisotropy of Photosystem I (PS I) particles, isolated from spinach chloroplasts and containing approximately 200 chlorophyll molecules per reaction center, is investigated at low temperatures. The particles are oriented by squeezing in polyacrylamid gels with different macroscopic deformation parameters. Fluorescence anisotropy is measured upon steady state excitation with a laser line at 632.8 nm. A formula for the fluorescence anisotropy in oriented Photosystem I particles is applied for a different polarization of the linearly polarized exciting light. Our calculations are based on the consideration of the Photosystem I complex as a triple-chromophore complex: the absorbing chlorophyll molecules (chl), belonging to the light-harvesting complex of PS I (LHC), and two fluorophores, emitting at 720 nm (F720) and at 735 nm (F735), respectively. Using polarized fluorescence spectroscopy with a different polarization of the linearly polarized exciting light, the experimental dependence of the fluorescence anisotropy on this polarization is obtained. Based on this dependence and applying the derived formula, as a first approximation, both the orientation of the photosynthetic pigments with respect to the membrane and their mutual orientation are determined in PS I particles. As the most probable average orientational angles in PS I particles, we obtained the values 35°÷ 50°, 50°÷ 60°, and 65°÷ 67° for the absorbing dipoles of chl and for the emission dipoles of F720 and F735, respectively, with the normal of the plane of the membrane. For their mutual orientation, the following limits are determined: 10°÷ 20°, 40 ± 2°, 20°÷ 30° for the angles between chl and F720, chl and F735; and F720 and F735, correspondingly. Of course, the values of the angles estimated as a result of our study are an average value of all angles of the excited transitions and must be considered as their first approximation valid for the idealized case when all PS I particles are oriented in gel.

Photoinduced changes in photosystem II pigments

The photosynthetic apparatus in higher plants performs two seemingly opposing tasks: efficient harvest of sunlight, but also rapid and harmless dissipation of excess light energy as heat to avoid deleterious photodamage. In order to study this process in pigment-protein supercomplexes of photosystem II (PSII), 77 K fluorescence and room temperature resonance Raman (RR) spectroscopy were applied to investigate the changes in structure and spectral properties of the pigments in spinach PSII membranes. The high-light treatment results in a strong quenching of the fluorescence (being largest when the excitation is absorbed by carotenoids) and a red-shift of the main maximum. Decomposition of the fluorescence spectra into four bands revealed intensive quenching of F685 and F695 bands, possible bleaching of chlorophyll a, enhanced extent of light harvesting complexes (LHCII) aggregation and increased energy transfer to aggregated LHCII. The analysis of RR spectra revealed the predominant contribution of s-carotene (s-Car) upon 457.8 and 488 nm excitations and lutein (Lut) at 514.5 nm. During prolonged exposure to strong light no significant bleaching of s-Car and weak photobleaching of Lut is observed. The results will contribute to the efforts to produce more efficient and robust solar cells when exposed to fluctuations in light intensity.

Light induced changes in Raman scattering of carotenoid molecules in Photosystem I particles

The photosynthetic antenna systems are able to regulate the light energy harvesting under different light conditions by dynamic changes in their protein structure protecting the reaction center complexes. The changes modulate the electronic structure of the main antenna pigments (chlorophylls and carotenoids) and distort the characteristic planar structure of carotenoids, allowing their forbidden out of plane vibrations. Electronic absorption and low-temperature resonance Raman spectroscopy were used to study the changes in composition and spectral properties of the major carotenoids in spinach Photosystem I particles due to high light treatment. The duration of the applied intensity of the white light (1800 &mgr;E m-2 s-1) was 30, 60 and 120 minutes. We used Raman scattering in an attempt to recognize the type and conformation of photobleached carotenoid molecules. The resonance Raman spectra were measured at 488 and 514.5 nm, coinciding with the absorption maximum positions of the carotenoids neoxanthin and lutein, correspondingly. The results revealed nearly a full photobleaching of the long wavelength lutein molecules, whereas the bleaching of neoxantin molecules is negligible. The involvement of these changes in the photoprotection and photoinactivation of the Photosystem I particles was discussed.

Relation Between Changes in Pigments’ Spectral Properties and Structural Distortions of Pigment Protein Complexes (abstract)

Scientists continue to investigate photosynthesis—nature’s process to efficiently regulate and store energy. To explore the mechanisms of regulation we used fluorescence, resonance Raman spectroscopy, and biochemical preparative methods. For detailed analysis we applied decomposition of the low temperature steady‐state fluorescence spectra. This allowed us to estimate the emission of distinct pigment protein complexes (PPC) and evaluate the fluorescence of various aggregation forms of the main light harvesting complex (LHCII), which plays a major role in the studied mechanisms. Resonance Raman spectroscopy revealed with precision the relation between changes in pigments’ spectral properties and structural distortions of PPC. It was shown that aggregation of LHCII led to out‐of‐plane distortion, not only of neoxanthin, but also of lutein molecules. This was enhanced when the complex was embedded in thylakoid membranes (TM). It was suggested that lutein molecules are more closely related to the process of a...