Jacek Goc

Blue-light-controlled photoprotection in plants at the level of the photosynthetic antenna complex LHCII

Plants have developed several adaptive regulatory mechanisms, operating at all the organization levels, to optimize utilization of light energy and to protect themselves against over-excitation-related damage. We report activity of a previously unknown possible regulatory mechanism that operates at the molecular level of the major photosynthetic pigment-protein complexes of plants, LHCII. This mechanism is driven exclusively by blue light, operates in the trimeric but not in the monomeric complex, and results in singlet excitation quenching leading to thermal energy dissipation. The conclusions are based on single molecule fluorescence lifetime analysis, direct measurements of thermal energy dissipation by photo-thermal spectroscopy, and on fluorescence spectroscopy. Possible molecular mechanisms involved in the blue-light-induced photoprotective effect are discussed, including xanthophyll photo-isomerization and the thermo-optic effect.

Interactions between chlorophyll a and β-carotene in nematic liquid crystals

Abstract Fluorescence lifetime in the ps range, polarized absorption, polarized fluorescence spectra and delayed luminescence time resolved spectra were measured for chlorophyll a solutions with and without β-carotene addition in nematic liquid crystal. Photoacoustic spectra of the same samples at various frequencies of light modulation were also taken. The frequency dependence of the photoacoustic spectra suggests that part of the excitation is converted into heat in a slow process (with a decay time of the order of ms). The lifetime results suggest that at used concentration some aggregation of the chlorophyll a occurs. The chlorophyll a molecules interact strongly with the β-carotene forming some nonfluorescent or weakly fluorescent aggregates characterized by having various thermal deactivation yields and orientations in anisotropic matrix when compared to those of separated pigments. It seems that the aggregated forms of the chlorophylls are partially disrupted as a result of the their interaction with the β-carotene. Singlet excitation of β-carotene is not transferred to the fluorescent form of chlorophyll a . Delayed (in μs time range) luminescence of chlorophyll a is quenched by β-carotene. This luminescence is located in the same spectral region as prompt fluorescence. Interactions between chlorophyll a and β-carotene depend on the degree of pigment orientation and their aggregation.

Aggregation of chlorophylls a and b in polymer films and monolayers

Abstract Polarized absorption and fluorescence spectra as well as lifetime of fluorescence (τ) of the mixtures of chlorophyll a (Chl a) and chlorophyll b (Chl b) in anisotropic polyvinyl alcohol (PVA) films were measured. Two mixtures with various concentrations ratios of Chl a to Chl b were investigated. For comparison, the fluorescence excitation and emission spectra of Chl b + phytol mixture in Langmuir—Blodgett films were also measured. At high Chl a and low Chl b concentrations (MI) the following three forms of Chl a aggregates occurred: ‘dry’ Chl a (aggregated with polymer) with a fluorescence maximum at 676 nm (F676), hydrated dimer (Chl a 2H2O)2 or (Chl a H2O)2 (F700–705 nm), and oligomers of hydrated dimers (F750–770 nm). Chl b in the same matrix also occurs in at least three forms: ‘dry’ (F653 nm), ‘wet’ - hydrated aggregates (F662 nm) and aggregated forms with the fluorescence at 710–720 nm and/or 730–740 nm regions. The orientation of various chlorophyll forms in the PVA matrix is different. The efficiency of excitation energy transfer from Chl b to various aggregates of Chl a is different. The yield of energy transfer from Chl b to Chl a oligomers is high. Fluorescence decay of various aggregates is different and the experimental decay can be analysed always on two or three exponential components. Results are discussed on the basis of previous data concerning the monolayers of Chl a with phytol and literature data concerning aggregation of chlorophylls in various model systems. In the spectra of chlorophyll in several model systems, as follows from the literature, it is possible to distinguish the contributions from the three or even more aggregated forms of pigment similar to those reported by us. Some similarities between aggregated forms or chlorophylls and pigment forms observed in organisms are noted. In polymer films, aggregated forms of pigments are stable and oriented; therefore, their properties and the condition of their formation can be established.

