Light Excitation Energy Research Articles

A Glycophorin A-Like Framework for the Dimerization of Photosynthetic Core Complexes

The core complex in photosynthetic bacteria plays a central role in photosynthesis. This molecular assembly is composed of two protein complexes, viz., the light-harvesting complex I (LH1), which absorbs sunlight by means of the protein-bound bacteriochlorophylls, and the reaction center (RC), which uses the light-excitation energy absorbed by the LH complexes to produce a transmembrane (TM) charge gradient, subsequently employed for energy conversion. In Rhodobacter (Rba.) sphaeroides, the core complex contains, in addition, two copies of the single TM alpha-helix protein, PufX, and forms a (RC-LH1-PufX)(2) dimer. To this date, no high-resolution structure has been reported for the entire core complex. In particular, the location of PufX within the (RC-LH1-PufX)(2) dimer is still the subject of much debate. Here, one of the proposed locations for PufX, requiring its dimerization, is examined. The PufX-dimer model on the basis of the glycophorin A (GpA) dimer was constructed, and its robustness was probed through a series of molecular dynamics (MD) simulations. The free-energy change due to the replacement of Gly35 by valine was also determined to assess whether this mutation is responsible for distinct PufX oligomerization states in different Rba. species. The present study shows that PufX helices form a stable GpA-like dimer with a helix-helix crossing angle that could constitute the molecular basis of the reported highly bent and V-shaped structure of the Rba. sphaeroides core complex dimer.

Self-Assembly Strategies for Integrating Light Harvesting and Charge Separation in Artificial Photosynthetic Systems

In natural photosynthesis, organisms optimize solar energy conversion through organized assemblies of photofunctional chromophores and catalysts within proteins that provide specifically tailored environments for chemical reactions. As with their natural counterparts, artificial photosynthetic systems for practical solar fuels production must collect light energy, separate charge, and transport charge to catalytic sites where multielectron redox processes will occur. While encouraging progress has been made on each aspect of this complex problem, researchers have not yet developed self-ordering and self-assembling components and the tailored environments necessary to realize a fully-functional artificial system. Previously researchers have used complex, covalent molecular systems comprised of chromophores, electron donors, and electron acceptors to mimic both the light-harvesting and the charge separation functions of photosynthetic proteins. These systems allow for study of the dependencies of electron transfer rate constants on donor-acceptor distance and orientation, electronic interaction, and the free energy of the reaction. The most useful and informative systems are those in which structural constraints control both the distance and the orientation between the electron donors and acceptors. Self-assembly provides a facile means for organizing large numbers of molecules into supramolecular structures that can bridge length scales from nanometers to macroscopic dimensions. The resulting structures must provide pathways for migration of light excitation energy among antenna chromophores, and from antennas to reaction centers. They also must incorporate charge conduits, that is, molecular "wires" that can efficiently move electrons and holes between reaction centers and catalytic sites. The central scientific challenge is to develop small, functional building blocks with a minimum number of covalent linkages, which also have the appropriate molecular recognition properties to facilitate self-assembly of complete, functional artificial photosynthetic systems. In this Account, we explore how self-assembly strategies involving pi-stacking can be used to integrate light harvesting with charge separation and transport. Our current strategy uses covalent building blocks based on chemically robust arylene imide and diimide dyes, biomimetic porphyrins, and chlorophylls. We take advantage of the shapes, sizes, and intermolecular interactions--such as pi-pi and/or metal-ligand interactions--of these molecules to direct the formation of supramolecular structures having enhanced energy capture and charge-transport properties. We use small- and wide-angle X-ray scattering (SAXS/WAXS) from a synchrotron source to elucidate the solution phase structures of these monodisperse noncovalent aggregates. We expect that a greater understanding of self-assembly using pi-stacking and molecular designs that combine those features with hydrogen bonding and metal-ligand bonding could simplify the structure of the building blocks for artificial photosynthetic complexes, while retaining their ability to assemble complex, photofunctional structures.

Cerium relieving the inhibition of photosynthesis and growth of spinach caused by lead

Chloroplasts were isolated from spinach cultured in lead chloride-present, Ce 3+-administered, cerium chloride-administered lead chloride-present Hoagland's media or that of Hoagland's media. The experimental study demonstrated the effects of cerium (Ce) on distribution of light energy and photochemical activities of spinach chloroplast grown in lead (Pb)-present media. It was observed that Pb 2+ significantly inhibited photosynthesis in spinach, including light absorption, energy transfer from LHCII to photosystem II, excitation energy distribution from photosystem I to photosystem II, and transformation from light energy to electron energy and oxygen evolution of chloroplasts, and decreased spinach growth. However, Ce 3+ treatment to Pb 2+-present chloroplasts could obviously improve light absorption and excitation energy distribution in both photosystems and increase activity of photochemical reaction and oxygen evolution of chloroplasts. The results suggested that Ce 3+ under Pb 2+ stress could maintain the stability of chloroplast membrane, and improve photosynthesis of spinach chloroplast, thus promote spinach growth.

