BBA - Bioenergetics (v.1837, #9)

Photosynthesis research for sustainability: Keys to produce clean energy by Suleyman I. Allakhverdiev; Jian-Ren Shen (1377-1383).

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo) at 460 nm is observed with rise time of ~ 11 ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (PheoD1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680+PheoDI . The subsequent electron transfer from PheoD1 to primary plastoquinone electron acceptor (QA) was accompanied by relaxation of the 460-nm band and occurred within ~ 250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680+ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of PheoDI , which is very similar to that measured earlier by accumulation method. The spectrum of PheoDI formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to PheoD1. According to previous measurements in the femtosecond–picosecond time range this Chl-670 was ascribed to ChlD1 [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45–50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~ 1 ps and ~ 14 ps. These components appear to reflect formation of P680+ChlD1 and P680+PheoD1 , respectively, as found earlier. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: P680; Chlorophyll; Pheophytin; Photosystem II core complex; Primary charge separation; Reaction center;

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn4CaO5-cluster (Mn-cluster) in four discrete oxidation steps [S1  − (S4  / S0)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: PSII; Mn-cluster; Protonation of the OEC; Redox states; pKa;

Water exchange in manganese-based water-oxidizing catalysts in photosynthetic systems: From the water-oxidizing complex in photosystem II to nano-sized manganese oxides by Mohammad Mahdi Najafpour; Mohsen Abbasi Isaloo; Julian J. Eaton-Rye; Tatsuya Tomo; Hiroshi Nishihara; Kimiyuki Satoh; Robert Carpentier; Jian-Ren Shen; Suleyman I. Allakhverdiev (1395-1410).
The water-oxidizing complex (WOC), also known as the oxygen-evolving complex (OEC), of photosystem II in oxygenic photosynthetic organisms efficiently catalyzes water oxidation. It is, therefore, responsible for the presence of oxygen in the Earth's atmosphere. The WOC is a manganese–calcium (Mn4CaO5(H2O)4) cluster housed in a protein complex. In this review, we focus on water exchange chemistry of metal hydrates and discuss the mechanisms and factors affecting this chemical process. Further, water exchange rates for both the biological cofactor and synthetic manganese water splitting are discussed. The importance of fully unveiling the water exchange mechanism to understand the chemistry of water oxidation is also emphasized here. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: Artificial photosynthesis; Manganese; Nano-sized manganese oxide; Oxygenic photosynthesis; Photosystem II; Water exchange; Water-oxidizing complex;

The main technique employed to characterize the efficiency of water-splitting in photosynthetic preparations in terms of miss and double hit parameters and for the determination of Si (i = 2,3,0) state lifetimes is the measurement of flash-induced oxygen oscillation pattern on bare platinum (Joliot-type) electrodes. We demonstrate here that this technique is not innocent. Polarization of the electrode against an Ag/AgCl electrode leads to a time-dependent formation of hydrogen peroxide by two-electron reduction of dissolved oxygen continuously supplied by the flow buffer. While the miss and double hit parameters are almost unaffected by H2O2, a time dependent reduction of S1 to S− 1 occurs over a time period of 20 min. The S1 reduction can be largely prevented by adding catalase or by removing O2 from the flow buffer with N2. Importantly, we demonstrate that even at the shortest possible polarization times (40 s in our set up) the S2 and S0 decays are significantly accelerated by the side reaction with H2O2. The removal of hydrogen peroxide leads to unperturbed S2 state data that reveal three instead of the traditionally reported two phases of decay. In addition, even under the best conditions (catalase + N2; 40 s polarization) about 4% of S− 1 state is observed in well dark-adapted samples, likely indicating limitations of the equal fit approach. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: Photosystem II (PSII); Oxygen evolving complex (OEC); Water oxidation; Manganese; Hydrogen peroxide (H2O2);

