PICOSECOND FLUORESCENCE STUDIES OF EXCITON MIGRATION AND ANNIHILATION IN PHOTOSYNTHETIC SYSTEMS. A REVIEW*

Abstract. In this paper we review picosecond fluorescence studies of exciton dynamics in photosynthesis. We discuss some of the exciton interactions that led to artifacts in early picosecond data and outline procedures for avoiding their presence. In the case of high intensity single pulse excitation (> 10¹³ photons cm²), the dominant mechanism is singlet-singlet fusion, manifesting itself by a decrease in the observed lifetime and quantum efficiency of fluorescence. The manner in which excitons interact in vivo provides an indicator of the topology of the photosynthetic unit (PSU). The shape of the fluorescence quenching curve, as a function of intensity, in particular, can be used to test various models. In addition to fluorescence quenching curves, we also report the results of fluorescence decay following ps laser flashes, using an ultrafast streak camera in four types of systems: (1) organic crystal anologues, (2) chromatophores of various mutants of the photosynthetic bacteria, Rhodopseudomonas sphaeroides, (3) intact cells of the green alga, Chlorella and (4) chloroplasts of higher plants (e.g. spinach).     

ENERGY TRANSFER IN TRIMERIC C-PHYCOCYANIN STUDIED BY PICOSECOND FLUORESCENCE KINETICS

Abstract— The excited state kinetics of trimeric C-phycocyanin from Mastigocladus laminosus has been measured as a function of the emission and excitation wavelength by the single-photon timing technique with picosecond resolution and simultaneous data analysis. A fast decay component of 22 ps (C-phycocyanin with linker peptides) and 36 ps (C-phycocyanin lacking linker peptides) is attributed to efficient energy transfer from sensitizing to fluorescing chromophores. At long detection wavelengths the fast decay components are found to turn into a rise term. This finding further corroborates the concept of intramolecular energy transfer. Previous reports on the conformational heterogeneity of the chromophores and/or proteins in C-phycocyanin are confirmed. Our data also provide indications for the importance of the uncoloured linker peptides for this heterogeneity.     

ENERGY LEVELS IN CHLOROPHYLL AND ELECTRON TRANSFER PROCESSES*,†

Abstract— A brief account of the localized model for charge carrier generation in solid dyes is given, and generalized to include transfers of electrons and holes between unlike molecules. A part of the experimental basis for the localized model is the finding that the dye molecule sees its environment as a classical dielectric medium, even when it is composed of like molecules; since the effects of the environment on the ionization energy of chlorophyll are small, a close relationship can be inferred between laboratory energy levels and those effective in the chloro-plast. The measured energy diagrams suggest a very low yield of charge carriers in either pure chlorophyll, but leave open the possibility of a substantial yield at an interface between chlorophylls a and b . The energy levels are consistent with the idea that charge carrier transfers between excited chlorophyll molecules and acceptors take place in the primary process.     

COHERENT MOTION AND TRANSFER OF EXCITATION ENERGY IN THE PRIMARY PROCESSES OF PHOTOSYNTHESIS

Fluorescence decay curves are calculated within the framework of the Liouville von Neumann equation for simple model systems of a photosynthetic unit consisting of a reaction center coupled to either a linear chain or a hexagonal array of identical antenna pigments. A comparison of the results with those obtained by solving the Pauli Master equations reveals that in the case of coherent excitation energy transfer the fluorescence decay kinetics exhibit a strong dependence on the topological array of the antenna pigments. For systems with a different pigment array, markedly different kinetics can be expected upon decreasing the pure dephasing time.     

