chlorophyll-a and pheophytin-a

The pathways of transformation of the chromophore of pigment-protein complexes have been studied at the terminal light-dependent stage of chlorophyll biosynthesis in plant leaves. The overall scheme of the sequence of photochemical and dark reactions of the pigment chromophore initiated by the reaction of photochemical hydration of a molecule of the precursor (protochlorophyllide) is presented. Schemes of the transformations of the components of the photoactive protochlorophyllide-oxidoreductase complex are discussed. Data are presented of features of the process at different stages of the formation of the pigment apparatus of plants.

Topics: Chlorophyll; Light; Oxidoreductases; Pheophytins; Photosynthesis; Pigments, Biological; Plant Leaves; Plant Proteins; Plants; Protochlorophyllide

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c

Topics: Chlorophyll; Cryoelectron Microscopy; Cyanobacteria; Electron Transport; Pheophytins; Photosystem I Protein Complex; Protein Structure, Quaternary

The identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale was determined. The ultrasound-assisted extraction was utilized for obtaining the chlorophyll compounds from biomass. A total of eleven molecules were separated in microalgae chlorophyll extract, with pheophytin a' (371μg·g

Topics: alpha-Tocopherol; Asphodelaceae; Chlorophyll; Chlorophyll A; Chromatography, High Pressure Liquid; Food Handling; Free Radical Scavengers; Microalgae; Peroxides; Pheophytins; Tandem Mass Spectrometry; Ultrasonics

The aims of this research were to evaluate the efficacy of copper oxychloride (CuCl2.3Cu(OH)2), copper hydroxide (Cu(OH)2) and diquat (1.1'-ethylene-2.2'-bipyridyldiylium dibromide), isolated and in association with 0.1% of both copper sources, in the control of the unicellular algae Ankistrodesmus gracilis and the filamentous algae Pithophora kewesis, and to determine the acute toxicity of the tested chemicals in Hyphressobrycon eques, Pomacea canaliculata, Lemna minor and Azolla caroliniana. The efficacy was estimated by the methods of chlorophyll a and pheophytin a readings, changed into growth inhibition percentage. Both algae were exposed to the following concentrations: 0.2; 0.4; 0.8; 1.2 mg L(-1) of diquat and its association with the copper sources; and 0.1; 0.3; 0.5; 0.7; 1.0 and 1.5 mg L(-1) in the isolated applications of copper hydroxide and copper oxychloride. An untreated control was kept. The acute toxicity was estimatedby 50% lethal concentration (LC50). The copper sources were effective for A. gracilis control, at rates as high as 0.1 mg L(-1) (>95% efficacy). Isolated diquat and its association with copper hydroxide were both effective at rates as high as 0.4 mg L(-1), with 95 and 88% control efficacy, respectively. The copper oxychloride was effective at 0.2 mg L(-1), with 93% efficacy. None of the tested chemicals and associations was effective on P. kewesis control. The most sensitive non target organism to the tested chemicals was L. minor; the less sensitive was H. eques.

Topics: Araceae; Chlorophyll; Chlorophyll A; Chlorophyta; Copper; Diquat; Ecotoxicology; Hydroxides; Pheophytins; Species Specificity; Toxicity Tests, Acute

Phosphorescence characterized by the main emission band at 952 ± 1 nm (1.30 eV), the lifetime of 1.5 ± 0.1 ms and the quantum yield nearly equal to that for monomeric chlorophyll a in aqueous detergent dispersions, has been detected in isolated reaction centers (RCs) of spinach photosystem II at 77 K. The excitation spectrum shows maxima corresponding to absorption bands of chlorophyll a, pheophytin a, and β-carotene. The phosphorescence intensity strongly depends upon the redox state of RCs. The data suggest that the phosphorescence signal originates from the chlorophyll triplet state populated via charge recombination in the radical pair [Formula: see text].

Topics: beta Carotene; Chlorophyll; Chlorophyll A; Cold Temperature; Luminescent Measurements; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spinacia oleracea

Biological photosynthetic machineries, such as photosystem I, photosystem II, or the bacterial reaction center, use cofactor molecules that absorb light or directly participate in chemical reactions. Accurate description of the structure of the cofactors, and of their interactions with protein groups, is an important step toward understanding how photosynthetic machineries work. Here we revisit the classical force field parameters for chlorophyll-a, pheophytin-a and plastoquinone-9. We present systematic quantum mechanical and classical mechanical computations that lead to a good description of the structure and non-bonded interactions of these cofactors.