The D1 protein of Photosystem II (PSII) is recognized as the main target of photoinhibitory damage and exhibits a high turnover rate due to its degradation and replacement during the PSII repair cycle. Damaged D1 is replaced by newly synthesized D1 and, although reasonable, there is no direct evidence for selective replacement of damaged D1. Instead, it remains possible that increased turnover of D1 subunits occurs in a non-selective manner due for example, to a general up-regulation of proteolytic activity triggered during damaging environmental conditions, such as high light. To determine if D1 degradation is targeted to damaged D1 or generalized to all D1, we developed a genetic system involving simultaneous dual expression of wild type and mutant versions of D1 protein. Dual D1 strains (nS345P:eWT and nD170A:eWT) expressed a wild type (WT) D1 from ectopic and a damage prone mutant (D1-S345P, D1-D170A) from native locus on the chromosome. Characterization of strains showed that all dual D1 strains restore WT like phenotype with high PSII activity. Higher PSII activity indicates increased population of PSII reaction centers with WT D1. Analysis of steady state levels of D1 in nS345P:eWT by immunoblot showed an accumulation of WT D1 only. But, in vivo pulse labeling confirmed the synthesis of both S345P (exists as iD1) and WT D1 in the dual strain. Expression of nS345P:eWT in FtsH2 knockout background showed accumulation of both iD1 and D1 proteins. This demonstrates that dual D1 strains express both forms of D1, yet only damage prone PSII complexes are selected for repair providing evidence that the D1 degradation process is targeted towards damaged PSII complexes. Since the N-terminus has been previously shown to be important for the degradation of damaged D1, the possibility that the highly conserved cysteine 18 residue situated in the N-terminal domain of D1 is involved in the targeted repair process was tested by examining site directed mutants of this and the other cysteines of the D1 protein. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: D1 protein; Photoinhibition; Photosystem II; Repair;

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II complex which bears together with PsbD, the D2 protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Photosystem II; PsbA; D1; Thermosynechococcus elongatus; Cyanobacterium;

The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II by Hao Luo; Simon A. Jackson; Robert D. Fagerlund; Tina C. Summerfield; Julian J. Eaton-Rye (1435-1446).
The PsbL protein is a 4.5 kDa subunit at the monomer–monomer interface of Photosystem II (PS II) consisting of a single membrane-spanning domain and a hydrophilic stretch of ~ 15 residues facing the cytosolic (or stromal) side of the photosystem. Deletion of conserved residues in the N-terminal region has been used to investigate the importance of this hydrophilic extension. Using Synechocystis sp. PCC 6803, three deletion strains: ∆(N6–N8), ∆(P11–V12) and ∆(E13–N15), have been created. The ∆(N6–N8) and ∆(P11–V12) strains remained photoautotrophic but were more susceptible to photodamage than the wild type; however, the ∆(E13–N15) cells had the most severe phenotype. The Δ(E13–N15) mutant showed decreased photoautotrophic growth, a reduced number of PS II centers, impaired oxygen evolution in the presence of PS II-specific electron acceptors, and was highly susceptible to photodamage. The decay kinetics of chlorophyll a variable fluorescence after a single turnover saturating flash and the sensitivity to low concentrations of PS II-directed herbicides in the Δ(E13–N15) strain indicate that the binding of plastoquinone to the QB-binding site had been altered such that the affinity of QB is reduced. In addition, the PS II-specific electron acceptor 2,5-dimethyl-p-benzoquinone was found to inhibit electron transfer through the quinone-acceptor complex of the ∆(E13–N15) strain. The PsbL Y20A mutant was also investigated and it exhibited increased susceptibility to photodamage and increased herbicide sensitivity. Our data suggest that the N-terminal hydrophilic region of PsbL influences forward electron transfer from QA through indirect interactions with the D–E loop of the D1 reaction center protein. Our results further indicate that disruption of interactions between the N-terminal region of PsbL and other PS II subunits or lipids destabilizes PS II dimer formation. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Electron transfer; Herbicide binding; Photosystem II; PsbL; QB; Synechocystis sp. PCC 6803;

