Energy transfer kinetics of phycoerythrocyanin trimer from cyanobacterium Anabaena 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. Project supported by the National Natural Science Foundation of China (Grant No. 39670065).     

Ultrafast energy transfer pathways in R-phycoerythrin from Polysiphonia urceolata

Energy transfer (ET) processes between chromophores in R-phycoerythrin (R-PE) from Polysiphonia urceolata were studied by use of ultrafast spectroscopic methods. Several primary ET pathways were elaborated. A fluorescence decay component with a time constant of several hundred picoseconds observed by streak camera is tentatively assigned to the reversible formation of exciton traps between α84 and β84 pigment pairs. In order to investigate much faster ET processes in R-PE, a noncollinear optical parametric amplifier based femtosecond time-resolved transient fluorescence spectrometer was employed. The results reveal that the ET between α84 and β84 pigment pair has a time constant of 1-2?ps; the energy migration between α84 and β84 pairs within the R-PE trimer has a time constant of 30-40?ps. We also demonstrated an ET process from phycourobilin to phycoerythrobilin with a time constant as fast as 2.5-3.0?ps, which was directly observed in fluorescence kinetics by selective excitation of the phycourobilin molecules acting as the energy donor.     

Studies on chromophore coupling in isolated phycobiliproteins: III. Picosecond excited state kinetics and time-resolved fluorescence spectra of different allophycocyanins from Mastigocladus laminosus

The excited state kinetics of three different allophycocyanin (AP) complexes has been studied by picosecond fluorescence spectroscopy. Both the fluorescence kinetics and the decay-associated fluorescence spectra of the different complexes can be understood on the basis of a structural model for AP which uses (a) an analogy to the known x-ray determined structure of C-phycocyanin, (b) the biochemical analogies of AP and C-phycocyanin, and (c) the biochemical composition of AP-B (AP-681). A model is developed that describes the excited state kinetics as a mixture of internal conversion processes within a coupled exciton pair and energy transfer processes between exciton pairs. We found excited state relaxation times in the range of 13 ps (AP with linker peptide) up to 66 ps (AP-B). The trimeric aggregates AP 660 and AP 665 show one fast relaxation component each, as was expected on the basis of their symmetry properties. The lower symmetry of AP-B (AP-681) gives rise to two fast lifetime components (τ₁ = 23 ps and τ₂ = 66 ps) which are attributed to internal conversion and/or energy transfer between excitonic states formed by the coupling of symmetrically and spectrally nonequivalent chromophores. It is proposed that the internal conversion between exciton states of strongly coupled chromophores fulfills the requirements of the small energy gap limit. Thus, internal conversion rates in the order of tens of picoseconds are feasible. The influence of the interaction of the linker peptide on the properties of the AP trimer are manifested in the fluorescence kinetics. Lack of the linker peptide in AP 660 gives rise to a heterogeneity in the chromophore conformations and chromophore-chromophore interactions.     

Picosecond study of energy-transfer kinetics in phycobilisomes of Synechococcus 6301 and the mutant AN 112

Excitation-energy-transfer kinetics in isolated phycobilisomes from the cyanobacterium Synechococcus 6301 (Anacystis nidulans) and the mutant AN 112 (rods containing one hexameric C-phycocyanin unit only) was investigated by picosecond absorption and fluorescence techniques. The different chromophores in the phycobilisomes were selectively excited. A lifetime component of about 10 ps was found for both C-phycocyanin and allophycocyanin in both types of phycobilisomes. We assign these signals to a transfer of excitation energy from sensitizing (‘s’) to fluorescing (‘f’) chromophores within C-phycocyanin and allophycocyanin units. A 10 ps component was also observed in the anisotropy relaxation measurements. The anisotropy decay is attributed mainly to differently oriented transition dipole moments of ‘s’- and ‘f’-chromophores and partially to ‘f’ → ‘f’ transfer. An absorption recovery signal of τ ≈ 90 ps at λ ≤ 630 nm in phycobilisomes of Synechococcus 6301 is reduced to 40–50 ps in AN 112 phycobilisomes. This is rationalized in terms of a decreased rod → core transfer time in the shorter rods of AN 112. The 40–50 ps lifetime of fluorescence and absorption recovery in AN 112 phycobilisomes is assigned mainly to a rate-limiting transfer step between C-phycocyanin and the allophycocyanin core. A decay component of allophycocyanin τ ≈ 50 ps was observed both in absorption recovery measurements and in fluorescence decay. It is assigned to energy transfer to the terminal chromophores. The final emitter(s) of the phycobilisomes from AN 112 have fluorescence lifetimes of 1.9 and 1.3 ns. We find a good correlation in the fluorescence kinetics between the decay times of phycocyanin and allophycocyanin and the fluorescence risetimes of the terminal emitters.     

