PHOTOCHEMISTRY AND SPECTROSCOPY OF CHLOROPHYLL, BACTERIOCHLOROPHYLL AND BACTERIOVIRIDIN IN MODEL SYSTEMS AND PHOTOSYNTHETIZING ORGANISMS

Abstract— In the system (electron donor—pigment—electron acceptor), the photosensitising pigment transfers an electron (hydrogen) from the donor to the acceptor by converting light energy, absorbed by the pigment, into the potential chemical energy of the products. The dependence of the reaction of excited pigments with electron donors or acceptors on the magnitude of the electron affinity of the reacting molecules, the nature of the medium, and the experimental conditions was observed. The initial photo-process was seen to involve the formation of free radicals, with the subsequent formation of compounds with saturated valencies. The reverse reaction of the photoproducts was accompanied by chemiluminescence. The study of systems containing pyridinenucleotides showed the possibility of photosensitised oxidation—reduction of these compounds by a pigment, and of direct photooxidation of reduced pyridinenucleotides by an electron acceptor of a different kind. In order to understand the specific photo-transfer of an electron in organisms, the spectral properties of pigments in organisms were compared with the properties of model pigments in monomeric and aggregated forms. It was established that the main role in the spectroscopic properties shown by bacteriochlorophyll, bacterioviridin and chloropyll in organisms is played by various types of intermolecular interactions (aggregations). Different forms of pigment may be involved in different stages of the phototransfer of an electron.     

SPECTROSCOPY AND PHOTOSENSITIZATION OF SAPPHYRINS IN SOLUTIONS AND BIOLOGICAL MEMBRANES

Abstract A spectroscopic and photophysical study of three new sapphyrin molecules is presented. The sapphyrin backbone that was derivatized to make them water soluble possesses an absorption band around 700 nm, a desired property for biological photosensitization. We studied the absorption and fluorescence spectra, from which evidence for aggregation in solvents of different polarities was obtained. The extent of aggregation is correlated with the nature of the attached moiety. The absolute quantum yields of singlet oxygen production were measured, with 1,3-diphenyl isobenzofuran as a model target, and were 0.13–0.18 in ethanol. The binding constants to liposomes and to cells were determined spectroscopically and were found to correspond to the hydrophobicities of the compounds, with an additional effect, ascribed to the sugar moiety, which was found in the case of one of the sapphyrins. The efficiency of photodamage to Staphylococcus aureus by sapphyrins and hematoporphyrin was equivalent, on the basis of cells killed per microgram of sensitizer in the incubation mixture.     

IN VIVO FLUORESCENCE SPECTROSCOPY OF CHLOROPHYLL IN VARIOUS UNRIPE AND RIPE FRUIT

—Low temperature (77 K) fluorescence emission spectra of slices obtained from the peel and various layers of the pericarp were recorded for fruits which remain green or undergo color break during ripening.
Fluorescence emission peaks characteristic of the photosystem II antennae (λ_F 686 nm) and reaction center (λ_F 696 nm), as well as of the photosystem I antenna (λ_F 730-740 nm), were present in the peel and all parts of the green pericarp of ripe kiwi, avocado and cantaloupe, as well as in ripe tomato and tangerine after color break. The pattern of the fluorescence emission spectra of all samples except that of the kiwi fruit was similar to that obtained from green photosynthetic tissue of leaves, indicating a normal organization of the chlorophyll-containing complexes of thylakoidal membranes. This pattern is characterized by a significantly higher emission at 730-740 nm relative to that of the 696 and 686 nm peaks. In contradistinction, the fluorescence emission at 686 and 696 nm was higher than that at 730 nm in the kiwi fruit, indicating a reduction in the size of the photosystem I antenna chlorophyll. In the innermost yellowish layers of the kiwi pericarp, a further loss of this antenna occurred, as well as disorganization of the photosystem II complex. The above conclusions are suggested also by measurements of variable fluorescence kinetics.
The results presented here indicate that fluorescence spectroscopy might be used as a tool for the study of chlorophyll organization during the growth and ripening periods of fruit.     

ENERGY TRANSFER IN SELF-ASSEMBLED CHLOROPHYLL a SYSTEMS *

Abstract Computer deconvolution of the optical absorption spectra has been used to follow the formation and transformation of various chlorophyll species formed by lowering the temperature in solutions of toluene or methylcyclohexane containing a hydrogen-bonding nucleophile such as ethanol. At cryogenic temperatures the self-assembled chlorophyll a-ethanol species has optical properties similar to those of special pair photoreaction center chlorophyll. Analysis of the absorption and fluorescence behavior of the self-assembled species suggests that chlorophyll-chlorophyll species are also formed in the self-assembly process. Fluorescence lifetime measurements indicate that the pathways for dissipation of excitation energy in these multicomponent systems are complex. Selective optical excitation at wavelengths corresponding to absorption of monomer, oligomer, etc. chlorophyll a species has been used to demonstrate the heterogeneous nature of these self-assembled systems.     