Identification of the basic amino acid residues on the PsbP protein involved in the electrostatic interaction with photosystem II by Taishi Nishimura; Chihiro Uno; Kunio Ido; Ryo Nagao; Takumi Noguchi; Fumihiko Sato; Kentaro Ifuku (1447-1453).
The PsbP protein is an extrinsic subunit of photosystem II (PSII) that is essential for photoautotrophic growth in higher plants. Several crystal structures of PsbP have been reported, but the binding topology of PsbP in PSII has not yet been clarified. In this study, we report that the basic pocket of PsbP, which consists of conserved Arg48, Lys143, and Lys160, is important for the electrostatic interaction with the PSII complex. Our release-reconstitution experiment showed that the binding affinities of PsbP-R48A, -K143A, and -K160A mutated proteins to PSII were lower than that of PsbP-WT, and triple mutations of these residues greatly diminished the binding affinity to PSII. Even when maximum possible binding had occurred, the R48A, K143A, and K160A proteins showed a reduced ability to restore the rate of oxygen evolution at low chloride concentrations. Fourier transform infrared resonance (FTIR) difference spectroscopy results were consistent with the above finding, and suggested that these mutated proteins were not able to induce the normal conformational change around the Mn cluster during S1 to S2 transition. Finally, chemical cross-linking experiments suggested that the interaction between the N-terminus of PsbP with PsbE was inhibited by these mutations. These data suggest that the basic pocket of PsbP is important for proper association and interaction with PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Extrinsic protein; Higher plants; Oxygen-evolving complex; Photosystem II; PsbP;

Proteomic characterization and three-dimensional electron microscopy study of PSII–LHCII supercomplexes from higher plants by Cristina Pagliano; Jon Nield; Francesco Marsano; Tillmann Pape; Simone Barera; Guido Saracco; James Barber (1454-1462).
In higher plants a variable number of peripheral LHCII trimers can strongly (S), moderately (M) or loosely (L) associate with the dimeric PSII core (C2) complex via monomeric Lhcb proteins to form PSII–LHCII supercomplexes with different structural organizations. By solubilizing isolated stacked pea thylakoid membranes either with the α or β isomeric forms of the detergent n-dodecyl-D-maltoside, followed by sucrose density ultracentrifugation, we previously showed that PSII–LHCII supercomplexes of types C2S2M2 and C2S2, respectively, can be isolated [S. Barera et al., Phil. Trans. R Soc. B 67 (2012) 3389–3399]. Here we analysed their protein composition by applying extensive bottom-up and top-down mass spectrometry on the two forms of the isolated supercomplexes. In this way, we revealed the presence of the antenna proteins Lhcb3 and Lhcb6 and of the extrinsic polypeptides PsbP, PsbQ and PsbR exclusively in the C2S2M2 supercomplex. Other proteins of the PSII core complex, common to the C2S2M2 and C2S2 supercomplexes, including the low molecular mass subunits, were also detected and characterized. To complement the proteomic study with structural information, we performed negative stain transmission electron microscopy and single particle analysis on the PSII–LHCII supercomplexes isolated from pea thylakoid membranes solubilized with n-dodecyl-α-D-maltoside. We observed the C2S2M2 supercomplex in its intact form as the largest PSII complex in our preparations. Its dataset was further analysed in silico, together with that of the second largest identified sub-population, corresponding to its C2S2 subcomplex. In this way, we calculated 3D electron density maps for the C2S2M2 and C2S2 supercomplexes, approaching respectively 30 and 28 Å resolution, extended by molecular modelling towards the atomic level. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Thylakoids; PSII–LHCII supercomplex; Proteomics; Transmission electron microscopy; Single particle analysis; Structure;

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle by Marjaana Suorsa; Marjaana Rantala; Ravi Danielsson; Sari Järvi; Virpi Paakkarinen; Wolfgang P. Schröder; Stenbjörn Styring; Fikret Mamedov; Eva-Mari Aro (1463-1471).
In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: PGR5; PGRL1; Photosystem II; PsbS; PsbW; PsbX;