PICOSECOND FLUORESCENCE KINETICS and ENERGY TRANSFER IN THE ANTENNA CHLOROPHYLLS OF GREEN ALGAE*

Single-photon timing measurements on flowing samples of Chlorella vulgaris and Chlamydomonas reinhardtii at low excitation intensities at room temperature indicate two main kinetic components of the fluorescence at open reaction centers (F₀) of photosystem II with lifetimes of approx. 130 and 500 ps and relative yields of about 30 and 70%. Closing the reaction centers progressively by preincubation of the algae with increasing concentrations of 3-(3′,4′-dichlorophenyl)-l,l-dimethylurea (DCMU) and hydroxylamine gave rise to a slow component with a lifetime increasing from 1.4 to 2.2 ns (F_max) The yield of the slow component increased to 65-68% of the total fluorescence yield in parallel to a decrease in the yield of the fast component to a value close to zero at the f_max-level. The 130 ps lifetime of the fast component remained unchanged. The middle component showed an increase of its lifetime from 500 to 1100 ps and of its yield by a factor of 1.5. Spacing of the ps laser pulses by 12 μs allowed us to resolve a new long-lived fluorescence component of very small amplitude which is ascribed to a small amount of chlorophyll not connected to functional antennae. The opposite dependence of the yield of the fast and the slow component on the state of the reaction centers at almost constant lifetimes is consistent with a mechanism of energy conversion in largely separately functioning photosystem II units. Yields and lifetimes of these two components are in agreement with the high quantum yield of photosynthesis. The lower lifetime limit of 1.4 ns of the slow component is assigned to the average transfer time of an excited state from a closed to a neighboring open reaction center and the increase in the lifetime to 2.2 ns is evidence for a limited energy transfer between photosystems II. Relative effects of changing the excitation wavelength from 630 to 652 nm on the relative fluorescence yields of the kinetic components were studied at the fluorescence wavelengths 682, 703 and 730 nm. Our data indicate that (i) the middle component has its fluorescence maximum at shorter wavelength than the fast component and (ii) that the antennae chlorophylls giving rise to the middle component are preferentially excited by 652 nm light. It is concluded that the middle component originates from the light-harvesting chlorophyll alb protein complexes and the major portion of the fast component from the chlorophyll a antennae of open photosystem II reaction centers.     

镥单酞菁和镥双酞菁电子转移过程的研究

本文用循环伏安法研究镥单酞菁和镥双酞菁在非水溶剂中的电化学行为,获得了相应的氧化还原反应半波电位E1/2,测定了其中四个氧化还原反应的电子转移速度常数ks。指出非水溶剂对镥单酞菁和镥双酞菁的电化学行为具有程度不同的影响。     

PICOSECOND FLUORESCENCE STUDY OF PHOTOSYNTHETIC MUTANTS OF Chlamydomonas reinhardii: ORIGIN OF THE FLUORESCENCE DECAY KINETICS OF CHLOROPLASTS

Abstract— The fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardii which lack photosystem II (PS II), photosystem I (PS I), and both PS II and PS I have been measured. The PS II mutant strain8–36C exhibits fluorescence decay lifetime components of 53, 424 and 2197 ps. The fluorescence decay of a PS I mutant strain12–7 contains two major fluorescence decay components with lifetimes of 152 and 424 ps. The fluorescence decay of mutant strain C2, which lacks both PS II and PS I, is nearly single exponential with a lifetime of 2561 ± 222 ps. In simulations in which it is assumed that wild-type decays are a simple sum of the major decay components of the isolated parts of the photosynthetic unit as measured in the mutants, curves are obtained that fit the wild-type C. reinhardii fluorescence decay data when the absorption cross-sections of PS II and PS I are weighted approximately equally. The 89 ps lifetime component in the wild-type is an average of 53 and 152 ps components arising from excitation transfer to and trapping in PS I and PS II. The single step transfer time in PS I is estimated to be between 100 and 700 fs depending on assumptions about array size. We find that between two and four visits to the PS I reaction center are required before final trapping.     

PICOSECOND TIME RESOLVED FLUORESCENCE OF CHLOROPHYLL IN VIVO

Abstract. The published data concerning the fluorescence kinetics of chlorophyll a in various photosynthetic species are reviewed. The effects of singlet-singlet and singlet-triplet annihilation induced by excessively high incident light intensities are discussed and related to the changes produced in the fluorescence lifetimes and quantum yields. We also review the fluorescence lifetimes of Chlorella pyrenoidosa and spinach chloroplast fragments under a variety of experimental conditions; these measurements were performed at single pulse excitation intensities of less than 5 × 10¹³ photons cm^–2 where distortion due to annihilation processes is negligible. Evidence for and against a time dependent rate equation for energy migration will be discussed with reference to the authors' work on in vitro systems.     