SITE-SELECTION SPECTROSCOPY OF CHLOROPHYLL b IN MEMBRANES OF LECITHIN VESICLES AND IN OTHER SOLVENTS

Abstract— Site-selection fluorescence spectra of chlorophyll b in membranes of lecithin vesicles and in the solvents ethanol, n -butanol, n -butyl acetate, 2-methyl tetrahydrofuran and toluene are presented. The spectra in vesicles display zero-phonon lines as pronounced as those in the best organic glasses. The characteristics of the distributions of O—O transition energies and of the electron-phonon couplings allow to infer the position of the chlorophyll molecule in the membrane of vesicles. It is thus found that the chromophore of chlorophyll b is situated in the layer formed by the ester moieties of the lecithins, i. e. close to the surface of the membrane but not in direct contact with the aqueous phase.     

POLARIZED SITE-SELECTION SPECTROSCOPY OF CHLOROPHYLL a IN DETERGENT

Monomeric chlorophyll a (Chl a ) was obtained from the isolated core antenna complex CP47 of photo-system II after incubation with the detergent triton X-100 and was studied by low-temperature polarized light spectroscopy with the aim to obtain model spectra for Chi a in intact photosynthetic complexes. Evidence is presented by circular dichroism and anisotropy measurements that the isolated chlorophyll is monomeric. The absorption bandwidths are relatively large compared to those found in photosynthetic complexes due to inhomogeneous broadening introduced by the detergent. By selective laser excitation at low temperature, considerable narrowing can be achieved. A number of vibrational bands are resolved in the site-selected, polarized absorption and fluorescence emission spectra. The emission spectrum of Chi a in detergent-damaged CP47 is compared with that of Chi a in the intact light-harvesting complex of photosystem II (LHC-II) from green plants. The spectra are remarkably similar indicating that the low-temperature thermal emitter in LHC-II has spectral properties that are very similar to those of monomeric Chl a .     

OPTICAL-MICROWAVE DOUBLE RESONANCE SPECTROSCOPY OF IN VIVO CHLOROPHYLL

At low temperatures, chloroplast and subchloroplast preparations exhibit complex fluorescence spectra. Emission bands can be attributed to photosystem (PS) particles and various antenna-chl proteins as well as solubilized chls. Initial results from a systematic study of the components of these fluorescence spectra via optical-microwave double resonance spectroscopy are presently reported. Conclusions regarding possible structural features are discussed. Experiments on triplet sublevel decay rates yielded data consistent with an interpretation of triplet energy transfer within antenna fragments.     

铜及其配合物在生物体系中的作用

自人类有历史记载以来，铜及其配合物即用作药物。铜是人体必需的金属元素。人体的正常代谢过程需要它们，但无法体内合成，因此，需要每天从饮食摄取和吸收。生物药学家和研究者以极大的努力彻底地了解铜及其配合物在生物体系中的作用，希望获得更多地信息以利于人类疾病的预防和治疗。本综述评述了铜在组织中的分配和代谢、铜依赖酶、在病态时铜的非正常代谢、铜配合物的药疗活性和抑制氧自由基的产生。     

THE SPECTROSCOPY AND PHOTOCHEMISTRY OF NATURALLY OCCURRING AND SYNTHETIC CHROMENES

Abstract— –All naturally occurring and synthetic chromenes studied exhibit photochromism at — 196°C and in some cases at room temperature. Chromenes containing a coumarin moiety have the least photochemistry and also exhibit the strongest emissions. Chromenes containing a hydroxyl group ortho to an acetyl group exhibit solely a phosphorescence. Lapachenole (a 7,8-benzochromene) shows the most rapid and highest percentage conversion to the colored form and no emission. Photocolored form of evodione can be produced by a triplet energy transfer from benzophenone.     