Functional assignment of chromophores and energy transfer in C phycocyanin isolated from the thermophilic cyanobacterium Mastigocladus laminosus

The optical characteristics and pathway of energy transfer in the C phycocyanin trimer isolated from the thermophilic cyanobacterium Mastigocladus laminosus were investigated at steady state by absorption, circular dichroism, fluorescence and fluorescence polarization spectroscopy. Based on the comparison of optical data with the 3-dimensional structure of the C-phycocyanin trimer determined by X-ray analysis (Schirmer, T., Bode, W., Huber, R., Sidler, W. and Zuber, H. (1984) in Proceedings of the Symposium on Optical Properties and Structure of Tetrapyrroles, (Blauer, G. and Sund, M., eds.), pp. 445–449, Walter de Gruyter, Berlin, and (1985) J. Mol. Biol. 184, 257–277), the functional assignment of three types of chromophore was established. An α subunit has an s chromophore and the chromophores at the positions 84 and 155 in the amino acid sequence of the β subunit are assigned as f and s chromophores, respectively. In the C phycocyanin trimer energy transfer occurs from the α chromophore in one monomer to the β_f chromophore in an adjacent monomer, and from the β_s chromophore to the β_f chromophore in the same monomer. The direction of energy flow is from the outside to the inside of the trimer, where the locus for the binding of a colourless polypeptide is postulated. In the phycobilisomes the energy concentrated at the β_f chromophores might be transferred toward the allophycocyanin core mainly by the β_f chromophores in the phycocyanin rods.     

Analysis of the Disk-to-Disk Energy Transfer Processes in C-Phycocyanin Complexes by Computer Simulation Technique

Based on the crystal structure and spectral properties of C-phycocyanin (C-PC) from cyanobacteria, models for complexes with 2 and 3 C-PC hexamer disks were built and the energy transfer dynamic properties were studied by the use of stochastic computer simulation approach. In addition, an experimental parameter of 0.056 ps^–1, corresponding to a time constant of 18 ps, derived from the previous time-resolved measurement, was used for simulation of the energy transfer process from the three terminal symmetrically equivalent ₈₄ chromophores of the core-linked disk to an ₈₄ chromophore of the allophycocyanin (APC) core. The simulation showed: (1) The disk-to-disk energy transfer can be as fast as several picoseconds. (2) The energy transfer efficiencies from the first disk to the core would depend on the length of the rod (i.e. the number of disks). Efficiencies of 0.95, 0.87, and 0.75 were found for the rods with 1, 2 and 3 hexamer disks, respectively. (3) The energy transfer along a rod in a native phycobilisome (PBS) is probably very close to the one-way manner. It is the core of PBS that makes the excitation energy be transferred fast in a nearly one-way manner.     

A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements

In this article we report the characterization of the energy transfer process in the reconstituted isoforms of the plant light-harvesting complex II. Homotrimers of recombinant Lhcb1 and Lhcb2 and monomers of Lhcb3 were compared to native trimeric complexes. We used low-intensity femtosecond transient absorption (TA) and time-resolved fluorescence measurements at 77 K and at room temperature, respectively, to excite the complexes selectively in the chlorophyll b absorption band at 650 nm with 80 fs pulses and on the high-energy side of the chlorophyll a absorption band at 662 nm with 180 fs pulses. The subsequent kinetics was probed at 30–35 different wavelengths in the region from 635 to 700 nm. The rate constants for energy transfer were very similar, indicating that structurally the three isoforms are highly homologous and that probably none of them play a more significant role in light-harvesting and energy transfer. No signature has been found in the transient absorption measurements at 77 K for Lhcb3 which might suggest that this protein acts as a relative energy sink of the excitations in heterotrimers of Lhcb1/Lhcb2/Lhcb3. Minor differences in the amplitudes of some of the rate constants and in the absorption and fluorescence properties of some pigments were observed, which are ascribed to slight variations in the environment surrounding some of the chromophores depending on the isoform. The decay of the fluorescence was also similar for the three isoforms and multi-exponential, characterized by two major components in the ns regime and a minor one in the ps regime. In agreement with previous transient absorption measurements on native LHC II complexes, Chl b → Chl a energy transfer exhibited very fast channels but at the same time a slow component (ps). The Chls absorbing at around 660 nm exhibited both fast energy transfer which we ascribe to transfer from ‘red’ Chl b towards ‘red’ Chl a and slow transfer from ‘blue’ Chl a towards ‘red’ Chl a. The results are discussed in the context of the new available atomic models for LHC II.     