Molecular dynamics study of the primary charge separation reactions in Photosystem I: Effect of the replacement of the axial ligands to the electron acceptor A0 by Georgy E. Milanovsky; Vasily V. Ptushenko; John H. Golbeck; Alexey Yu. Semenov; Dmitry A. Cherepanov (1472-1483).
Molecular dynamics (MD) calculations, a semi-continuum (SC) approach, and quantum chemistry (QC) calculations were employed together to investigate the molecular mechanics of ultrafast charge separation reactions in Photosystem I (PS I) of Thermosynechococcus elongatus. A molecular model of PS I was developed with the aim to relate the atomic structure with electron transfer events in the two branches of cofactors. A structural flexibility map of PS I was constructed based on MD simulations, which demonstrated its rigid hydrophobic core and more flexible peripheral regions. The MD model permitted the study of atomic movements (dielectric polarization) in response to primary and secondary charge separations, while QC calculations were used to estimate the direct chemical effect of the A0A/A0B ligands (Met or Asn in the 688/668 position) on the redox potential of chlorophylls A0A/A0B and phylloquinones A1A/A1B. A combination of MD and SC approaches was used to estimate reorganization energies λ of the primary (λ1) and secondary (λ2) charge separation reactions, which were found to be independent of the active branch of electron transfer; in PS I from the wild type, λ1 was estimated to be 390 ± 20 mV, while λ2 was estimated to be higher at 445 ± 15 mV. MD and QC approaches were used to describe the effect of substituting Met688PsaA/Met668PsaB by Asn688PsaA/Asn668PsaB on the energetics of electron transfer. Unlike Met, which has limited degrees of freedom in the site, Asn was found to switch between two relatively stable conformations depending on cofactor charge. The introduction of Asn and its conformation flexibility significantly affected the reorganization energy of charge separation and the redox potentials of chlorophylls A0A/A0B and phylloquinones A1A/A1B, which may explain the experimentally observed slowdown of secondary electron transfer in the M688NPsaA variant. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.Display Omitted
Keywords: Photosystem I; Molecular dynamics; Quantum chemistry; Primary reactions; Charge separation; Reorganization energy;

Energy transfer processes in chlorophyll f-containing cyanobacteria using time-resolved fluorescence spectroscopy on intact cells by Tatsuya Tomo; Toshiyuki Shinoda; Min Chen; Suleyman I. Allakhverdiev; Seiji Akimoto (1484-1489).
We examined energy transfer dynamics in the unique chlorophyll (Chl) f-containing cyanobacterium Halomicronema hongdechloris. The absorption band of Chl f appeared during cultivation of this organism under far-red light. The absorption maximum of Chl f in organic solvents occurs at a wavelength of approximately 40 nm longer than that of Chl a. In vivo, the cells display a new absorption band at approximately 730 nm at 298 K, which is at a significantly longer wavelength than that of Chl a. We primarily assigned this band to a long wavelength form of Chl a. The function of Chl f is currently unknown. We measured the fluorescence of cells using time-resolved fluorescence spectroscopy in the picosecond-to-nanosecond time range and found clear differences in fluorescence properties between the cells that contained Chl f and the cells that did not. After excitation, the fluorescence peaks of photosystem I and photosystem II appeared quickly but diminished immediately. A unique fluorescence peak located at 748 nm subsequently appeared in cells containing Chl f. This finding strongly suggests that the Chl f in this alga exists in photosystem I and II complexes and is located close to each molecule of Chl a. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Chlorophyll f; Energy transfer; Fluorescence; Photosynthesis;

In adaption to its specific environmental conditions, the cyanobacterium Acaryochloris marina developed two different types of light-harvesting complexes: chlorophyll-d-containing membrane-intrinsic complexes and phycocyanobilin (PCB) — containing phycobiliprotein (PBP) complexes. The latter complexes are believed to form a rod-shaped structure comprising three homo-hexamers of phycocyanin (PC), one hetero-hexamer of phycocyanin and allophycocyanin (APC) and probably a linker protein connecting the PBPs to the reaction centre. Excitation energy transfer and electron-vibrational coupling in PBPs have been investigated by selectively excited fluorescence spectra. The data reveal a rich spectral substructure with a total of five low-energy electronic states with fluorescence bands at 635 nm, 645 nm, 654 nm, 659 nm and a terminal emitter at about 673 nm. The electronic states at ~ 635 and 645 nm are tentatively attributed to PC and APC, respectively, while an apparent heterogeneity among PC subunits may also play a role. The other fluorescence bands may be associated with three different isoforms of the linker protein. Furthermore, a large number of vibrational features can be identified for each electronic state with intense phonon sidebands peaking at about 31 to 37 cm− 1, which are among the highest phonon frequencies observed for photosynthetic antenna complexes. The corresponding Huang-Rhys factors S fall in the range between 0.98 (terminal emitter), 1.15 (APC), and 1.42 (PC). Two characteristic vibronic lines at about 1580 and 1634 cm− 1 appear to reflect C―NH+ and C―C stretching modes of the PCB chromophore, respectively. The exact phonon and vibrational frequencies vary with electronic state implying that the respective PCB chromophores are bound to different protein environments. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: Acaryochloris marina; Phycobiliproteins; Excitation energy transfer; Electron phonon coupling; Spectral hole-burning; Difference fluorescence line-narrowing;