Analysis of the Chloroplast Protein Kinase Stt7 during State Transitions

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b 6 f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b 6 f assembly and heme biogenesis.

Absorption and Transfer of Light and Photoreduction Activities of Spinach Chloroplasts under Calcium Deficiency: Promotion by Cerium

Chloroplasts were isolated from spinach cultured in calcium-deficient, cerium-chloride-administered calcium-present Hoagland's media or that of calcium-deficient Hoagland's media and demonstrated the effects of cerium on distribution of light energy between photosystems II and I and photochemical activities of spinach chloroplast grown in calcium-deficient media. It was observed that calcium deprivation significantly inhibited light absorption, energy transfer from LHCII to photosystemII, excitation energy distribution from PSI to PSII, and transformation from light energy to electron energy and oxygen evolution of chloroplasts. However, cerium treatment to calcium-deficient chloroplasts could obviously improve light absorption and excitation energy distribution from photosystem I to photosystem II and increase activity of whole chain electron transport, photosystems II and I DCPIP photoreduction, and oxygen evolution of chloroplasts. The results suggested that cerium under calcium deficiency condition could substitute for calcium in chloroplasts, maintain the stability of chloroplast membrane, and improve photosynthesis of spinach chloroplast, but the mechanisms still need further study.

Role of thylakoid protein kinases in photosynthetic acclimation

Photosynthetic organisms are able to adjust to changes in light quality through state transition, a process which leads to a balancing of the light excitation energy between the antennae systems of photosystem II and photosystem I. A genetic approach has been used in Chlamydomonas with the aim of elucidating the signaling chain involved in state transitions. This has led to the identification of a small family of Ser–Thr protein kinases associated with the thylakoid membrane and conserved in algae and land plants. These kinases appear to be involved both in short and long term adaptations to changes in the light environment.

Transient photoresponse in Pr0.7Ca0.3MnO3 thin films at room temperature

Photoresponse characteristics of Pr0.7Ca0.3MnO3 thin films were investigated under different applied voltages and excitation light energy densities at room temperature.The transient resistance change of 92.3% and response time of～36ns were observed under the excitation with a 532 nm laser pulse of 7ns duration at a light energy density of 275.16mJ/cm2.Both the resistance change and response time showed nonlinear behavior as a function of the light energy density, and the resistance and the response time were reduced when the intensity increased. However, the applied voltage had almost no effect on the photoresponse at room temperature. The phenomena are explained in terms of the photoinduced nonequilibrium insulator-to-metal transition in the film, which may have potential applications in optoelectronic devices.

Photogeneration of holes and electrons in amorphous molecular semiconductors with neutral and ionic organic dyes

Photoconducting properties of amorphous molecular semiconductors based on polystyrene films doped with tetranitrofluorenone and merocyanine or an anionic polymethine dye, or with epoxypropylcarbazole and merocyanine or a cationic polymethine dye were studied. The former type of the films is characterized by electron conductivity, whereas the latter type by hole conductivity. The activation energy for photogeneration of mobile charge carriers increases on passing from a merocyanine dye to ionic dyes and decreases with a growth in quantum energy of excitation light for the films of the former type, but does not depend on the light quantum energy for the films of the latter type. It was concluded that the activation energy of photogeneration is determined by electrostatic interaction of a photogenerated charge carrier with the ionized photogeneration site or a counterion for neutral and ionic dyes, respectively. At low dissipation rates of the excess thermal energy of excited dye molecules via electron-nucleus interaction, photogenerated electrons have a possibility to travel over a long distance from the photogeneration site as compared with holes.

State transitions and light adaptation require chloroplast thylakoid protein kinase STN7

Photosynthetic organisms are able to adjust to changing light conditions through state transitions, a process that involves the redistribution of light excitation energy between photosystem II (PSII) and photosystem I (PSI). Balancing of the light absorption capacity of these two photosystems is achieved through the reversible association of the major antenna complex (LHCII) between PSII and PSI (ref. 3). Excess stimulation of PSII relative to PSI leads to the reduction of the plastoquinone pool and the activation of a kinase; the phosphorylation of LHCII; and the displacement of LHCII from PSII to PSI (state 2). Oxidation of the plastoquinone pool by excess stimulation of PSI reverses this process (state 1). The Chlamydomonas thylakoid-associated Ser-Thr kinase Stt7, which is required for state transitions, has an orthologue named STN7 in Arabidopsis. Here we show that loss of STN7 blocks state transitions and LHCII phosphorylation. In stn7 mutant plants the plastoquinone pool is more reduced and growth is impaired under changing light conditions, indicating that STN7, and probably state transitions, have an important role in response to environmental changes.