Aggregation of chlorophyll b in model systems

Abstract Chlorophyll b (Chl b ) in model systems occures in several forms. The relationship between these forms and the forms occurring in organisms is not yet clear. Chl b aggregation has been investigated less than that of Chl a . In this paper, several spectral features, e.g. fluorescence lifetimes in the picosecond time range, time-resolved delayed luminescence spectra, photoacoustic spectra and photopotential generation of Chl b embedded in model systems (poly(vinylalcohol) film or nematic liquid crystal), were measured. The decay of the fluorescence of Chl b in polymer films can be analyzed, to a good approximation, on the basis of the following three exponential components: 3300–4300 ps, 400–900 ps and 40–71 ps. These decays are tentatively related to the emission of “dry” monomers and dry and wet dimers and oligomers respectively. The delayed luminescence spectra of the same samples in the microsecond range (at 8–295 K) are located in a similar spectral range to the fluorescence spectra. The intensity ratio between the delayed luminescence and prompt fluorescence is higher in the long-wavelength region, in which the oligomer emission is observed, than in the short-wavelength region, in which the emission of monomers and small aggregates predominates. The yield of thermal deactivation of the aggregated forms is higher than that of the monomers. The differences between the lifetimes of the various forms can be explained by the competition between the emission of prompt fluorescence, thermal deactivation and energy trapping (which is, in part, later deactivated as delayed luminescence). The excitation energy transfer from “dry” monomers to aggregated forms is not very effective. The most effective process of excitation trapping followed by delayed luminescence emission occurs in oligomers of Chl b . On the basis of photopotential generation, the delayed luminescence is due, at least in part, to pigment ionization followed by slow charge recombination. The kinetics of photopotential generation and decay depend on the aggregation of the pigment.

The interactions between bacteriochlorophyll c and amphiphilic peptides

Abstract Absorption, fluorescence, photoacoustic and delayed luminescence spectra of bacteriochlorophyll c with and without peptides located in dimethyl sulfoxide were measured. For most molecules, the hydrophobic interactions between peptide and pigment in the ground state seems not to be very strong, as it follows from the absorption spectra of the samples with and without peptides. From photoacoustic spectra, it seems that at least part of the illuminated pigment molecules are forming complexes with peptides. These complexes are very efficient in thermal deactivation of excitation. The occurrence of the interactions between peptides and bacteriochlorophyll c follows also from delayed luminescence spectra, which are different for the sample with and without peptides. The observed delayed luminescence is so-called delayed fluorescence or E-type delayed luminescence because it is generated by thermal activation from the triplet to first excited state followed by emission from this state.

Photoreaction and thermal deactivation of excitation in purple bacteria light harvesting complexes (LH2) with and without reaction centres

Light harvesting peripheral complexes (LH2) and carotenoids free-reaction centres were isolated from purple photosynthetic bacteria Rhodospirillum molischianum and Rhodobacter sphaeroides, respectively. The LH2 complexes without and with various amounts of reaction centres were immobilized in polyacrylamide gel. The absorption and photoacoustic spectra of such samples were measured. The changes due to continuous light illumination in absorption and photoacoustic spectra were investigated. The changes in absorption generated by flash were also measured. The conclusions concerning the photoreactions in antenna and reaction centre chromophores, the storage of energy in reaction centres as well as the protection of LH2 chromophores against overexcitation by heat emission and excitation transfer to reaction centres are discussed.

The influence of the presence of lipid on the aggregation of 8,12-diethyl farnesyl bacteriochlorophyll c located in adsorbed layers and monolayers

The photoacoustic spectra and time-resolved delayed luminescence spectra in the microsecond time range were measured for layers of 8,12-diethyl farnesyl bacteriochlorophyll c adsorbed on quartz supports by solvent evaporation and as Langmuir-Blodgett monolayers. Both types of model system were also investigated with the addition of lipid. The data showed a very strong influence of lipid addition on pigment aggregation. In samples with synthetic and natural lipid addition, the pigments were found to be predominantly in the monomeric and dimeric states, whereas in the same type of sample without lipid, the pigments were aggregated to a higher degree. The influence of the presence of lipid on the aggregation of bacteriochlorophyll c in monolayers and adsorbed layers may also suggest that the contact of various pigment molecules with the lipids surrounding the chlorosome may influence the formation of various pigment aggregates in vivo. The synthetic lipid L-alpha-phosphatidylcholine dipalmitoyl and the natural lipid L-alpha-phosphatidylcholine type IVS from soy beans were used. In the latter case, only adsorbed layers were investigated. Our interpretation is preliminary as only one 8,12-diethyl farnesyl bacteriochlorophyll c homologue was present in our systems.