In the last ten years, a large series of studies have targeted antenna complexes of plants (Lhc) with the aim of understanding the mechanisms of light harvesting and photoprotection. Combining spectroscopy, modeling and mutation analyses, the role of individual pigments in these processes has been highlighted in vitro. In plants, however, these proteins are associated with multiple complexes of the photosystems and function within this framework. In this work, we have envisaged a way to bridge the gap between in vitro and in vivo studies by knocking out in vivo pigments that have been proposed to play an important role in excitation energy transfer between the complexes or in photoprotection. We have complemented a CP24 knock-out mutant of Arabidopsis thaliana with the CP24 (Lhcb6) gene carrying a His-tag and with a mutated version lacking the ligand for chlorophyll 612, a specific pigment that in vitro experiments have indicated as the lowest energy site of the complex. Both complexes efficiently integrated into the thylakoid membrane and assembled into the PSII supercomplexes, indicating that the His-tag does not impair the organization in vivo. The presence of the His-tag allowed the purification of CP24-WT and of CP24-612 mutant in their native states. It is shown that CP24-WT coordinates 10 chlorophylls and 2 carotenoid molecules and has properties identical to those of the reconstituted complex, demonstrating that the complex self-assembled in vitro assumes the same folding as in the plant. The absence of the ligand for chlorophyll 612 leads to the loss of one Chl a and of lutein, again as in vitro, indicating the feasibility of the method. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Light-harvesting complex; In vivo mutation analysis; Reconstituted complex; Lhcb6; Photosystem II; Arabidopsis thaliana;

Exploring the mechanism(s) of energy dissipation in the light harvesting complex of the photosynthetic algae Cyclotella meneghiniana by Charusheela Ramanan; Rudi Berera; Kathi Gundermann; Ivo van Stokkum; Claudia Büchel; Rienk van Grondelle (1507-1513).
Photosynthetic organisms have developed vital strategies which allow them to switch from a light-harvesting to an energy dissipative state at the level of the antenna system in order to survive the detrimental effects of excess light illumination. These mechanisms are particularly relevant in diatoms, which grow in highly fluctuating light environments and thus require fast and strong response to changing light conditions. We performed transient absorption spectroscopy on FCPa, the main light-harvesting antenna from the diatom Cyclotella meneghiniana, in the unquenched and quenched state. Our results show that in quenched FCPa two quenching channels are active and are characterized by differing rate constants and distinct spectroscopic signatures. One channel is associated with a faster quenching rate (16 ns− 1) and virtually no difference in spectral shape compared to the bulk unquenched chlorophylls, while a second channel is associated with a slower quenching rate (2.7 ns− 1) and exhibits an increased population of red-emitting states. We discuss the origin of the two processes in the context of the models proposed for the regulation of photosynthetic light-harvesting. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Photosynthesis; Photoprotection; Non-photochemical quenching; Light-harvesting; Diatoms; Transient absorption;

