Energy transfer and bacteriochlorophyll fluorescence in purple bacteria at low temperature

Emission spectra of bacteriochlorophyll a fluorescence and absorption spectra of various purple bacteria were measured at temperatures between 295 and 4 K. For Rhodospirillum rubrum the relative yield of photochemistry was measured in the same temperature region. In agreement with earlier results, sharpening and shifts of absorption bands were observed upon cooling to 77 K. Below 77 K further sharpening occurred. In all species an absorption band was observed at 751-757 nm. The position of this band and its amplitude relative to the concentration of reaction centers indicate that this band is due to reaction center bacteriopheophytin. The main infrared absorption band of Rhodopseudomonas sphaeroides strain R26 is resolved in two bands at low temperature, which may suggest that there are two pigment-protein complexes in this species. Emission bands, like the absorption bands, shifted and sharpened upon cooling. The fluorescence yield remained constant or even decreased in some species between room temperature and 120 K, but showed an increased below 120 K. This increase was most pronounced in species, such as R. rubrum, which showed single banded emission spectra. In Chromatium vinosum three (835, 893 and 934 nm) and in Rps. sphaeroides two (888 and 909 nm) emission bands were observed at low temperature. The temperature dependence of the amplitudes of the short wavelength bands indicated the absence of a thermal equilibrium for the excitation energy distribution in C. vinosum and Rps. sphaeroides. In all species the increased in the yield was larger when all reaction centers were photochemically active than when the reaction centers were closed. In R. rubrum the increase in the fluorescence yield was accompanied by a decrease of the quantum yield of charge separation upon excitation of the antenna but not of the reaction center chlorophyll. Calculation of the F?rster resonance integral at various temperatures indicated that the increase in fluorescence yield and the decrease in the yield of photochemistry may be due to a decrease in the rate of energy transfer between antenna bacteriochlorophyll molecules. The energy transfer from carotenoids to bacteriochlorophyll was independent of the temperature in all species examined. The results are discussed in terms of existing models for energy transfer in the antenna pigment system.     

Energy transfer among the chromophores in phycocyanins measured by picosecond kinetics.

Energy-transfer processes in the algal light-harvesting proteins, the phycocyanins, have been studied by means of picosecond absorption spectroscopy. After excitation at 530 nm, the absorption at several wavelengths in the range 480--669 nm decayed with a short time constant (picosecond) and a long time constant (greater than 1 ns). For C-phycocyanin, energy transfer from the beta to the alpha subunits is interpreted as being a likely candidate for the short time constant; the long time constant probably is the excitation lifetime of the chromophore on the alpha subunits. The time constants for energy transfer in monomers, trimers, and hexamers of C-phycocyanin extracted from a blue-green alga, Phormidium luridum, were measured as approximately 85, approximately 56, and approximately 32 ps, respectively. The corresponding time constant in the cryptomonad phycocyanin 645 from Chroomonas species was found to be less than 5 ps.     

Energy transfer in spectrally inhomogeneous light-harvesting pigment-protein complexes of purple bacteria.

Energy transfer within the peripheral light-harvesting antenna of the purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris was studied by one- and two-color pump-probe absorption spectroscopy with approximately 100-fs tunable pulses at room temperature and at 77 K. The energy transfer from B800 to B850 occurs with a time constant of 0.7 +/- 0.05 ps at room temperature and 1.8 +/- 0.2 ps at 77 K and is similar in both species. Anisotropy measurements suggest a limited but fast B800 <--> B800 transfer time (tau approximately 0.3 ps). This is analyzed as incoherent hopping of the excitation in a system of spectrally inhomogeneous antenna pigment-protein complexes, by a master equation approach. The simulations show that the measured B800 dynamics is well described as energy transfer with a characteristic average nearest-neighbor pairwise transfer time of 0.35 ps among approximately 10 Bchl molecules in a circular arrangement, in good agreement with the recent high-resolution structure of LH2. The possible presence of fast intramolecular relaxation processes within the Bchl a molecule was investigated by measurement of time-resolved difference absorption spectra and kinetics of Bchl a in solution and in low-temperature glasses. From these measurements it is concluded that fast transients observed at room temperature are due mainly to solvation processes, whereas at 77 K predominantly slower (> 10-ps) relaxation occurs.     