多管藻中R-藻蓝蛋白能量传递途径及动力学

以蛋白亚基复性技术和皮秒级时间分辨荧光光谱,研究海洋红藻多管藻中R－藻蓝蛋白（R－PC）单体和三聚体内能量传递过程。利用亚基复性技术对分离后的β亚基复性,以R－藻蓝蛋白单体和β亚基之间的差谱获得α亚基的吸收光谱。皮秒级时间分辨三维谱图（时间、波长和强度）直观地显示出藻红胆素发色团向藻蓝胆素发色团的能量传递;根据时间分辨测量结果的组份解析,对R藻蓝蛋白单体和三聚体内能量传递途径和相关传递参数进行了指认和讨论;对观察到的单体与三聚体能量传递组份特性的差别提出了解释。与C－藻蓝蛋白光谱对比,R－藻蓝蛋白独特的色团组成使其更有效地捕获与传递光能。     

蓝藻(A.variabilis)藻胆体核结构与功能的研究II.变藻蓝蛋白六聚体内能量传递过程的物理机制

以变藻蓝蛋白的晶体结构和光谱性质为基础,利用密度矩阵理论对变藻蓝蛋白六聚体内的激发能传递物理机制进行分析,并利用时间分辨荧光光谱技术对其能量传递途径进行实时探测。结果表明：在变藻蓝蛋白六聚体内,色素对（毗邻单体上的色素αｉ８４βｊ８４,其中ｊ＝ｉ±１,和β＊ＬＣＭ４２）内的能量传递服从激子偶极－偶极相互作用机制;而色素对之间的能量传递机制则为Ｆｒｓｔｅｒ偶极－偶极相互作用机制,并且其能量传递途径分为两类：（１）．两个变藻蓝蛋白三聚体之间色素对的能量传递,其时间常数大约为１５ｐｓ左右;（２）．同一变藻蓝蛋白三聚体内色素对间的能量传递,在ＡＰＩＩ三聚体内,其能量传递时间大约为４５ｐｓ左右,而在ＡＰＩ三聚体内,其能量传递时间常数为４５ｐｓ和６５ｐｓ。     

蓝藻(A.variabilis)藻胆体核结构与功能的研究II.变藻蓝蛋白六聚体内能量传递过程的物理机制

以变藻蓝蛋白的晶体结构和光谱性质为基础,利用密度矩阵理论对变藻蓝蛋白六聚体内的激发能传递物理机制进行分析,并利用时间分辨荧光光谱技术对其能量传递途径进行实时探测。结果表明：在变藻蓝蛋白六聚体内,色素对（毗邻单体上的色素αｉ８４βｊ８４,其中ｊ＝ｉ±１,和β＊ＬＣＭ４２）内的能量传递服从激子偶极－偶极相互作用机制;而色素对之间的能量传递机制则为Ｆｒｓｔｅｒ偶极－偶极相互作用机制,并且其能量传递途径分为两类：（１）．两个变藻蓝蛋白三聚体之间色素对的能量传递,其时间常数大约为１５ｐｓ左右;（２）．同一变藻蓝蛋白三聚体内色素对间的能量传递,在ＡＰＩＩ三聚体内,其能量传递时间大约为４５ｐｓ左右,而在ＡＰＩ三聚体内,其能量传递时间常数为４５ｐｓ和６５ｐｓ。     

Studies on the Kinetic Properties of Photosystem Ⅱ Primary Reaction Using Fluorescence Spectroscopy with 470 fs Time Resolution

The primary reaction kinetics of the isolated photosystem Ⅱ particles and photosystem Ⅱ core complexes from spinach ( Spinacia deracea Mill. ) was investigated using the time-resolved fluorescence spectroscopy with 470 fs time resolution. 2 to 4 lifetime components were detected by the multi-exponential curve fining method. These components were analyzed and discussed in terms of different kinetic processes. It is suggested that 3 ps component is attributed to the charge separation and 0.8 ps, 12 ps, 25 ps and 100 ps components are related to the energy transfer processes. A possible kinetic scheme in photosystem Ⅱ reaction center was proposed based upon the reported previously result.     

