Energy transfer in the inhomogeneously broadened core antenna of purple bacteria: a simultaneous fit of low-intensity picosecond absorption and fluorescence kinetics.

The excited state decay kinetics of chromatophores of the purple photosynthetic bacterium Rhodospirillum rubrum have been recorded at 77 K using picosecond absorption difference spectroscopy under strict annihilation free conditions. The kinetics are shown to be strongly detection wavelength dependent. A simultaneous kinetic modeling of these experiments together with earlier fluorescence kinetics by numerical integration of the appropriate master equation is performed. This model, which accounts for the spectral inhomogeneity of the core light-harvesting antenna of photosynthetic purple bacteria, reveals three qualitatively distinct stages of excitation transfer with different time scales. At first a fast transfer to a local energy minimum takes place (approximately 1 ps). This is followed by a much slower transfer between different energy minima (10-30 ps). The third component corresponds to the excitation transfer to the reaction center, which depends on its state (60 and 200 ps for open and closed, respectively) and seems also to be the bottleneck in the overall trapping time. An acceptable correspondence between theoretical and experimental decay kinetics is achieved at 77 K and at room temperature by assuming that the width of the inhomogeneous broadening is 10-15 nm and the mean residence time of the excitation in the antenna lattice site is 2-3 ps.     

Energy transfer pathways among phycobilin chromophores and fluorescence emission spectra of the phycobilisome core at 293 and 77 K

Energy transfer pathways between phycobiliproteins chromophores in the phycobilisome (PBS) core of the cyanobacterium Synechocystis sp. PCC 6803 were investigated. The computer 3D model of the PBS core with determination of chromophore to chromophore distance was created. Our kinetic equations based on this model allowed us to describe the relative intensities of the fluorescence emission of the short(peaked at 665 nm) and long-wavelength (peaked at 680 nm) chromophores in the PBS core at low and room temperatures. The difference of emissions of the PBS core at 77 and 293 K are due to the back energy transfer, which is observed at room temperature and is negligible at 77 K.     

Energy transfer and distribution in the red alga Porphyra perforata studied using picosecond fluorescence spectroscopy

The detailed process of excitation transfer among the antenna pigments of the red alga Porphyra perforata was investigated by measuring time-resolved fluorescence emission spectra using a single-photon timing system with picosecond resolution. The fluorescence decay kinetics of intact thalli at room temperature revealed wavelength-dependent multi-component chlorophyll a fluorescence emission. Our analysis attributes the majority of chlorophyll a fluorescence to excitation originating in the antennae of PS II reaction centers and emitted with maximum intensities at 680 and 740 nm. Each of these fluorescence bands was characterized by two kinetic decay components, with lifetimes of 340-380 and 1700-2000 ps and amplitudes varying with wavelength and the photochemical state of the PS II reaction centers. In addition, a small contribution to the long-wavelength fluorescence band is proposed to arise from chlorophyll a antennae coupled to PS I. This component displays fast decay kinetics with a lifetime of approx. 150 ps. Desiccation of the thalli dramatically increases the contribution of this fast decay component.     

Energy transfer kinetics in C-phycocyanin from cyanobacterium Westiellopsis prolifica studied by pump-probe techniques

The relaxation processes of C-phycocyanin at different aggregates have been investigated by pump-probe techniques. The lifetimes of ground state recovery measured at various wavelengths are analyzed by computer fitting of the kinetic data to a sum of three and four exponentials for monomers and trimers according to the nonlinear least-square principle, respectively. The shortest lifetime (about 56ps) is due to beta s----beta f transfer in one monomer, that decreases to 31ps in trimer due to the opening of new transfer channels. The second fastest component (about 151ps) in monomer is attributed tentatively to distribution of excitation energy between alpha and beta f chromophores, that decreases to about 117ps in trimer caused by redistribution of excitation energy between them. The two long-lived components (about 690ps and 1385ps for monomer, 620ps and 1320ps for trimer) from some kinds of heterogeneity in some chromophores, such as alpha and beta 1 chromophores which are emitting, show an equal amplitude ratio of 1:2 in both monomer and trimer.     

Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry

Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.     

Modification of room-temperature picosecond chlorophyll fluorescence kinetics in Photosystem-II-enriched particles by photochemistry

Room-temperature single photon timing measurements on Photosystem-II (PS II-) enriched thylakoid fragments at low excitation energies indicate the presence of three kinetic decay components of chlorophyll fluorescence arising from PS-II-associated pigments. Closing the PS II reaction centres produced three variable components, with lifetime values of 0.02–0.25, 0.15–0.90 and 0.35–2.0 ns, between the initial (F₀) and maximal (F_m) fluorescence levels. The yield of each component paralleled the changes in their respective lifetimes, indicating the presence of well-connected PS II reaction centres favouring energy transfer between each other. These changes show that variable chlorophyll fluorescence (F_v) does not arise from one specific origin. The extent of the modifications and the observed relationship between component lifetime and yield, on closing PS II reaction centres, cannot be explained by either the delayed fluorescence (charge recombination) hypothesis of Klimov and co-workers (Klimov, V.V. et al. (1978) Dokl. Akad. Nauk. SSSR 242, 1204–1207) or the proposed changes and origins put forward by Holzwarth and co-workers (Holzwarth, A.R. (1986) Photochem. Photobiol. 43, 707–725; Holzwarth, A.R. et al. (1985) Biochim. Biophys. Acta 807, 155–167).     