ON THE SPATIAL CORRELATION BETWEEN THE PROTEIN SURFACE AND THE ELECTRON TRANSFER CYCLE IN BACTERIAL PHOTOSYNTHETIC REACTION CENTERS

Abstract— n -Doxylstearic acids are shown to be electron acceptors in reaction centers from R. spheroides R-26 mutant illuminated with 860 nm light. The electron accepting efficiency varies with n , thus with the location of the nitroxide moiety on the stearic acid chain, and is maximum for 12-doxylstearic acid. In this molecule the nitroxide moiety is 1.4 nm from the polar carboxyl carbon based on molecular models. If the nitroxide moiety is located in or near the polar reaction center protein surface, the results suggest that the quinone-iron complex, which is the ultimate electron acceptor from a bacterio-chlorophyll dimer in the reaction center, is located ∼ 1.4 nm from the protein surface. The protein itself is estimated to have a diameter of 5.4 nm assuming spherical symmetry, so it is postulated that the quinone-iron complex is located on one side of the protein.     

EXCITATION ENERGY TRANSFER IN THE CRYPTOPHYTES. FLUORESCENCE EXCITATION SPECTRA AND PICOSECOND TIME-RESOLVED EMISSION SPECTRA OF INTACT ALGAE AT 77 K

Excitation spectra of chlorophyll- a (Chl- a ) fluorescence in intact cells of Cryptomonas ovata, Chroomonas pauciplastida and Chroomonas salina were determined at 77 K. For all species the excitation spectra for emission from Chl- a associated with photosystem II (PSII) showed increased contributions by a carotenoid (493 nm) and phycobiliproteins, and decreased contributions by carotenoid (417 nm, 505 nm) and Chl- a (445 nm) as compared to excitation spectra for emission from Chl- a associated with photosystem I (PSI). Excitation spectra of C. salina and C. ovata showed an increased contribution by Chl- c ² to PSII Chl- a fluorescence emission. In all three species the absorbance band positions of Chl- a , as determined from the excitation spectra, were similar to those previously described in green plants. green algae and phycobilisome-containing organisms. Time-resolved 77 K fluorescence emission spectra of C. ovata and C. salina showed successive emission from both phycoerythrin and Chl- c ², PSII Chl- a , and PSI Chl- a. C. pauciplastida showed successive emission from phycocyanin, PSII Chl- a , and PSI Chl- a. Spectral red-shifts with time were observed for the phycobiliprotein peaks in all three species. The fluorescence decay of phycoerythrin in C. ovata and C. salina was faster than that of phycocyanin in C. pauciplastida. The results are discussed in relation to the organization of the antenna pigments of PSII and PSI in the cryptophyte algae.     

PICOSECOND FLUORESCENCE KINETICS IN SPINACH CHLOROPLASTS AT LOW TEMPERATURE

Abstract— At 77 K the fluorescence from spinach chloroplasts excited using picosecond mode-locked laser pulses at 620 nm is made up of 5 separate kinetic components. Three of these are predominant at short wavelengths. between 650 and 690 nm, and they appear to correspond to the 3 decay phases seen at room temperature. The 2 new components. a 100 ps rise and a 3000 ps decay, characterize the longer (730–770 nm) wavelength fluorescence. The temperature dependence of the kinetic components of the long wavelength fluorescence shows that the 3000 ps decay accounts for essentially all of the large increase in fluorescence yield observed at low temperature. Furthermore, it appears that this increase does not result entirely from an increase in the fluorescence lifetime, as has been proposed. The dependences of these 2 new components (the 100 ps rise and 3000 ps decay) on emission wavelength and temperature are similar enough to suggest they have a common origin, presumably the chlorophyll pigment component C705. The amplitudes (yield/lifetime) of these 2 phases are approximately equal, and they are opposite in sign. Thus. we see evidence of time-resolved excitation transfer from those pigment molecules that absorb the 620 nm radiation to those that give rise to the long wavelength fluorescence at low temperature.     

PICOSECOND FLUORESCENCE SPECTROSCOPY ON INCORPORATION PROCESSES OF HEMATOPORPHYRIN DERIVATIVE INTO MALIGNANT TUMOR CELLS in vitro

Abstract— By using a highly sensitive streak-camera technique, we investigate incorporation processes of HpD into malignant tumor m-KSA cells in vitro. The picosecond decays of the total fluorescence spectra, the wavelength-resolved fluorescence decays and the time-resolved fluorescence spectra from HpD in the cells are measured as a function of the incubation time. The results show that the aggregate component of HpD which has a fast fluorescence lifetime of 100 ps and a red-shifted band of ∼ 660 nm selectively accumulates more and more in the cells with the increase of the incubation time.     

