INCREASED ENERGY TRANSFER FROM PHYCOBILISOMES TO PHOTOSYSTEM II IN HIGH LIGHT ADAPTED Anabaena cylindrica

Excitation energy transfer from phycobilisomes to photosystem II in high-light adapted cells of Anabaena cylindrica was studied by fluorescence spectroscopy and compared to that of low-light adapted cells. Measurements were made on membrane fragments containing phycobilisomes, photosystem I and II, isolated in 0.75 M K-phosphate. Relative efficiency of 430 to 590 nm light in the excitation of F₆₈₀ chlorophyll fluorescence was compared in low and high light adapted cells, respectively. The values indicate that light energy absorbed by phycobilisomes is transferred to photosystem II antenna chlorophylls with higher efficiency in high-light adapted cells than in low-light adapted cells. Partial dissociation and uncoupling of energy transfer caused by low ion concentration were different in the membrane fragments isolated from the two kinds of cells and indicated a higher aggregation state of pigment-protein complexes of phycobilisomes in high-light adapted A. cylindrica cells.     

HOLE-BURNING SPECTROSCOPY OF ACTIVE AND INACTIVATEE) PHOTOSYSTEM II PARTICLES

This paper reports transient and persistent hole-burning of photosynthetically active as well as chemically reduced and heat inactivated photosystem II particles isolated from cyanobacteria. Transient spectra of active and non-active particles are significantly different. For both, the possible origin of the bottle-neck state is discussed. Persistent holes were ascribed to the antenna complex of photosystem II. From their width the energy transfer rate was estimated to be 4.8 ps at 4.2 K.     

PIGMENT ORGANIZATION IN THE LIGHT-HARVESTING CHLOROPHYLL-a/b PROTEIN COMPLEX OF LETTUCE CHLOROPLASTS. EVIDENCE OBTAINED FROM PROTECTION OF THE CHLOROPHYLLS AGAINST PROTON ATTACK and FROM EXCITATION ENERGY TRANSFER

Abstract— The light-harvesting Chl-a/b protein complex (LHC) of Lactuca sativa L. was examined for pigment content, excitation energy transfer and behavior under acidic conditions:
(1) Lettuce LHC contains Chl-a, Chl-b and xanthophylls (lutein, neoxanthin, lactucaxanthin, viola-xanthin) at a molar ratio of 6:4:3; their contribution to the absorbance of the LHC between 390 and 530 nm is estimated to be about 31% (Chl-a), 26% (Chl-h) and 43% (xanthophylls).
(2) Energy transfer from xanthophylls and Chl-fe to Chl-a takes place at 100% transfer efficiency.
(3) LHC exhibits an unusual acid stability: in contrast to complexes of photosystem I or II, LHC-bound chlorophylls are not converted to phaeophytin and LHC apoprotein is not denatured at pH 1.5; also, energy transfer is maintained.
(4) Pronase or trypsin treatment do not affect acid stability and energy transfer.
(5) Treatments that break down acid stability (heat, urea or TritonX–100) also inhibit energy transfer.
The coincidental breakdown of energy transfer and acid stability points at one underlying process, namely, the breakdown of a structure that enables protection of chlorophylls from proton attack and close contiguity of xanthophylls and chlorophylls as required for energy transfer. Dense packing of xanthophylls and chlorophylls within lipophilic crevices of the LHC is suggested.     

CHLOROPHYLL a FLUORESCENCE OF GONYAULAX POLYEDRA GROWN ON A LIGHT-DARK CYCLE AND AFTER TRANSFER TO CONSTANT LIGHT

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL. After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a-c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl a_II complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a₁, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU-treated to non-treated cells. Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light-harvesting peridinin-Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin-Chl a complex to other Chl a complexes than do cells in LD.     

Ultrafast carotenoid band shifts correlated with ChlZ excited states in the photosystem II reaction center: are the carotenoids involved in energy transfer?

We show a correlation between the electronic excitation of the peripheral chlorophylls (Chls(Z)) of the photosystem II reaction center and a shift of the S(2) absorption bands of β-carotene, and suggest that the carotenoids may enhance the excitation energy transfer rate from these chlorophylls to the central cofactors.     