Time-resolved absorption and emission show that the CP43' antenna ring of iron-stressed synechocystis sp. PCC6803 is efficiently coupled to the photosystem I reaction center core

Excitation energy transfer and trapping processes in an iron stress-induced supercomplex of photosystem I from the cyanobacterium Synechocystis sp. PCC6803 were studied by time-resolved absorption and fluorescence spectroscopy on femtosecond and picosecond time scales. The data provide evidence that the energy transfer dynamics of the CP43'-PSI supercomplex are consistent with energy transfer processes that occur in the Chl a network of the PSI trimer antenna. The most significant absorbance changes in the CP43'-PSI supercomplex are observed within the first several picoseconds after the excitation into the spectral region of CP43' absorption (665 nm). The difference time-resolved spectra (DeltaDeltaA) resulting from subtraction of the PSI trimer kinetic data from the CP43'-PSI supercomplex data indicate three energy transfer processes with time constants of 0.2, 1.7, and 10 ps. The 0.2 ps kinetic phase is tentatively interpreted as arising from energy transfer processes originating within or between the CP43' complexes. The 1.7 ps phase is interpreted as possibly arising from energy transfer from the CP43' ring to the PSI trimer via closely located clusters of Chl a in CP43' and the PSI core, while the slower 10 ps process might reflect the overall excitation transfer from the CP43' ring to the PSI trimer. These three fast kinetic phases are followed by a 40 ps overall excitation decay in the supercomplex, in contrast to a 25 ps overall decay observed in the trimer complex without CP43'. Excitation of Chl a in both the CP43'-PSI antenna supercomplex and the PSI trimer completely decays within 100 ps, resulting in the formation of P700(+). The data indicate that there is a rapid and efficient energy transfer between the outer antenna ring and the PSI reaction center complex.     

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.     

PSI-SMALP,a Detergent-free Cyanobacterial Photosystem I,Reveals Faster Femtosecond Photochemistry

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c₆-ferredoxin/oxidoreductase located in the thylakoid membrane. In this work, the energy and charge transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-β-D-maltoside solubilization (DM-PSI) and a, to our knowledge, new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll forms within 0.1–20 ps and revealed little or no charge separation and oxidation of the special pair, P700. The formation of ion-radical pair P700⁺A1⁻ occurred with a characteristic time of 36 ps, being kinetically controlled by energy transfer from the long-wavelength chlorophyll to P₇₀₀. Quite surprisingly, the detergent-free SMA-PSI complexes upon excitation by these long-wave pulses undergo an ultrafast (<100 fs) charge separation in ∼45% of particles. In the remaining complexes (∼55%), the energy transfer to P₇₀₀ occurred at ∼36 ps, similar to the DM-PSI. Both isolation methods result in a trimeric form of PSI, yet the SMA-PSI complexes display a heterogenous kinetic behavior. The much faster rate of charge separation suggests the existence of an ultrafast pathway for charge separation in the SMA-PSI that may be disrupted during detergent isolation.     

Lateral energy transfer model for adjacent light-harvesting antennae rods of C-phycocyanins

Modeling of excitation transfer pathways have been carried out for the structure of Spirulina platensis C-phycocyanin. Calculations by Förster mechanism using the crystal structure coordinates determined in our laboratory indicate ultra-fast lateral energy transfer rates between pairs of chromophores attached to two adjacent hexamer disks. The pairwise transfer times of the order of a few pico-seconds correspond to resonance transitions between peripheral β155 chromophores. A quantitative lateral energy transfer model for C-phycocyanin light-harvesting antenna rods that is suggestive to its native structural organization emerges from this study.     