The role of photochemical quenching and antioxidants in photoprotection of Deschampsia antarctica.

Deschampsia antarctica Desv. (Poaceae) is the only grass that grows in the maritime Antarctic. Constant low temperatures and episodes of high light are typical conditions during the growing season at this latitude. These factors enhance the formation of active oxygen species and may cause photoinhibition. Therefore, an efficient mechanism of energy dissipation and / or scavenging of reactive oxygen species (ROS) would contribute to survival in this harsh environment. In this paper, non-acclimated and cold-acclimated D. antarctica were subjected to high light and / or low temperature for 24 h. The contribution of non-photochemical dissipation of excitation light energy and the activities of detoxifying enzymes in the development of resistance to chilling induced photoinhibition were studied by monitoring PSII fluorescence, total soluble antioxidants, and pigments contents and measuring variations in activity of superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2). The photochemical efficiency of PSII, measured as Fv / F m, and the yield of PSII electron transport (ΦPSII) both decreased under high light and low temperatures. In contrast, photochemical quenching (qP) in both non-acclimated and cold-acclimated plants remained relatively constant (approximately 0.8) in high-light-treated plants. Unexpectedly, qP was lower (0.55) in cold-acclimated plants exposed to 4°C and low light intensity. Activity of SOD in cold-acclimated plants treated with high light at low temperature showed a sharp peak 2-4 h after the beginning of the experiment. In cold-acclimated plants APX remained high with all treatments. Activity of GR decreased in cold-acclimated plants. Compared with other plants, D. antarctica exhibited high levels of SOD and APX activity. Pigment analyses show that the xanthophyll cycle is operative in this plant. We propose that photochemical quenching and particularly the high level of antioxidants help D. antarctica to resist photoinhibitory conditions. The relatively high antioxidant capacity of D. antarctica may be a determinant for its survival in the harsh Antarctic environment.

Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy.

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.

Role of chloroplast protein kinase Stt7 in LHCII phosphorylation and state transition in Chlamydomonas.

Photosynthetic organisms adapt to changes in light quality by redistributing light excitation energy between two photosystems through state transition. This reorganization of antenna systems leads to an enhanced photosynthetic yield. Using a genetic approach in Chlamydomonas reinhardtii to dissect the signal transduction pathway of state transition, we identified a chloroplast thylakoid-associated serine-threonine protein kinase, Stt7, that has homologs in land plants. Stt7 is required for the phosphorylation of the major light-harvesting protein (LHCII) and for state transition.

The composition and spectral properties of three different forms of light-harvesting complex II

Different aggregates of LHC II play a very important role in regulating the light absorption and excitation energy transfer of plant. Trimeric LHC II was purified from spinach thylakoid membrane. In order to obtain the dimeric and monomeric LHC II, the trimer was treated with the mixture of 2% OGP and 10 microg/mL PLA(2), then loaded onto the sucrose density gradient in the presence of 0.06% triton X-100. The LHC II trimer, dimer and monomer isolated by sucrose density gradient all contained three polypeptides with molecular weight of 29, 28 and 26 kd respectively. The pigment composition showed much difference in the content of Chl b and xanthophyll among three forms of LHC II. To study the light capture and excitation energy transfer in different forms of LHC II, the absorption and fluorescence spectra were analyzed. The results clearly showed that the efficiency of energy absorption and transfer was different in the three kinds of LHC II, the highest for trimeric LHC II, intermediate for dimeric LHC II, and the lowest for monomeric LHC II. It was suggested that there might be a physiological homeostasis of different aggregates of LHC II in plants, which is significant for the plant self-regulating upon exposure to variable light environment.

Effects of the Structural Rigidity of Polymethine Dye Molecules on Quenching of Their Photoluminescence by an Electric Field and Charge Photogeneration in Poly(N-epoxypropylcarbazole)

For photoconducting poly(N-epoxypropylcarbazole) films doped with cationic polymethine dyes used as an example, it was found that an increase in dye molecular rigidity increased the photoconductivity of the films in the region of dye absorption, quenching effect of an electric field on photoluminescence, and concentration ratio of photogenerated triplet electron–hole pairs to singlet pairs. The effects were enhanced by an increase in the quantum energy of excitation light. The results are explained by a decrease in the dissipation rate of the electronic excitation energy of dye molecules upon growing their rigidity, which accelerates singlet–triplet intersystem crossing in the photogenerated electron–hole pairs via the spin–lattice relaxation mechanism.

Density of phonon states in the light-harvesting complex II of green plants

In photosynthetic antenna complexes, the coupling of electronic transitions to low-frequency vibrations of the protein matrix (phonons) plays an essential role in light absorption and ultra-fast excitation energy transfer (EET). The model calculations presented here indicate that inelastic neutron scattering experiments provide invaluable information on the phonon density of states for light-harvesting complex II, which may permit a consistent interpretation of contradictory results from high-resolution optical spectroscopy.