Excitation energy transfer in the photosystem II core antenna complex CP43 studied by femtosecond visible/visible and visible/mid-infrared pump probe spectroscopy

Excitation energy transfer in the Photosystem II core antenna complex CP43 has been investigated by vis/vis and vis/mid-IR pump-probe spectroscopy with the aim of understanding the relation between the dynamics of energy transfer and the structural arrangement of individual chlorophyll molecules within the protein. Energy transfer was found to occur on time scales of 250 fs, 2-4 ps, and 10-12 ps. The vis/mid-IR difference spectra show that the excitation is initially distributed over chlorophylls located in environments with different polarity, since two 9-keto C=O stretching bleachings, at 1691 and 1677 cm-1, are observable at early delay times. Positive signals in the initial difference spectra around 1750 and 1720 cm-1 indicate the presence of a charge transfer state between strongly interacting chlorophylls. We conclude, both from the spectral behavior in the visible when the annihilation processes are increased and from the vis/mid-IR data, that there are two pigments (one absorbing around 670 nm and one at 683 nm) which are not connected to the other pigments on a time scale faster than 10-20 ps. Since, in the IR, on a 10 ps time scale the population of the 1691 cm-1 mode almost disappears, while the 1677 cm-1 mode is still significantly populated, we can conclude that at least some of the red absorbing pigments are located in a polar environment, possibly forming H-bonds with the surrounding protein.     

Characterization and deposition of various light-harvesting antenna complexes by electrospray atomization

Photosynthetic organisms have light-harvesting complexes that absorb and transfer energy efficiently to reaction centers. Light-harvesting complexes (LHCs) have received increased attention in order to understand the natural photosynthetic process and also to utilize their unique properties in fabricating efficient artificial and bio-hybrid devices to capture solar energy. In this work, LHCs with different architectures, sizes, and absorption spectra, such as chlorosomes, Fenna–Matthews–Olson (FMO) protein, LH2 complex, and phycobilisome have been characterized by an electrospray-scanning mobility particle-sizer system (ES-SMPS). The size measured by ES-SMPS for FMO, chlorosomes, LH2, and phycobilisome were 6.4, 23.3, 9.5, and 33.4?nm, respectively. These size measurements were compared with values measured by dynamic light scattering and those reported in the literature. These complexes were deposited onto a transparent substrate by electrospray deposition. Absorption and fluorescence spectra of the deposited LHCs were measured. It was observed that the LHCs have light absorption and fluorescence spectra similar to that in solution, demonstrating the viability of the process.     

The electronic structure of Mn in oxides, coordination complexes, and the oxygen-evolving complex of photosystem II studied by resonant inelastic X-ray scattering

Resonant inelastic X-ray scattering (RIXS) was used to collect Mn K pre-edge spectra and to study the electronic structure in oxides, molecular coordination complexes, as well as the S1 and S2 states of the oxygen-evolving complex (OEC) of photosystem II (PS II). The RIXS data yield two-dimensional plots that can be interpreted along the incident (absorption) energy or the energy transfer axis. The second energy dimension separates the pre-edge (predominantly 1s to 3d transitions) from the main K-edge, and a detailed analysis is thus possible. The 1s2p RIXS final-state electron configuration along the energy transfer axis is identical to conventional L-edge absorption spectroscopy, and the RIXS spectra are therefore sensitive to the Mn spin state. This new technique thus yields information on the electronic structure that is not accessible in conventional K-edge absorption spectroscopy. The line splittings can be understood within a ligand field multiplet model, i.e., (3d,3d) and (2p,3d) two-electron interactions are crucial to describe the spectral shapes in all systems. We propose to explain the shift of the K pre-edge absorption energy upon Mn oxidation in terms of the effective number of 3d electrons (fractional 3d orbital population). The spectral changes in the Mn 1s2p(3/2) RIXS spectra between the PS II S1 and S2 states are small compared to that of the oxides and two of the coordination complexes (Mn(III)(acac)3 and Mn(IV)(sal)2(bipy)). We conclude that the electron in the step from S1 to S2 is transferred from a strongly delocalized orbital.     

Core mutations of Synechococcus sp. PCC 7002 phycobilisomes: a spectroscopic study.

Three cyanobacterial strains harboring mutations affecting phycobilisome (PBS) cores were studied using steady state absorption and fluorescence and time-resolved fluorescence. The apcF mutant, missing beta 18, and the apcDF mutant, missing both alpha APB and beta 18, showed only small spectroscopic differences from the wild-type strain; their PBS emission was blue shifted by 10 nm, whereas their absorption spectra and time-resolved fluorescence kinetics were virtually unchanged. The third mutant studied was the apcE/C186S mutant in which the chromophore-binding cysteine-186 in the LCM99 polypeptide has been substituted with serine. The apcE/C186S mutant contained a modified chromophore which significantly changed the spectroscopic properties of the PBS complex. The apcE/C186S PBS absorbed more than the wild-type strain at 705 nm, and the emission spectrum gave two peaks at 660 nm and 715 nm. The time-resolved kinetics of the apcE/C186S mutant PBS were also significantly altered from those of the wild-type strain.     

