J. Hála

Excitation Energy Transfer Dynamics and Excited-State Structure in Chlorosomes of Chlorobium phaeobacteroides

The excited-state relaxation within bacteriochlorophyll (BChl) e and a in chlorosomes of Chlorobium phaeobacteroides has been studied by femtosecond transient absorption spectroscopy at room temperature. Singlet-singlet annihilation was observed to strongly influence both the isotropic and anisotropic decays. Pump intensities in the order of 10(11) photons x pulse(-1) x cm(-2) were required to obtain annihilation-free conditions. The most important consequence of applied very low excitation doses is an observation of a subpicosecond process within the BChl e manifold (approximately 200-500 fs), manifesting itself as a rise in the red part of the Q(y) absorption band of the BChl e aggregates. The subsequent decay of the kinetics measured in the BChl e region and the corresponding rise in the baseplate BChl a is not single-exponential, and at least two components are necessary to fit the data, corresponding to several BChl e-->BChl a transfer steps. Under annihilation-free conditions, the anisotropic kinetics show a generally slow decay within the BChl e band (10-20 ps) whereas it decays more rapidly in the BChl a region ( approximately 1 ps). Analysis of the experimental data gives a detailed picture of the overall time evolution of the energy relaxation and energy transfer processes within the chlorosome. The results are interpreted within an exciton model based on the proposed structure.

Kinetics and efficiency of excitation energy transfer from chlorophylls, their heavy metal-substituted derivatives, and pheophytins to singlet oxygen

Time-resolved measurements of the singlet oxygen infrared (1269 nm) luminescence were used to follow the kinetics and efficiency of excitation energy transfer (EET) between chlorophyll (Chl) derivatives and oxygen in acetone. The studied pigments were Mg-Chl a and b and their heavy metal (Cu(2+) and Zn(2+))-substituted analogues, as well as pheophytin (Pheo) a and b. The efficiency of EET from chlorophyll to oxygen was highly dependent on the central ion in the pigment. Cu-Chl a and Cu-Chl b had the lowest efficiencies of singlet oxygen production, while Pheo a had a higher one, and Zn-Chl a had a similar one compared to Mg-Chl a. Also the side chain (position C-7, i.e. Chl a vs. Chl b) influenced the efficiency of singlet oxygen formation. In the case of square-planar complexes like Cu-Chl and Pheo, EET was more efficient in the Chl a derivatives than in those of Chl b; the opposite effect was observed in the case of the five- or six-coordinated Mg-Chl and Zn-Chl. As for the lifetime of the Chl triplet state, the most striking difference to Mg-Chl again was found in the case of Cu-Chls, which had much shorter lifetimes. Furthermore, the central ion in Chl affected the physical quenching of singlet oxygen: its efficiency was decreasing from Mg-Chl through Zn-Chl over Cu-Chl to Pheo. The results are discussed in the context of the oxidative stress accompanying heavy metal-induced stress in plants.

Phosphorescence of singlet oxygen and meso-tetra(4-sulfonatophenyl)porphin: time and spectral resolved study

Highly sensitive spectroscopic experimental setup was built to detect infrared luminescence with both time and spectral resolution. In this contribution, phosphorescence of meso-tetra(4-sulphonatophenyl)porphin, which is used as a photosensitizer for photodynamic therapy of cancer, and emission of singlet oxygen were studied. Two-dimensional matrices of data (counts as a function of time and wavelength) were obtained. From these matrices, 1.7 μs lifetime of the photosensitizer triplet state and the same rise-time of the singlet oxygen emission were resolved. Further, 3.7 μs lifetime of singlet oxygen phosphorescence was determined.

Time-resolved Luminescence and Singlet Oxygen Formation After Illumination of the Hypericin–Low-density Lipoprotein Complex

Time‐resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low‐density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self‐quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime (∼0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime (∼8 μs) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.