MULTIPLE EXCITATIONS AND THE YIELD OF CHLOROPHYLL a FLUORESCENCE IN PHOTOSYNTHETIC SYSTEMS

Abstract. Analysis of the effect of multiple excitations on chlorophyll a fluorescence yields in the green alga Chlorella reveals several distinct reactions. The first excitation in dark-z-adapted units produces photochemistry and the high yield state with a rise time of 35 ns. It is ascribed to a change in coupling between the antenna pigments and the photochemical trap. The second hit produces with the same quantum yield a quenched state which changes to the high yield state with a rise time of 4 μ s. This is ascribed to the formation and the decay of a particular carotenoid triplet state near the funnel or antenna-trap junction. Further hits produce enhanced quenching assigned to mobile triplets with lifetimes in the order of 100 ns. The fluorescence yield decreases monotonically with increasing excitations during the 7 ns pulse. This effect can be adequately ascribed to annihilation of excitations with lifetimes longer than the trapping time, or by a unique model of a multi-trapped unit. The latter model is favored by arguments based both on the absence of a local maximum in the graph of fluorescence yield vs excitation energy and on the fact that the high yield state shows a different behaviour on multiple excitation, fit by a single-trapped unit. This analysis is related to that used in experiments with ps flashes and is applied to the qualitatively different bacterial system.     

REACTION OF EXCITED CHLOROPHYLL MOLECULES AT ELECTRODES AND IN PHOTOSYNTHESIS*

Abstract— Excited molecules can exchange electrons with suitable electrodes in an electrochemical cell. Excitation energy of molecules can therefore be converted into electrochemical energy. Electrochemical reactions of excited chlorophyll molecules have been investigated with the help of semiconductor electrodes. It is suggested that this type of reaction also occurs during the primary steps of photosynthesis. Consequently, concepts of electrochemical kinetics would have to be applied in order to elucidate chlorophyll-sensitized reactions in photosynthetic membranes. In order to provide evidence for this conclusion, electrochemical kinetics is applied to the calculation of decay of delayed light from photosynthetic membranes, and the result is compared with experimental data from Chlorella pyrenoidosa. The conversion of light into electrochemical energy via photoelectrochemical reactions of organic dyes in an electrochemical cell is demonstrated, and the postulated analogous electrochemical mechanism for photosynthesis is discussed.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: EFFECTS OF CHOLESTEROL ON RADICAL YIELDS AND KINETIC PARAMETERS*

Abstract The quenching of the triplet state of chlorophyll a (Chl) by asymmetrically located electron acceptors was examined in vesicle systems containing egg yolk phosphatidylcholine and 0–50 mole % cholesterol. The incorporation of cholesterol had two main effects: (1) the distribution of Chl within the vesicle wall shifted from one favoring the inner monolayer to one favoring the outer monolayer, and (2) the Chi molecules (both ground and excited states) became more accessible to water and to the quencher molecules. This latter property was probably due to the creation of space between the phospholipid head groups by insertion of cholesterol. These phenomena required cholesterol concentrations in excess of 15 mol %. In general, the addition of cholesterol caused increases in the apparent bimolecular rate constant for triplet quenching, in the probability that quenching produced radicals, and in the rate of radical recombination. Some of the specific effects of cholesterol depended upon whether or not the quencher molecules were amphiphilic.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: CORRELATIONS BETWEEN KINETIC PARAMETERS AND SOLUBILITIES OF VIOLOGEN ACCEPTORS *

Abstract— Twelve viologens (quaternary 4,4′-bipyridinium ions) are investigated as electron acceptors in vesicle suspensions containing chlorophyll-a (Chl) using laser flash photolysis. The structures of the viologens, which determine how they and their reduced radicals partition between the water and bilayer membrane phases, are systematically varied in order to probe the effects of acceptor solubility on the kinetics of photosensitized electron transfer reactions. The effectiveness of the viologens as quenchers of the Chl triplet excited state increases as they become less soluble in water, because the viologens are more likely to be incorporated into the membrane, but the efficiency of radical formation decreases and the rate of the reverse electron transfer reaction increases. The data obtained with a homologous series of di-alkyl viologens are analyzed quantitatively using a kinetic model which includes the partition coefficients of the viologens and their radicals. For the most part, the reactivities of the viologen species are proportional to their concentrations in the membrane phase, but the locations and mobilities of the viologens in the membrane phase also have to be considered. In order for the Chl and viologen radicals to separate efficiently. the viologen radical has to be able to diffuse from the membrane to the water phase at a rate that competes effectively with reverse electron transfer within the radical pair complex (> 10⁵ s^?1).     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID VESICLE SYSTEMS: COMPARISON OF INSIDE-OUTSIDE ASYMMETRY BETWEEN LARGE AND SMALL UNILAMELLAR VESICLES*