PHOTOSYSTEM II HETEROGENEITY IN THE MARINE DIATOM Phaeodactylum tricornutum

Abstract— The kinetics of photosystem II photochemistry are analyzed in the marine diatom Phaeodacfylum tricornutum by measurement of fluorescence induction in cell suspensions treated with 3–(3,4-dichlorophenyl)-1,1-dimethylurea. Photosystem II kinetics are found to be biphasic, the sum of two exponential components, suggesting that biphasic energy conversion in photosystem II may be a general consequence of thylakoid membrane appression. The emission wavelength-dependence of fluorescence induction suggests that the two photosystem II components have different variable fluorescence emission spectra. The slower component exhibits characteristic emission of the diatom light-harvesting complexes while emission from the faster component resembles that of the photosystem II reaction center. Variable fluorescence emission (293 K) at wavelengths > 700 nm is assigned to photosystem II. Application of model equations indicates that the two photosystem II unit types differ primarily in antenna size. A new analytical procedure is presented which eliminates ambiguities in the kinetic analysis associated with the incorrect assignment of the maximal fluorescence yield.     

P680: what is it and where is it?

Electron and X-ray crystallography have provided intermediate structural models for photosystem II (PSII), the membrane located multisubunit complex which uses light energy to split water into its elemental constituents. This reaction is thermodynamically demanding and involves the production of redox potentials in excess of 1 V. Structural analyses have now shown that the primary oxidant, P680, is not a 'special pair' of chlorophylls, as in other types of photosynthetic reaction centres, but a tetramer of equally spaced chlorophyll a molecules. Its high redox potential, and the involvement of four weakly coupled isoenergetic monomers rather than a strongly excitonically coupled 'special pair', has implications for redox mechanisms which are unique to PSII, and therefore not found in any other photosynthetic system. The importance of these features is discussed.     

How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem II in cryptophyte Rhodomonas CS24 cells

We report an investigation of energy migration dynamics in intact cells of the photosynthetic cryptophyte Rhodomonas CS24 using analyses of steady-state and time-resolved fluorescence anisotropy measurements. By fitting a specific model to the fluorescence data, we obtain three time scales (17, 58, and 113 ps) by which the energy is transferred from phycoerythrin 545 (PE545) to the membrane-associated chlorophylls (Chls). We propose that these time scales reflect both an angular distribution of PE545 around the photosystems and the relative orientations of the donor dihydrobiliverdin (DBV) bilin and the acceptor Chl. Contrary to investigations of the isolated antenna complex, it is demonstrated that energy transfer from PE545 does not occur from a single-emitting bilin, but rather both the peripheral dihydrobiliverdin (DBV) chromophores in PE545 appear to be viable donors of excitation energy to the membrane-bound proteins. The model shows an almost equal distribution of excitation energy from PE545 to both photosystem I (PSI) and photosystem II (PSII), whose trap times correspond well to those obtained from experiments on isolated photosystems.     

Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77K emission spectra revealed that Mg2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.     

气相水杨酸激发态分子内质子转移特性分析

采用密度泛函理论(DFT)和含时密度泛函理论(TD-DFT)研究了气相水杨酸(SA)分子的激发态氢键动力学过程。通过对水杨酸分子基态和激发态结构的优化,以及对其稳态吸收和发射光谱特性、前线分子轨道、红外振动光谱和势能曲线的计算分析,阐明水杨酸分子内质子转移可在激发态下自发地发生,导致其激发态可存在烯醇式和酮式两种异构体结构,并揭示了这种质子转移源于分子内电荷转移的激发态氢键的加强机制。     