DELAYED EMISSION OF CHLOROPHYLL a AGGREGATES AND RHODAMINE 6G EMBEDDED IN POLYMER MATRIX

Delayed luminescence (in the microsecond time range) of the chlorophyll (Chl) a“dry” form as well as hydrated dimers located in a polyvinylalcohol film was measured from room temperature down to 8 K. In the same matrix the delayed luminescence of rhodamine 6G (Rhod) was investigated. The delayed emission both of Chl a and Rhod is probably due to the formation and delayed recombination of a radical pair. It seems that this process occurs without participation of triplet states, as it does not reflect their well‐known sensitivity to oxygen. The temperature dependence of the delayed luminescence of vanous Chl forms is different. In the region around 678 nm (dry monomer) delayed luminescence needs a thermal activation energy of about 0.03 eV, whereas at 740 nm (wet aggregates) delayed luminescence intensity increases linearly with decreasing temperature. Its assignment as a‐type delayed luminescence from the low‐lying triplet state can consistently be excluded from both the weak temperature dependence of the delayed fluorescence and its large intensity as compared to the prompt fluorescence. Delayed luminescence of Rhod is almost independent of temperature between 8 K and 300 K. The dependence of delayed luminescence intensity on exciting light intensity is linear at lower intensities and tends to saturation at higher. Therefore the delayed luminescence is not related to exciton annihilation. Positions and intensities of the Chl delayed luminescence bands show that it is not phosphorescence (β‐type delayed luminescence). The aggregation of both Chl and Rhod molecules strongly influences delayed luminescence since it differs in several properties if excited in the monomer or in the aggregate absorption range. Every aggregational form of dye emits its characteristic delayed luminescence band.

Deactivation of excitation energy in bacterial photosynthetic reaction centres in Langmuir–Blodgett films

Abstract Absorption, photoacoustic and time-resolved in μs time range delayed luminescence spectra have been measured in order to follow the interaction among chromophores when Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centres are closely packed in a form of Langmuir–Blodgett multilayers. Two types of Langmuir–Blodgett samples have been prepared and investigated: multilayers consist of one type of reaction centre ( Rhodobacter sphaeroides or Rhodopseudomonas viridis ) and multilayers composed of mixed reaction centres ( Rhodobacter sphaeroides mixed with Rhodopseudomonas viridis ). Using the Langmuir–Blodgett multilayers composed of two types of bacteria reaction centres mixture, we were able to extend the spectral region of the light/solar energy absorbed by the system. It was shown that each form of pigment participates in thermal dissipation but to a different degree. A special pair (bacteriochlorophyll dimer) does not contribute to delayed luminescence. Delayed luminescence in Rhodopseudomonas viridis and Rhodobacter sphaeroides differs very significantly from each other. Bacteriopheophytin as well as dihydromesochlorophyll contribute to delayed luminescence but the degree of their participation in this radiative process depends strongly on the type of reaction centre. Delayed luminescence and thermal processes have been indicated as important processes of deactivation of the photoexcited chromophores in reaction centres.

Spectral Properties of Bacteriochlorophyll c in Organisms and in Model Systems

Polarized absorption and fluorescence spectra of bacteriochlorophyll c and green photosynthetic bacterium Prostheecochloris aestuarii cells and cell fragments embedded in stretched polymer film were measured. In pigment samples the artificial oligomers of bacteriochlorophyll c (with absorption about 750 nm) and other forms of this pigment and bacteriopheophytin (with absorption at 670 nm) were present. In bacteria samples, embedded in polymer, oligomers were in high degree disaggregated and as a result the absorption about 670 nm was observed. Previously for similar sets of samples the decay of fluorescence excited only at one wavelength was analyzed on three exponential components, but exact lifetime values of these components for various samples were different. The aim of present paper was to check if these differences occur because of various contributions to decay from three well defined forms or if they were related to the existence of several pigment forms with slightly different lifetimes. The global analysis of data obtained for various excitation and observation wavelengths of fluorescence were done. From this analysis it follows that the second situation occurs. For a model system containing artificial oligomers the largest component of decay has a τ4 of about 0.183 ns or 0.136 ns depending on observation wavelength. For the bacteria sample, in which the emission at 680 nm is the superposition from various pigments, global analysis done for various excitation wavelengths shows also that the τ values differ depending on the regions of fluorescence observation. From polarized spectra, it follows that in the model system the pigments absorbing at 670 nm are randomly distributed whereas oligomers are highly oriented. In bacteria fragments absorbing at 670 nm pigment molecules can be divided into two groups: one oriented along the axis of film stretching and the second practically randomly distributed. In living organisms, under some conditions, small amount of 670 nm pigments can be present and can work as excitation energy traps or as antenna transferring the excitation. Present results show that the role of various pools of 670 nm absorbing pigments can be different because of their differing orientation.