Chlamydomonas, a model system for studying the assembly and dynamics of photosynthetic complexes

The green unicellular alga Chlamydomonas reinhardtii has emerged as a powerful model system for studying the biosynthesis of the photosynthetic apparatus and the acclimation of this system to changes in light conditions. The assembly of the photosynthetic complexes involves the coordinate interaction between the nuclear and chloroplast genetic systems. Many factors involved in specific chloroplast post-transcriptional steps have been identified and characterized. Chlamydomonas is able to adapt to changes in light quality and in cellular ATP content by performing state transition, a process that leads to a redistribution of light excitation energy between photosystem II and photosystem I and that involves the redox state of the plastoquinone pool, the cytochrome b 6 f complex and one or several kinases specific for the light-harvesting system. Genetic approaches have provided new insights into this process.

Photosystem II and the Evolution of the Light-harvesting Antennae of Vascular Plants: A New Concept

Cryo-electron crystallography of grana membranes has led toa new concept with regards to the positioning of photosystem II (PSII) relative to its peripheral light-harvesting complex (LHCII). In projection, the structural data reveals small domains surrounding the central PSII core which are compatible with the size and expected stoichiometry of the LHCII proteins. When viewed in 3D, however, these small domains are shown to occupy a membrane separate from the membrane plane that houses the PSII core region. This observation fits with the known morphology of the grana membrane preparation which consists of paired, tightly appressed membranes. The structural data has been confirmed by separate biochemical experiments where LHCII-enriched and core PSII-enriched membrane fractions have been isolated. A vertical segregation of LHCII and PSII within the grana will be of wide significance: (a) Optimisation of light harvesting capacity (packing one membrane with LHCII, whilst at the same time maintaining efficient diffusion of plastoquinone in an adjacent membrane loosely packed with PSII core complexes). (b) Rapid adaptation to changes in light quality and intensity (via physical separation/appression of membrane pairs). (c) Cooperativity (PSII core complexes can tap into a large LHCII antenna located in an adjacent membrane). (d) New understanding of the evolution of light harvesting in plants (cyanobacteria also move light excitation energy vertically from the phycobilisome to PSII core via linker proteins).

Intravital fluorescence microscopy: impact of light-induced phototoxicity on adhesion of fluorescently labeled leukocytes.

Alterations in leukocyte/endothelium interaction due to phototoxic effects of the fluorescent dyes acridine orange (AO) and rhodamine 6G (Rh6G) were studied by intravital microscopy using the dorsal skinfold model in awake Syrian golden hamsters. AO (0.5 mg/kg/min; constant IV infusion) and Rh6G (0.1 micromol/kg; bolus IV) were administered via an indwelling venous catheter. Five to seven arterioles (35-55 microm) and postcapillary venules (30-65 microm) were investigated in each animal. Vessels were exposed four times for 30 sec to continuous light of the appropriate excitation wavelength with a 10-15-min time interval between exposures. Animals were randomly assigned to five experimental groups (five distinct light energy levels). AO and Rh6G induced leukocyte rolling/sticking in postcapillary venules and arterioles when exposed to high light energy levels. AO, but not Rh6G, induced arteriolar vasospasm when exposed to high light energies. The potential phototoxic effect of AO and Rh6G is demonstrated, as assessed by the stimulation of leukocyte-endothelium interaction and arteriolar vasospasm in vivo. This study underscores the necessity to optimize microscopic set-ups for intravital microscopy, to reduce the excitation light energy level significantly, and to perform stringent control experiments, ruling out an artificial phototoxicity-induced stimulation of leukocyte adhesion.

Cytochrome b 6 f complex: Dynamic molecular organization, function and acclimation

The cytochrome b 6 f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP(+), and around PS I for cyclic electron flow. Cytochrome b 6 f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b 6 f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b 6 f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b 6 f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b 6 f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b 6 f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b 6 f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b 6 f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.

Mechanisms of photosystem I activity stimulation in trypsin-treated pea chloroplast membranes

Trypsin-induced effects on the PSI-photochemical activity of pea chloroplast membranes have been measured under saturating light conditions. It was found that incubation of isolated chloroplasts with trypsin in concentration range of 20–60 /smg trypsin per mg Chl leads to a linear increase of the rate of PSI-mediated electron transport, measured by O 2-uptake with methyl viologen as an electron acceptor and DCPIP·H 2 as electron donor. Low temperature (77 K) chlorophyll fluorescence measurements have indicated that a remarkable redistribution of excitation light energy in favor of PSI occurs under these conditions. The results are interpreted in terms of trypsin-induced lateral reorganization of the chlorophyll-protein complexes within the thylakoid membranes and specific alteration at the level of LHC a/b-PSII supramolecular organization.