CHARGE TRANSFER FROM PHOTOSYSTEM I TO THE CYTOCHROME b/f COMPLEX: DIFFUSION AND MEMBRANE LATERAL HETEROGENEITY

Abstract— We discuss here the minimum requirements for diffusion of a charge carrier between appressed and stroma-exposed membrane regions of chloroplasts based on recent models of the thylakoid membrane and flash-induced kinetic data. We have investigated the kinetics of the transfer of a positive charge from photosystem I to the cytochrome b/f complex in spinach chloroplasts by measuring the light-induced oxidation of cytochrome f. The rate and extent of cytochrome f oxidation were measured spectrophotometrically using either long actinic flashes that induced several turnovers of photosystem I or short actinic flashes that induced a single turnover of photosystem I. In the long actinic flashes, in the electron transfer reaction from water to methyl viologen, we observed the rapid oxidation of all of the cytochrome f present in the membrane. The half-time of the oxidation was 1.0 ± 0.1 ms. The total amount of the cytochrome was determined by chemical difference spectra to be one molecule of cytochrome f per 650 – 30 chlorophyll molecules. Using short actinic flashes we studied the photosystem I-driven electron transfer reaction from duroquinol to methyl viologen in the presence of the inhibitor 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Under these conditions a single turnover flash induced the oxidation of 62 ± 5% of cytochrome f with a half-time of 240 ± 30 μs. An Arrhenius plot of the temperature dependence of the cytochrome f oxidation rate revealed an activation energy between 16 and 21 kJ/mol, a value consistent with a diffusion-controlled reaction. These kinetic data are considered in the context of two models of the thylakoid membrane.     

Chlorophyll excitation energies and structural stability of the CP47 antenna of photosystem II: a case study in the first-principles simulation of light-harvesting complexes

Natural photosynthesis relies on light harvesting and excitation energy transfer by specialized pigment–protein complexes. Their structure and the electronic properties of the embedded chromophores define the mechanisms of energy transfer. An important example of a pigment–protein complex is CP47, one of the integral antennae of the oxygen-evolving photosystem II (PSII) that is responsible for efficient excitation energy transfer to the PSII reaction center. The charge-transfer excitation induced among coupled reaction center chromophores resolves into charge separation that initiates the electron transfer cascade driving oxygenic photosynthesis. Mapping the distribution of site energies among the 16 chlorophyll molecules of CP47 is essential for understanding excitation energy transfer and overall antenna function. In this work, we demonstrate a multiscale quantum mechanics/molecular mechanics (QM/MM) approach utilizing full time-dependent density functional theory with modern range-separated functionals to compute for the first time the excitation energies of all CP47 chlorophylls in a complete membrane-embedded cyanobacterial PSII dimer. The results quantify the electrostatic effect of the protein on the site energies of CP47 chlorophylls, providing a high-level quantum chemical excitation profile of CP47 within a complete computational model of “near-native” cyanobacterial PSII. The ranking of site energies and the identity of the most red-shifted chlorophylls (B3, followed by B1) differ from previous hypotheses in the literature and provide an alternative basis for evaluating past approaches and semiempirically fitted sets. Given that a lot of experimental studies on CP47 and other light-harvesting complexes utilize extracted samples, we employ molecular dynamics simulations of isolated CP47 to identify which parts of the polypeptide are most destabilized and which pigments are most perturbed when the antenna complex is extracted from PSII. We demonstrate that large parts of the isolated complex rapidly refold to non-native conformations and that certain pigments (such as chlorophyll B1 and β-carotene h1) are so destabilized that they are probably lost upon extraction of CP47 from PSII. The results suggest that the properties of isolated CP47 are not representative of the native complexed antenna. The insights obtained from CP47 are generalizable, with important implications for the information content of experimental studies on biological light-harvesting antenna systems.

Advanced QM/MM simulations explore the excited states of a photosynthetic light-harvesting antenna in its physiologically complexed state and model the consequences of extraction on conformational and electronic properties.     

Primary processes in plant photosynthesis: photosystem I reaction center

The photosystem I (PSI) pigment-protein complex of plants converts light energy into a transmembrane charge separation, which ultimately leads to the reduction of carbon dioxide. Recent studies on the dynamics of primary energy transfer, charge separation, and following electron transfer of the reaction center (RC) of the PSI prepared from spinach are reviewed. The main results of femtosecond transient absorption and fluorescence spectroscopies as applied to the P700-enchied PSI RC are summarized. This specially prepared material contains only 12–14 chlorophylls per P700, which is a special pair of chlorophyll a and has a significant role in primary charge separation. The P700-enriched particles are useful to study dynamics of cofactors, since about 100 light-harvesting chlorophylls are associated with wild PSI RC and prevent one from observing the elementary steps of the charge separation. In PSI RC energy and electron transfer were found to be strongly coupled and an ultrafast up-hill energy equilibration and charge separation were observed upon preferential excitation of P700. The secondary electron-transfer dynamics from the reduced primary electron acceptor chlorophyll a to quinone are described. With creating free energy differences (ΔG₀) for the reaction by reconstituting various artificial quinones and quinoids, the rate of electron transfer was measured. Analysis of rates versus ΔG₀ according to the quantum theory of electron transfer gave the reorganization energy, electronic coupling energy and other factors. It was shown that the natural quinones are optimized in the photosynthetic protein complexes. The above results were compared with those of photosynthetic purple bacteria, of which the structure and functions have been studied most.