Effect of Carotenoids and Monogalactosyl Diglyceride on Bacteriochlorophyll c Aggregates in Aqueous Buffer: Implications for the Self-assembly of Chlorosomes¶

Aggregation of bacteriochlorophyll (BChl) c from chlorosomes, the main light‐harvesting complex of green bacteria, has been studied in aqueous buffer. Unlike other chlorophyll‐like molecules, BChl c is rather soluble in aqueous buffer, forming dimers. When BChl c is mixed with carotenoids (Car), the BChl c Qy transition is further redshifted, in respect to that of monomers and dimers. The results suggest that Car are incorporated in the aggregates and induce further aggregation of BChl c. The redshift of the BChl c Qy band is proportional to the Car concentration. In contrast, the mixture of bacteriochlorophyllide (BChlide) c, which lacks the nonpolar esterifying alcohol, does not form aggregates with Car in aqueous buffer or nonpolar solvents. Instead, the position of the BChlide c Qy transition remains unshifted in respect to that of the monomeric molecule, and Car precipitates with the course of time in aqueous buffer. Similar effects on both BChl c and BChlide c are also observed when monogalactosyl diglyceride (MGDG), which forms the monolayer envelope of chlorosomes, is used instead of (or together with) Car. The results show that the hydrophobic interactions of the BChl c esterifying alcohols with themselves and the nonpolar carbon skeleton of Car, or the fatty acid tails of MGDG, are essential driving forces for BChl aggregation in chlorosomes.

Fluorescence detected magnetic resonance (FDMR) of green sulfur photosynthetic bacteria Chlorobium sp.

Fluorescence Detected Magnetic Resonance (FDMR) spectra have been measured for whole cells and isolated chlorosomal fractions for the green photosyntheic bacteria Chlorobium phaeobacteroides (containing bacteriochlorophyll e, and isorenieratene as major carotenoid) and Chlorobium limicola (containing bacteriochlorophyll c, and chlorobactene as major carotenoid). The observed transition at 237 MHz (identical in both bacteria) and > 1100 MHz can be assigned, by analogy with published data on other carotenoids, to the 2E and D + E transitions, respectively, of Chlorobium carotenoids. Their zero field splitting (ZFS) parameters are estimated to be: |D|=0.0332 cm−1 and |E|=0.0039 cm−1 (chlorobactene), and |D|=0.0355 cm−1 and |E|=0.0039 cm−1 (isorenieratene). In the intermediate frequency range 300–1000 MHz the observed transitions can be assigned to chlorosomal bacteriochlorophylls c and e, and to bacteriochlorophyll a located in the chlorosome envelope and water-soluble protein. The bacteriochlorophyll e triplet state measured in 750 nm fluorescence (aggregated chlorosomal BChl e) is characterised by the ZFS parameters: |D|=0.0251 cm−1 and |E|=0.0050 cm−1.

Luminescence Study of Singlet Oxygen Production by Meso-Tetraphenylporphine

The research in the field of the photodynamic therapy of cancer (PDT) is focused on a development of photosensitizers exhibiting high quantum yield of singlet oxygen production. Direct time-resolved spectroscopic observation of singlet oxygen phosphorescence can provide time constants of its population and depopulation as well as photosensitizer phosphorescence lifetime and relative quantum yields. In our contribution, a study of time and spectral resolved phosphorescence of singlet oxygen photosensitized by meso-tetraphenylporphine in acetone together with the photosensitizer phosphorescence is presented. Time constants of singlet oxygen population and depopulation were determined at wide range of photosensitizer concentrations. The time constant of singlet oxygen generation (0.28 ± 0.01) μs is slightly shorter then the lifetime of photosensitizers triplet state (0.32 ± 0.01) μs. It is caused by lower ability of TPP aggregates to transfer excitation energy to oxygen. The lifetime of singlet oxygen (≈50 μs) decreases with increasing photosensitizer concentration. Therefore, the photosensitizer acts also as a quencher of oxygen singlet state, similarly to the effects observed in [A. A. Krasnovsky, P. Cheng, R. E. Blankenship, T. A. Moore, and D. Gust (1993). Photochem. Photobiol.57, 324–330; H. Küpper, R. Dědic, A. Svoboda, J. Hála, and P. M. H. Kroneck (2002). Biochim. Biophys. Acta Gen. Subj.1572, 107–113]. Moreover, the increasing concentraion of the photosensitizer causes a slight hypsochromic shift of the singlet oxygen luminescence maximum.