A Kinetic Model for the Energy Transfer in Phycobilisomes

A kinetic model for the energy transfer in phycobilisome (PBS) rods of Synechococcus 6301 is presented, based on a set of experimental parameters from picosecond studies. It is shown that the enormous complexity of the kinetic system formed by 400-500 chromophores can be greatly simplified by using symmetry arguments. According to the model the transfer along the phycocyanin rods has to be taken into account in both directions, i.e., back and forth along the rods. The corresponding forward rate constants for single step energy transfer between trimeric disks are predicted to be 100-300 ns^-1. The model that best fits the experimental data is an asymmetric random walk along the rods with overall exciton kinetics that is essentially trap-limited. The transfer process from the sensitizing to the fluorescing C-PC phycocyanin chromophores (τ ≈ 10 ps) is localized in the hexamers. The transfer from the innermost phycocyanin trimer to the core is calculated to be in the range 36-44 ns^-1. These parameters lead to calculated overall rod-core transfer times of 102 and 124 ps for rods containing three and four hexamers, respectively. The model calculations confirm the previously suggested hypothesis that the energy transfer from the rods to the core is essentially described by one dominant exponential function. Extension of the model to heterogeneous PBS rods, i.e., PBS containing also phycoerythrin, is straightforward.     

Comparison of excitation energy transfer in cyanobacterial photosystem I in solution and immobilized on conducting glass

Excitation energy transfer in monomeric and trimeric forms of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 in solution or immobilized on FTO conducting glass was compared using time-resolved fluorescence. Deposition of PSI on glass preserves bi-exponential excitation decay of ~4–7 and ~21–25 ps lifetimes characteristic of PSI in solution. The faster phase was assigned in part to photochemical quenching (charge separation) of excited bulk chlorophylls and in part to energy transfer from bulk to low-energy (red) chlorophylls. The slower phase was assigned to photochemical quenching of the excitation equilibrated over bulk and red chlorophylls. The main differences between dissolved and immobilized PSI (iPSI) are: (1) the average excitation decay in iPSI is about 11 ps, which is faster by a few ps than for PSI in solution due to significantly faster excitation quenching of bulk chlorophylls by charge separation (~10 ps instead of ~15 ps) accompanied by slightly weaker coupling of bulk and red chlorophylls; (2) the number of red chlorophylls in monomeric PSI increases twice—from 3 in solution to 6 after immobilization—as a result of interaction with neighboring monomers and conducting glass; despite the increased number of red chlorophylls, the excitation decay accelerates in iPSI; (3) the number of red chlorophylls in trimeric PSI is 4 (per monomer) and remains unchanged after immobilization; (4) in all the samples under study, the free energy gap between mean red (emission at ~710 nm) and mean bulk (emission at ~686 nm) emitting states of chlorophylls was estimated at a similar level of 17–27 meV. All these observations indicate that despite slight modifications, dried PSI complexes adsorbed on the FTO surface remain fully functional in terms of excitation energy transfer and primary charge separation that is particularly important in the view of photovoltaic applications of this photosystem.     

Energy transfer processes in Gloeobacter violaceus PCC 7421 that possesses phycobilisomes with a unique morphology

We examined energy transfer dynamics in phycobilisomes (PBSs) of cyanobacteria in relation to the morphology and pigment compositions of PBSs. We used Gloeobacter violaceus PCC 7421 and measured time-resolved fluorescence spectra in three types of samples, i.e., intact cells, PBSs, and rod assemblies separated from cores. Fremyella diplosiphon, a cyanobacterial species well known for its complementary chromatic adaptation, was used for comparison after growing under red or green light. Spectral data were analyzed by the fluorescence decay-associated spectra with components common in lifetimes with a time resolution of 3 ps/channel and a spectral resolution of 2 nm/channel. This ensured a higher resolution of the energy transfer kinetics than those obtained by global analysis with fewer sampling intervals. We resolved four spectral components in phycoerythrin (PE), three in phycocyanin (PC), two in allophycocyanin, and two in photosystem II. The bundle-like PBSs of G. violaceus showed multiple energy transfer pathways; fast (≈ 10 ps) and slow (≈ 100 ps and ≈ 500 ps) pathways were found in rods consisting of PE and PC. Energy transfer time from PE to PC was two times slower in G. violaceus than in F. diplosiphon grown under green light.