Abstract— We have determined the chlorophyll triplet quenching efficiencies, the chlorophyll cation radical yields and the conversion efficiencies of chlorophyll triplet to radical in large and small unilamellar phosphatidylcholine vesicles (LUV and SUV, respectively) in the presence of electrically-charged electron acceptors (ferricyanide and oxidized cytochrome c) located in either the inner or outer aqueous compartments of the vesicles. Both types of vesicles displayed inside-outside asymmetry, although the properties were reversed. Triplet quenching in SUV was more efficient when ferricyanide was located within the vesicle interior, whereas the reverse was true in LUV. When ferricyanide was located on the outside of the vesicles, the extent of triplet quenching in LUV was about two times that in SUV and the amount of cation radical formed in LUV was about two times that in SUV. Under these conditions, the conversion efficiencies of chlorophyll triplet to radical were 12.2% for LUV and 8.5% for SUV. With cytochrome c as an electron acceptor in negatively charged vesicles (25 mol per cent dixhexadecylphosphate incorporated) similar results were obtained. Again, the triplet quenching and radical yield inside-outside asymmetry properties were reversed between the two types of vesicles, and radical formation efficiencies when cyt c was located outside the vesicles were higher in LUV (11.7%) than in SUV (4.2%). We conclude that the inside-outside asymmetric photochemical behavior of unilamellar phosphatidylcholine vesicles is influenced by factors in addition to the difference in radius of curvature between the inside and outside surfaces. It is suggested that transmembrane electrostatic potentials may be involved. Furthermore, in the present system the properties of LUV were more favorable to photochemical electron transfer product formation than those of SUV.     

PICOSECOND ABSORPTION SPECTROSCOPY OF CHLOROPHYLL A DIHYDRATE

Abstract— The solution of chlorophyll a in hexane with more than 70% of chlorophyll molecules in the form of the dihydrate (CHl-a.2H₂O)_n is investigated at room temperature by picosecond absorption technique. The transient difference spectra and the kinetic dependences of the absorption changes are measured for several excitation and probe wavelengths. Creation of a new state with blue-shifted absorption band is observed in the system after excitation. A model of the behaviour of the system after excitation is proposed and checked by comparison of the computer-simulated spectral and time dependences with the experimental data. According to the model, about 30% of excited molecules are in the state with the 0.046 eV shift of the absorption band in comparison with the ground state absorption. The lifetime of this state has 170-ps component. The rest of the excited molecules are in the first excited state with the decay time component of 37 ps.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-I. PHOTOPRODUCTION OF THE CATION RADICALS OF CHLOROPHYLL AND BACTERIOCHLOROPHYLL IN SOLUTIONS AT LOW TEMPERATURES*

Abstract— Electron spin resonance studies have shown that chlorophyll and bacteriochlorophyll can be photo-oxidized in a variety of solvents via their lowest excited singlet states to produce cation radicals. Pheophytin does not undergo this reaction. The mechanism of this photoprocess and its implications for photosynthesis are discussed.     

ON THE NATURE OF CHLOROPHYLL a IN AQUEOUS MICELLAR SYSTEMS

Abstract— Absorption and fluorescence spectra of chlorophyll a in aqueous micellar solutions were studied. Solutions in anionic micelles gave an absorption band at 740 nm, attributable to microcrystal- line chlorophyll a. Solutions in cationic and nonionic micelles do not show this band. The positions of the spectral bands in anionic micelles and the possible structure of microcrystalline form are discussed. A comparison is made of the behaviour of microcrystalline form in hydrocarbon solvents and in micellar solutions.     

FLUORESCENCE AND LUMINESCENCE STUDIES OF IN VIVO CHLOROPHYLL WITH A LASER PHOSPHOROSCOPE*

Abstract— A simple apparatus using a He-Ne laser and a rotating sector gives repetitive light pulses with rise and fall times of a few μsec and intensities of up to 0.65 W/cm². Fluorescence during the actinic pulse and luminescence following it (after 20 μsec) may be recorded and compared. Sampling of the suspension of photosynthetic cells or chloroplasts by intermittent flow permits the averaging of 25 such measurements. With Chlorella, the fast (30 μsec) and the medium (30 msec) components of luminescence do not saturate at the same light intensity. The former saturates in the same range as the photochemical fluorescence rise, whereas the latter saturates at higher intensities. Under a variety of conditions, the decay of luminescence intensity is always strongly polyphasic. Analysis of the kinetics discloses a dependency with the log concentration of the hypothetical precursor, suggesting a connection of luminescence with electrochemical phenomena. The shape of the luminescence integral, as well as the light saturation pattern, suggests another kind of interpretation, in terms of several pools, possibly two of equal size. In the presence of hydroxylamine, only one pool is seen. The stimulating effect of preillumination is only observed for the medium component. However, a stimulation of short duration also exists for the fast component. The problems of number of pools, mechanism, and stimulation are briefly discussed.