Singlet oxygen-sensitized delayed fluorescence of common water-soluble photosensitizers

Six common water-soluble singlet oxygen ((1)O2) photosensitizers - 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphine (TMPyP), meso-tetrakis(4-sulfonathophenyl)porphine (TPPS4), Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4), eosin Y, rose bengal, and methylene blue - were investigated in terms of their ability to produce delayed fluorescence (DF) in solutions at room temperature. All the photosensitizers dissolved in air-saturated phosphate buffered saline (PBS, pH 7.4) exhibit easily detectable DF, which can be nearly completely quenched by 10 mM NaN3, a specific (1)O2 quencher. The DF kinetics has a biexponential rise-decay character in a microsecond time domain. Therefore, we propose that singlet oxygen-sensitized delayed fluorescence (SOSDF), where the triplet state of a photosensitizer reacts with (1)O2 giving rise to an excited singlet state of the photosensitizer, is the prevailing mechanism. It was confirmed by additional evidence, such as a monoexponential decay of triplet-triplet transient absorption kinetics, dependence of SOSDF kinetics on oxygen concentration, absence of SOSDF in a nitrogen-saturated sample, or the effect of isotopic exchange H2O-D2O. Eosin Y and AlPcS4 show the largest SOSDF quantum yield among the selected photosensitizers, whereas rose bengal possesses the highest ratio of SOSDF intensity to prompt fluorescence intensity. The rate constant for the reaction of triplet state with (1)O2 giving rise to the excited singlet state of photosensitizer was estimated to be ~/>1 × 10(9) M(-1) s(-1). SOSDF kinetics contains information about both triplet and (1)O2 lifetimes and concentrations, which makes it a very useful alternative tool for monitoring photosensitizing and (1)O2 quenching processes, allowing its detection in the visible spectral region, utilizing the photosensitizer itself as a (1)O2 probe. Under our experimental conditions, SOSDF was up to three orders of magnitude more intense than the infrared (1)O2 phosphorescence and by far the most important pathway of DF. SOSDF was also detected in a suspension of 3T3 mouse fibroblast cells, which underlines the importance of SOSDF and its relevance for biological systems.

Fluorescence detected magnetic resonance of monomers and aggregates of bacteriochlorophylls of green sulfur bacteria Chlorobium sp.

Fluorescence detected magnetic resonance (FDMR) was used to study the lowest triplet state of bacteriochlorophylls (BChls) c and d in Chlorobium (Chl.) tepidum and Chl. vibrioforme, respectively. These pigments were studied both in the oligomeric form (in whole cells) and in the monomeric form (after conversion using a 1% 1-hexanol treatment). Fluorescence spectra show the presence of lower-state aggregates, apart from monomers, in samples treated with 1-hexanol. Values of the zero field splitting (ZFS) parameter D, obtained from FDMR spectra, were found to decrease with an increasing aggregate size. The observed ZFS trends are explained by a delocalization of the triplet spins, including a charge resonance (CR) contribution, over the aggregate. A simple model is presented relating the changes of D and E as a result of monomer aggregation to the aggregate geometry. Application of this model to BChls c and d indicates approximately diagonal stacking of the monomers in the dimer. Results for oligomeric BChl c and d were compared with those previously obtained for oligomeric BChl e. FDMR transitions of BChls c, d and e differ both in frequencies and in signs. The D and E values of Cars and BChl a (in whole cells) agree well with those reported for Chl. phaeobacteroides and Chl. limicola.

Site-selection and Shpolskii spectroscopy of model photosynthetic systems

Low temperature (T= 10 K) site‐selection fluorescence and excitation spectra of both isolated pheophorbide‐a molecules (I PHEO) and pheophorbide covalently bonded to the synthetic l‐lysyl‐l‐alanyl‐l.‐alanine polypeptide (B PHEO) were measured in dimethylformamide (DMF) matrices. The fluorescence spectra display sharp vibronic lines at different wavenumbers for different dye laser excitation wavelengths, superimposed on a broad band background. Spectral analysis provides frequencies of normal vibrations (FNV) and site distribution functions (SDF). The FNV and SDF of I PHEO in DMF have been found to show good agreement with other chlorophyll‐like molecules. A covalent binding between the polypeptide and PHEO molecules produces only slight changes in FNV but significant broadenings and shifts to lower energies in SDF. The fluorescence spectra of I PHEO in a typical Shpolskii matrix (n‐octane) were also measured and found to exhibit sharp non‐shifting vibronic lines for all wavelengths and kinds of excitation. A general model explaining both site‐selection and Shpolskii spectroscopy is presented.