Biochemical characterization of the dissociated forms from the core antenna proteins from purple bacteria

The core light-harvesting protein from Rhodospirillum rubrum is of particular interest for studying membrane polypeptide association, as it can be reversibly dissociated in the presence of n-octyl-beta-D-glucopyranoside (betaOG) into smaller subunit forms, which exhibit dramatically blue-shifted absorption properties (Miller et al. (1987) Biochemistry 26, 5055-5062). During this dissociation/reassociation process, two main spectroscopic forms are observed, absorbing at 820 (B820) and 777 (B777) nm, respectively. By using polyacrylamide gel electrophoresis in the presence of betaOG, these forms were characterized from a biochemical point of view. B777 consist of a mixture of alpha or beta polypeptide chains, retaining their bound bacteriochlorophyll (BChl) molecules. The absorption properties of the BChl molecules bound to the monomeric polypeptides do not depend on the chemical nature of the polypeptides they are bound to. B820 is more complex and consist of equilibrium between alphabeta-containing oligomers and beta only containing dimers, all exhibiting very similar electronic absorption properties. Resonance Raman spectroscopy indicates that the binding site provided by the beta-only B820 to the BChl molecules is very similar to that provided by the alphabeta B820. This, together with the observation that the alpha polypeptide alone is unable to form B820, suggests that the local organization of the BChl molecules tightly depends on BChl-protein interactions. On the other hand, our results suggest that the affinity of the beta-BChl complexes for itself and for the alpha-BChl ones are of the same order of magnitude, the formation of heterodimeric complexes being mainly driven by the inability of alpha-BChl complexes to self-associate.     

Energy transfer and trapping in the photosystem I core antenna. A temperature study.

The fluorescence decay kinetics of the photosystem I-only mutant strain of Chlamydomonas reinhardtii, A4d, are used to study energy transfer and structural organization in photosystem I (PSI). Time-resolved measurements over a wide temperature range (36-295 K) have been made both on cells containing approximately 65 core chl a/P700 and an additional 60-70 chl a + b from LHC proteins and on PSI particles containing 40-50 chl a/P700. In each case, the fluorescence decay kinetics is dominated by a short component, tau 1 which is largely attributed to the lifetime of the excitations in the core complex. The results are discussed in terms of simulations of the temperature dependence of tau 1 in model systems. Spectral inhomogeneity and the temperature dependence of the spectral lineshapes are included explicitly in the simulations. Various kinds of antenna arrangements are modeled with and without the inclusion of pigments with lower absorption energies than the trap (red pigments). We conclude that funnel arrangements are not consistent with our measurements. A random model that includes one or two red pigments placed close to the trap shows temperature and wavelength dependence similar to that observed experimentally. A comparison of the temperature dependence of tau 1 for cells and PSI particles is included.     

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.     

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λ_em = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λ_exc = 630 or λ_exc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F₀), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τ_m and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the F_max state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.     

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 in the purple membrane of Halobacterium halobium.

The absorption spectrum of the primary photoproduct (the bathoproduct, or K) of the purple membrane protein (PM) at-196 degrees C has a maximum at 628 nm and an extinction coefficient of 87,000. Knowing the absorption spectrum allowed us to calculate the quantum efficiencies for PM to K and K to PM conversion at -196 degrees C. Direct measurements of these quantum yeilds at -196 degrees C gave 0.33 +/- 0.05 and 0.67 +/- 0.04, respectively. Determination of relative quantum efficiencies for PM to K and K to PM conversion by analysis of the absorption spectra of several photostationary-state mixtures of PM and K at -196 degrees C, however, gave wavelength-dependent quantum efficiencies that appear to be greater than 1. These anomolous results can be readily explained in terms of energy transfer from PM to K within the trimer clusters of pigment molecules which exist in the purple membrane. A model for such a transfer predicts an efficiency of energy transfer from PM to K of about 43%.     

Exciton dynamics in circular aggregates: application to antenna of photosynthetic purple bacteria.

A theoretical model of exciton dynamics in circular molecular aggregates of light-harvesting bacteriochlorophyll of photosynthetic bacteria is proposed. The spectra and anisotropy of photoinduced absorption changes in the femto- and picosecond time domain are under its scope. The excited state of aggregate was treated due to the standard exciton theory, taking into account a pigment inhomogeneity. Dephasing processes via the exciton-phonon interactions were described by means of the Haken-Strobl equation. It was shown that only two exciton levels are dipole-allowed in the case of homogeneous circular aggregate. The pigment inhomogeneity results in the appearance of several weak transitions to higher exciton levels. It was proposed that the minor band (B896) in an absorption spectrum of the B875 complex as well as the similar minor band in spectra of B800-850 complex correspond to electron transition from the ground to the lowest exciton level, whereas the major band corresponds to transition to the higher exciton level. The proposed model shows the subpicosecond decay of anisotropy at the short-wavelength side of absorption band and a high degree of anisotropy at the long-wavelength side, even at high temperatures.     