Intramembrane positions of membrane-bound chromophores determined by excitation energy transfer

A detailed theory has been derived to evaluate the efficiency of nonradiative transfer of electronic excitation energy between nonassociated membrane-bound chromophores. Two different approaches are presented and shown to lead to identical numerical results. In the first of these the efficiency of transfer is computed from the decay with time of the donor excited state. In the second approach, the efficiency is calculated directly, demonstrating that to a high degree of accuracy the array of acceptors can be represented as consisting of a single nearest acceptor plus a continuum of secondary acceptors. A general expression is derived for the dipole-dipole orientation factor as a function of the position of an acceptor. It is shown that, by invoking the range of orientations that must be present at the very least in a particular case, the expected values of transfer efficiency may be limited to a relatively narrow band of uncertainty about those predicted for total randomization. In the limit of total randomization, the theory reduces to functions of but two dimensionless parameters: an effective number of acceptors and a normalized distance of closest approach, a parameter which in turn is a function of an excluded surface area and the depth in the membrane of a donor relative to that of an acceptor. Finally, data analysis procedures are presented whereby one can determine the surface density of acceptors for a known geometry or, alternatively, determine the distance of closest approach for known surface densities.     

Inhibition of NADH oxidation by chloramphenicol in the freely moving rat measured by picosecond time-resolved emission spectroscopy

Owing to the lack of methods capable to monitor the energetic processes taking place within small brain regions (i.e. nucleus raphe dorsalis, nRD), the neurotoxicity of various categories of substances, including antibiotics and psycho-active drugs, still remains difficult to evaluate. Using an in vivo picosecond optical spectroscopy imaging method, we report that chloramphenicol (CAP), besides its well-known ability to inhibit the mitochondria protein synthesis, also influences the NADH/NAD+ redox processes of the respiratory chain. At a 200-mg/kg dose, CAP indeed produces a marked increase in the fluorescent signal of the nRD which, according to clear evidence, is likely to be related to the NADH concentration. This effect also implies an efficient inhibition of complex I of the respiratory chain by CAP. It refers to the mechanism through which the adverse effects of the antibiotic may take place. It could explain why paradoxical sleep, a state needing aerobic energy to occur, is suppressed after CAP administration. The present approach constitutes the first attempt to determine by fluorescence methods the effects of substances on deep brain structures of the freely moving animal. It points out that in vivo ultrafast optical methods are innovative and adequate tools for combined neurochemical and behavioural approaches.     

Polarized absorption picosecond kinetics as a probe of energy transfer in phycobilisomes of Synechococcus 6301

The energy transfer between C-phycocyanin chromophores in intact phycobilisomes of Synechococcus 6301 is shown to lead to an anisotropy relaxation with a lifetime of 10 ± 2 ps. However, due to the molecular order within the hexameric units of C-phycocyanin the anisotropy does not decay to zero. The Förster dipole-dipole mechanism of energy transfer can qualitatively explain these data provided that there is no back transfer of excitation energy and that the chromophore distribution is non-random. The rate of energy transfer in phycobilisomes between C-phycocyanin and allophycocyanin can best be described by a double exponential with lifetimes of 12 ± 3 and 84 ± 8 ps.     

Energy transfer by chlorophyll beta in detergent micelles.

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyll beta-containing detergent micelles with Triton X-100 were studied in a concentration range of c equal to 0.4 muM-0.6mM chlorophyll beta and cd equal to 0.4-7.0 mM Triton X-100. The concentration-dependent depolarization obeys F?rster's theory of depolarization of fluorescence with a transfer distance parameter R0 equal to 43 plus or minus 2 A. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yield n/n0 equal to 1/[1+(c/c1/2)-2] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149-161). With increasing chlorophyll beta concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyll beta molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyll beta yields chlorophyll beta homotransfer may be about 3-26% of the homotransfer chlorophyll alpha yields chlorophyll-alpha depending on the ratio of their concentrations.     