2-芳基苯并噁唑的荧光猝灭和光诱导电子转移研究

本工作对两种不同的2-芳基苯并 唑化合物溶液荧光被四氯化碳所猝灭的机理进行了详细研究,通过多种途径研究表明该猝灭过程具有光诱导电子转移性质,工作还利用此电子转移所形成的活泼自由基来引发烯类单体的聚合,得到了有一定聚合度的聚甲基丙烯酸甲酯。     

酪胺酸-色胺酸缩聚二肽分子内电子转移速率常数的从头算研究

用量子化学从头算方法对色氨酸－酪氨酸缩聚二肽体系进行电子转移动力学参数的计算。用ＵＨＦ／６－３１Ｇ方法分别优化给体,受体和桥体的几何构型,用线性反应坐标构造了给体和受体分子间电子转移的双势阱,得到两透热势能面在Ｒｃ约为０处交叉,表面气相反应为无能垒过程。     

PICOSECOND FLUORESCENCE FROM PHYCOCYANIN 612

The biliprotein, phycocyanin 612, was purified from a cryptomonad, Hemiselmis virescens. The protein, which is an α₂β₂ dimer having four spectrally different tetrapyrrole chromophores, was studied using picosecond fluorescence by exciting the various chromophores at three wavelengths, 565, 585 and 615 nm. These wavelengths were chosen to excite selectively the three highest energy chromophores. Decay times were measured as the excitation energy migrated from each of the three excited chromophores to the lowest-energy chromophore. The ps decay times were found to be 9, 13, and 12 ps for excitations at 565, 585, and 615 nm, respectively. A comparison is made between phycocyanin 612 and phycocyanin 645 with regard to the causes of their differing absorption maxima.     

以共价键相连的卟啉-酞菁化合物分子内能量传递及光诱导电子转移

合成了叶啉与酞菁以共价键连接起来的双发色团分子。测定了它们的吸收光谱,荧光光谱,荧光寿命等。计算了分子内能量传递过程的效率(φ_EnT)及速率常数(κ_EnT)。结果表明:在稀溶液中,卟啉与酞菁等克分子混合时,观察不到分子间能量传递过程现象的发生;而双发色团分子的分子内能量传递过程则明显发生了,其效率(φ_EnT=13～70%)与速率常数(κ_EnT=1.2×10⁷～2.0×10⁸s^-1)取决于分子的结构类型。电子转移与能量传递过程与介质性质有关。在极性溶剂中有利于电子转移过程的进行,而不利于能量传递过程;在非极性溶剂中,则有利于能量传递过程的进行,而不利于电子转移。 选择性激发酞菁发色团,观测到了只有电子转移发生的过程,其电子转移效率达到38%。     

RELATIONS BETWEEN PHOTOCHEMISTRY AND FLUORESCENCE IN CELLS AND EXTRACTS OF PHOTOSYNTHETIC BACTERIA*

Abstract— Light-induced changes in the yield of bacteriochlorophyll fluorescence have been measured in cells and chromatophores of photosynthetic bacteria, and coordinated with light-induced absorbancy changes. Comparisons were drawn during transitions between dark and light steady states and also between steady states established at different light intensities. Aerobic cell suspensions of Rhodospirillum rubrum, Rhodopseudomonas spheroides, Chromatium and Rhodopseudomonas sp. NHTC 133 showed a strict correspondence between changes in the fluorescence yield and the bleaching of P870 (P985 in Rps. sp. NHTC 133), as reported by Vredenberg and Duysens for R. rubrum cells. The relationship shows that singlet excitation energy in bacteriochlorophyll is quenched by P870 at a rate proportional to the concentration of unbleached P870. This implies that the photosynthetic units are not independent with respect to energy transfer. In anaerobic cell suspensions the change in fluorescence did not follow the bleaching of P870 in the manner described by Vredenberg and Duysens. Here a change in fluorescence may have resulted from the reduction of a primary photochemical electron acceptor as well as from the oxidation (bleaching) of P870. In chromatophore preparations there were further deviations from the Vredenberg and Duysens relationship which could be attributed to changes in the rate constants for quenching of singlet excitation energy. Finally there was a light-induced increase in the fluorescence yield which was related to a band shift of bacteriochlorophyll and not to the bleaching of P870. Aerobic cell suspensions presented a limiting case in which these complications were absent. No change in the fluorescence was associated uniquely with the oxidation of cytochrome or band shifts of carotenoid pigments. These results, when coordinated with earlier findings about the fluorescence of bacteriochlorophyll and P870, indicate that the singlet excitation quantum is the only energy carrier linking the absorption of light with the initiation of photochemistry in bacterial photosynthesis.