Excitation relaxation dynamics and energy transfer in fucoxanthin–chlorophyll a/c-protein complexes, probed by time-resolved fluorescence by Seiji Akimoto; Ayaka Teshigahara; Makio Yokono; Mamoru Mimuro; Ryo Nagao; Tatsuya Tomo (1514-1521).
In algae, light-harvesting complexes contain specific chlorophylls (Chls) and keto-carotenoids; Chl a, Chl c, and fucoxanthin (Fx) in diatoms and brown algae; Chl a, Chl c, and peridinin in photosynthetic dinoflagellates; and Chl a, Chl b, and siphonaxanthin in green algae. The Fx–Chl a/c-protein (FCP) complex from the diatom Chaetoceros gracilis contains Chl c 1, Chl c 2, and the keto-carotenoid, Fx, as antenna pigments, in addition to Chl a. In the present study, we investigated energy transfer in the FCP complex associated with photosystem II (FCPII) of C. gracilis. For these investigations, we analyzed time-resolved fluorescence spectra, fluorescence rise and decay curves, and time-resolved fluorescence anisotropy data. Chl a exhibited different energy forms with fluorescence peaks ranging from 677 nm to 688 nm. Fx transferred excitation energy to lower-energy Chl a with a time constant of 300 fs. Chl c transferred excitation energy to Chl a with time constants of 500–600 fs (intra-complex transfer), 600–700 fs (intra-complex transfer), and 4–6 ps (inter-complex transfer). The latter process made a greater contribution to total Chl c-to-Chl a transfer in intact cells of C. gracilis than in the isolated FCPII complexes. The lower-energy Chl a received excitation energy from Fx and transferred the energy to higher-energy Chl a. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Fucoxanthin–chlorophyll a/c protein; Light harvesting; Photosynthesis; Energy transfer; Time-resolved spectroscopy;

Fluorescence yield relaxation following a light pulse was studied in various cyanobacteria under aerobic and microaerobic conditions. In Synechocystis PCC 6803 fluorescence yield decays in a monotonous fashion under aerobic conditions. However, under microaerobic conditions the decay exhibits a wave feature showing a dip at 30–50 ms after the flash followed by a transient rise, reaching maximum at ~ 1 s, before decaying back to the initial level. The wave phenomenon can also be observed under aerobic conditions in cells preilluminated with continuous light. Illumination preconditions cells for the wave phenomenon transiently: for few seconds in Synechocystis PCC 6803, but up to one hour in Thermosynechocystis elongatus BP-1. The wave is eliminated by inhibition of plastoquinone binding either to the QB site of Photosystem-II or the Qo site of cytochrome b6f complex by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, respectively. The wave is also absent in mutants, which lack either Photosystem-I or the NAD(P)H-quinone oxidoreductase (NDH-1) complex. Monitoring the redox state of the plastoquinone pool revealed that the dip of the fluorescence wave corresponds to transient oxidation, whereas the following rise to re-reduction of the plastoquinone pool. It is concluded that the unusual wave feature of fluorescence yield relaxation reflects transient oxidation of highly reduced plastoquinone pool by Photosystem-I followed by its re-reduction from stromal components via the NDH-1 complex, which is transmitted back to the fluorescence yield modulator primary quinone electron acceptor via charge equilibria. Potential applications of the wave phenomenon in studying photosynthetic and respiratory electron transport are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Photosystem II; Variable Chl fluorescence; Linear electron transport; Cyclic electron transport; NDH-1 complex;

Here we show how the protein environment in terms of detergent concentration/protein aggregation state, affects the sensitivity to pH of isolated, native LHCII, in terms of chlorophyll fluorescence quenching. Three detergent concentrations (200, 20 and 6 μM n-dodecyl β-d-maltoside) have been tested. It was found that at the detergent concentration of 6 μM, low pH quenching of LHCII is close to the physiological response to lumen acidification possessing pK of 5.5. The analysis has been conducted both using arbitrary PAM fluorimetry measurements and chlorophyll fluorescence lifetime component analysis. The second led to the conclusion that the 3.5 ns component lifetime corresponds to an unnatural state of LHCII, induced by the detergent used for solubilising the protein, whilst the 2 ns component is rather the most representative lifetime component of the conformational state of LHCII in the natural thylakoid membrane environment when the non-photochemical quenching (NPQ) was absent. The 2 ns component is related to a pre-aggregated LHCII that makes it more sensitive to pH than the trimeric LHCII with the dominating 3.5 ns lifetime component. The pre-aggregated LHCII displayed both a faster response to protons and a shift in the pK for quenching to higher values, from 4.2 to 4.9. We concluded that environmental factors like lipids, zeaxanthin and PsbS protein that modulate NPQ in vivo could control the state of LHCII aggregation in the dark that makes it more or less sensitive to the lumen acidification. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: LHCII; Non-photochemical quenching; ∆pH; Chlorophyll fluorescence lifetime; Detergent; Protein aggregation;