Temperature and detection-wavelength dependence of the picosecond electron-transfer kinetics measured in Rhodopseudomonas sphaeroides reaction centers. Resolution of new spectral and kinetic components in the primary charge-separation process

We have examined the temperature dependence of the rate of electron transfer to ubiquinone from the bacteriopheophytin (BPh) that serves as an initial electron acceptor (I) in reaction centers of Rhodopseudomonas sphaeroides. The kinetics were measured from the decay of the 665-nm absorption band of the reduced BPh (BPh⁻ or I⁻) and from the recovery of the BPh band at 545 nm, following excitation of reaction centers in polyvinyl alcohol films with 30-ps flashes. The measured time constant decreases from 229 ± 25 ps at 295 K to 97 ± 8 ps near 100 K and then remains constant down to 5 K. The temperature dependence of the kinetics can be rationalized on the assumption that the reaction results in changes in the frequencies of numerous low-energy nuclear (vibrational) modes of the electron carriers and/or the protein. The kinetics measured in the absorption bands near 765 and 795 nm show essentially the same temperature dependence as those measured at 545 or 665 nm, but the time constants vary with detection wavelength. The time constant measured in the 795-nm region (70 ± 10 ps at 5 and 76 K) is shorter than that seen in the absorption bands of the BPh; the time constant measured at 758 nm is longer. Time constants measured with reaction centers in solution at 288 K also vary with the detection wavelength. These results can be explained on the assumption that the absorption changes measured at some wavelengths reflect nuclear relaxations rather than electron transfer. The absorption changes at 795 nm probably reflect a relaxation of the bacteriochlorophyll molecules that are near neighbors of the BPh and the primary electron donor (P). Those near 530 and 755 nm probably are due to the second BPh molecule, which does not appear to undergo oxidation or reduction.     

Energy transfer in lipid bilayers.

The quenching of fluorescence due to energy transfer between a dilute, random array of donor and acceptor chromophores in lipid bilayer was measured and compared to theoretical expressions developed to predict the decrease in emission intensity under these circumstances. The observed intensity was found to be the same function of quencher concentration in both planar, multilamellar dispersions and small, spherical vesicles. The degree of quenching was accurately predicted by a simple relation derived in this paper, as well as a more complex equation previously developed by Tweet, et al. The results suggest that significant quenching may be observed even when the average donor-acceptor separation exceeds the F?rster critical distance by severalfold. Application of these results to problems of current interest in membrane research are discussed.     

Low-Intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts

The fluorescence decays of barley chloroplasts have been measured by single-photon counting with tunable picosecond dye laser excitation. The fluorescence decays of dark-adapted chloroplasts are best fitted to a sum of three exponential lifetime components with lifetimes of 112, 380 and 2214 ps. The relative magnitude of each component is shown to be dependent on the excitation wavelength and collected emission wavelength. The excitation wavelength dependence is correlated with the Photosystem (PS) I and PS II action study of Ried [36] and with the measured pigment distributions in the photosynthetic unit [37,41]. Experiments varying the single excitation pulse intensity from 10⁸ to 10¹² photons/cm² pulse show that our results are not distorted by singlet-singlet annihilation. Unflowed samples where the cloroplasts are under constant illumination show 2-fold increases in quantum yield of fluorescence primarily in the two longer lifetime components. Theoretical calculations of Shipman [31] on an isolated reaction center with a homogeneous antenna are discussed and the principles extended to discussion of the measured barley chloroplast fluorescence decay components in terms of photosynthetic unit light-harvesting array models and earlier experimental work. Our data support a photosynthetic unit model in which 70–90% of the photons absorbed are quenched by either PS I or efficiently quenching PS II in a process where the fluorescence lifetime is 100 ps. The origin of the intermediate 380 ps. component is probably due to excitation transfer to a PS II reaction center in a redox state which quenches less efficiently.     

Energy transfer kinetics of phycoerythrocyanin trimer from cyanobacteriumAnabaena variabilis (II)

The excitation energy transfer processes in trimeric PEC have been studied by using steady-state and time-resolved fluorescence spectra techniques in detail. The results indicate that the energy transfer processes should take place between α₈₄-PVB and β₈₄-or β₁₅₅-PCB chromophores with the time constants 34.7 ps and 175–200 ps individually; in contrast with monomeric PEC, from time-resolved fluorescence anisotropic spectrum technique, the decay constant of 45 ps which was assigned to the energy transfer time among three β₈₄-PCB chromophores was observed and the energy levels of β₈₄-and/or β₁₅₅-PCB chromophores were confirmed to turn over in trimeric PEC.     

Comparison of sickle cell hemoglobin gelation kinetics measured by NMR and optical methods.

We describe a technique for monitoring the kinetics of sickle cell hemoglobin gelation by observing the change in the amplitude and linewidth of the water proton magnetic resonance. The resulting kinetic progress curves are very similar to those obtained by optical birefringence and turbidity methods. The curves consist of a delay, followed by a rapidly accelerating signal change which terminates quickly. From a study of the temperature dependence of the delay time, it is shown that all three techniques see the onset of gelation simultaneously. The origin of the change in physical properties upon gelation is briefly discussed in relation to the component steps of the reaction.     

The ligand affinity of proteins measured by isothermal denaturation kinetics

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.