Singlet-singlet annihilation at low temperatures in the antenna of purple bacteria

Chromatophores of the purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter (Rhodopseudomonas) sphaeroides were excited by means of 35-ps flashes at 532 nm of varying intensities, both at room temperature and at 4 K. With increasing exciting energy densities the integrated yield of fluorescence produced by these flashes was found to decrease considerably due to singlet-singlet annihilation. An analysis of the results showed that in R. rubrum the number of connected antenna molecules between which energy transfer is possible decreases from about 1000 to about 150 when the temperature is lowered from 298 to 4 K. In Rb. sphaeroides the B875 light-harvesting complex appears to contain about 100 connected bacteriochlorophyll (BChl) 875 molecules at 4 K, while the B800–850 complex contains about 45 BChl 850 molecules. The data are explained by a model for the antenna of Rb. sphaeroides in which units of B875, containing about four reaction centres, are separated by an array of B800–850 units that surrounds B875. By applying a random walk model we found that in both species the rate of energy transfer between neighbouring antenna molecules decreased about 10-fold upon lowering the temperature. The rate of energy transfer from antenna molecules to either open or closed reaction centres decreased only 3- to 4-fold in R. rubrum and remained approximately constant in Rb. sphaeroides upon cooling. A blue shift of the emission spectra at 4 K of both species was observed when the excitation energy density was increased to a level where singlet-singlet annihilation plays a significant role. This observation appears to support the notion that an additional long-wave pigment exists in the antenna of these bacteria.     

Energy transfer and charge separation kinetics in photosystem I: Part 1: Picosecond transient absorption and fluorescence study of cyanobacterial photosystem I particles

The energy transfer and charge separation kinetics of a photosystem I (PS I) core particle of an antenna size of 100 chlorophyll/P700 has been studied by combined fluorescence and transient absorption kinetics with picosecond resolution. This is the first combined picosecond study of transient absorption and fluorescence carried out on a PS I particle and the results are consistent with each other. The data were analyzed by both global lifetime and global target analysis procedures. In fluorescence major lifetime components were found to be 12 and 36 ps. The shorter-lived one shows a negative amplitude at long wavelengths and is attributed to an energy transfer process between pigments in the main antenna Chl pool and a small long-wavelength Chl pool emitting around 720 nm whereas the longer-lived component is assigned to the overall charge separation lifetime. The lifetimes resolved in transient absorption are 7-8 ps, 33 ps, and [unk]1 ns. The shortest-lived one is assigned to energy transfer between the same pigment pools as observed also in fluorescence kinetics, the middle component of 33 ps to the overall charge separation, and the long-lived component to the lifetime of the oxidized primary donor P700⁺. The transient absorption data indicate an even faster, but kinetically unresolved energy transfer component in the main Chl pool with a lifetime <3 ps. Several kinetic models were tested on both the fluorescence and the picosecond absorption data by global target analysis procedures. A model where the long-wave pigments are spatially and kinetically connected with the reaction center P700 is favored over a model where P700 is connected more closely with the main Chl pool. Our data show that the charge separation kinetics in these PS I particles is essentially trap limited. The relevance of our data with respect to other time-resolved studies on PS I core particles is discussed, in particular with respect to the nature and function of the long-wave pigments. From the transient absorption data we do not see any evidence for the occurrence of a reduced Chl primary electron acceptor, but we also can not exclude that possibility, provided that reoxidation of that acceptor should occur within a time <40 ps.     

PSII核心复合物能量传递的飞秒时间分辩荧光光谱学研究

运用稳态、瞬态荧光光谱技术对光系统Ⅱ核心复合物的能量传递动力学进行研究。分别用436nm光脉冲激发叶绿素a分子、451nm光激发叶绿素a和β-胡萝卜素分子、473和481nm光激发β-胡萝卜素分子,得到5组反应能量传递、电荷重组等过程的寿命组分：8～40 ps为核心天线中β-胡萝卜素分子通过相邻β-胡萝卜素分子或中间叶绿素a向叶绿素a分子传递能量的时间;85～152 ps为核心天线色素分子激发能传递时间;201～925ps反映部分电荷重组过程;1．031～1．21ns为参与能量传递的色素分子从激发态衰退回到基态的时间;6．17～18、13 ns的长寿命时间组分归因于P680^ Pheo^-的重组过程。将荧光发射谱进行高斯解析,发现在核心复合物中还至少存在Chla683^685、Chla680^682、Chla673,677^679三种特征叶绿素a分子。     

Energy transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II

The energy equilibration and transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II have been studied by steady-state and ultrafast (femto- to nanosecond) time-resolved spectroscopy at room temperature. The annihilation-free femtosecond absorption data can be described by surprisingly simple sequential kinetic models, in which the excitation energy transfer between blue and red states in both antenna complexes is dominated by sub-picosecond processes and is completed in less than 2 ps. The slowest energy transfer steps with lifetimes in the range of 1-2 ps are assigned to transfer steps between the chlorophyll layers located on the stromal and lumenal sides. We conclude that these ultrafast intra-antenna energy transfer steps do not represent a bottleneck in the rate of the primary processes in intact photosystem II. Since the experimental energy equilibration rates are up to a factor of 3-5 higher than concluded previously, our results challenge the conclusions drawn from theoretical modeling.     

Probing the kinetics of photosystem I and photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer.

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.     

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.