The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry by E.G. Maksimov; F.-J. Schmitt; E.A. Shirshin; M.D. Svirin; I.V. Elanskaya; T. Friedrich; V.V. Fadeev; V.Z. Paschenko; A.B. Rubin (1540-1547).
As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures — from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmol photons m− 2  s− 1. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
Keywords: Phycobilisome; Energy migration; Non-photochemical quenching; Fluorescence lifetime; Temperature;

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus by Emine Dinc; Silvia Ramundo; Roberta Croce; Jean-David Rochaix (1548-1552).
A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: Chloroplast; Gene expression; Photosystem degradation; Chlamydomonas;

Oxygenic photosynthesis is driven via sequential action of Photosystem II (PSII) and (PSI)reaction centers via the Z-scheme. Both of these pigment–membrane protein complexes are found in cyanobacteria, algae, and plants. Unlike PSII, PSI is remarkably stable and does not undergo limiting photo-damage. This stability, as well as other fundamental structural differences, makes PSI the most attractive reaction centers for applied photosynthetic applications. These applied applications exploit the efficient light harvesting and high quantum yield of PSI where the isolated PSI particles are redeployed providing electrons directly as a photocurrent or, via a coupled catalyst to yield H2. Recent advances in molecular genetics, synthetic biology, and nanotechnology have merged to allow PSI to be integrated into a myriad of biohybrid devices. In photocurrent producing devices, PSI has been immobilized onto various electrode substrates with a continuously evolving toolkit of strategies and novel reagents. However, these innovative yet highly variable designs make it difficult to identify the rate-limiting steps and/or components that function as bottlenecks in PSI-biohybrid devices. In this study we aim to highlight these recent advances with a focus on identifying the similarities and differences in electrode surfaces, immobilization/orientation strategies, and artificial redox mediators. Collectively this work has been able to maintain an annual increase in photocurrent density (A cm− 2) of ~ 10-fold over the past decade. The potential drawbacks and attractive features of some of these schemes are also discussed with their feasibility on a large-scale. As an environmentally benign and renewable resource, PSI may provide a new sustainable source of bioenergy. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: Photosystem I; Photocurrent; Photovoltaic; Biohybrid;

Bio-inspired photoresponse of porphyrin-attached gold nanoparticles on a field-effect transistor by Mariko Miyachi; Yoshinori Yamanoi; Kazuo Nakazato; Hiroshi Nishihara (1567-1571).
A bio-inspired photoresponse was engineered in porphyrin-attached Au nanoparticles (AuNPs) on a field-effect transistor (FET). The system mimics photosynthetic electron transfer, using porphyrin derivatives as photosensitizers and AuNPs as photoelectron counting devices. Porphyrin-protected AuNPs were immobilized onto the gate of an FET via the formation of self-assembled monolayers. Photoinduced electron transfer from the porphyrin led to single electron transfer at the Au nanoparticles, which was monitored via a changing gate voltage on the FET in the presence of organic electrolyte. The further attachment of other functional molecules to this system should enable various other potential functionalities. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: Bio-inspired photosensor; Porphyrin; Au nanoparticles; Quantum size effect; Field-effect transistor;

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering by R. Ünnep; O. Zsiros; K. Solymosi; L. Kovács; P.H. Lambrev; T. Tóth; R. Schweins; D. Posselt; N.K. Székely; L. Rosta; G. Nagy; G. Garab (1572-1580).
We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 Å− 1, corresponding to spatially and statistically averaged repeat distance values of about 275–330 Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D2O in dim laboratory light or transpired with D2O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.
Keywords: Electron microscopy; Granum; Lamellar repeat distance; Small-angle neutron scattering; Thylakoid membrane;

eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM) by Franz-Josef Schmitt; Bastian Thaa; Cornelia Junghans; Marco Vitali; Michael Veit; Thomas Friedrich (1581-1593).
The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H+ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.Display Omitted
Keywords: GFP; FLIM; pH determination; M2 proton channel; Xanthophyll cycle; pH-dependent ROS;