Topics: Chlorophyll; Models, Molecular; Molecular Structure; Pheophytins; Photosystem II Protein Complex; Plastoquinone; Quantum Theory

Microglia-mediated inflammation is associated with pathogenesis of various neuronal disorders. This study investigated inhibitory effects of pheophytin a (PP) and chlorophyll a (CP) on neuroinflammation and underlying cellular mechanisms in microglia cells.. BV2 murine microglia cells were stimulated by lipopolysaccharide (LPS, 100 ng/mL) and interferon (IFN)-γ (10 U/mL). The productions of nitric oxide (NO) and expressions of proinflammatory cytokines and chemokines were determined by ELISA and RT-PCR. Western blot and confocal microscopy were applied to analyze activation of transcription factors and mitogen activated protein kinase (MAPK).. PP and CP significantly reduced the levels of NO, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and chemokines including macrophage inhibitory protein (MIP)-1α, macrophage chemoattractant protein (MCP)-1 and IFN-γ inducible protein (IP)-10 in BV2 cells stimulated with LPS and IFN-γ (LI). The nuclear expression of p65 NF-κB was significantly suppressed, which was accompanied by reduced the levels of IFN-β, phospho-STAT-1, and interferon regulatory factor (IRF)-1. Activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not p38 MAPK were prominently suppressed by PP and/or CP.. PP and CP may suppress inflammatory responses by inhibiting NF-κB activation and type I IFN signaling pathway. These result suggested that PP and CP have potential as anti-inflammatory agents for microglia-mediated neuroinflammatory disorders.

Topics: Animals; Cell Line; Cell Survival; Chlorophyll; Chlorophyll A; Inflammation Mediators; Interferon-gamma; Lipopolysaccharides; Mice; Microglia; Pheophytins

Anti-inflammatory activity of Saccharina japonica and its active components was evaluated via in vitro inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression in RAW 264.7 murine macrophage cells. Since the methanolic extract of S. japonica showed strong anti-inflammatory activity, it was fractionated with several solvents. Among the fractions, the ethyl acetate fraction demonstrated the highest inhibition of LPS-induced NO production (IC50=25.32μg/mL), followed by the CH2Cl2 fraction (IC50=75.86μg/mL). Considering the yield and anti-inflammatory potential together, the CH2Cl2 fraction was selected for chromatographic separation to yield two active porphyrin derivatives, pheophorbide a and pheophytin a, together with an inactive fucoxanthin. In contrast to fucoxanthin, pheophorbide a and pheophytin a showed dose-dependent inhibition against LPS-induced NO production at nontoxic concentrations in RAW 264.7 cells. Both compounds also suppressed the expression of iNOS proteins, while they did not inhibit the COX-2 expression in LPS-stimulated macrophages. These results indicate that pheophorbide a and pheophytin a are two important candidates of S. japonica as anti-inflammatory agents which can inhibit the production of NO via inhibition of iNOS protein expression. Thus, these compounds hold great promise for use in the treatment of various inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Chlorophyll; Cyclooxygenase 2; Lipopolysaccharides; Macrophage Activation; Macrophages; Magnetic Resonance Spectroscopy; Mice; Nitric Oxide Synthase Type II; Phaeophyceae; Pheophytins

The oral consumption of capsicum has been reported to increase interleukin (IL)-2 and interferon (IFN)-γ production in Peyer's patches (PP); however, the active components responsible for these effects have not been completely identified. The beneficial biological effects of green peppers cultivated under environmentally friendly farming conditions (ECP), without the use of chemical pesticides, have rarely been compared with those of green peppers cultivated under conventional farming conditions (CCP). Oral administration of ECP extract significantly induced the production of IL-2 and IFN-γ in concanavalin A-treated cells from PP ex vivo; their levels were much higher than those in the CCP extract-treated group. A comparative analysis of the HPLC profiles indicated a 1.7-fold increase of a peak, named EF-1, at 415 nm in the ECP extract. The major component of EF-1 was identified as pheophytin a, which is a chlorophyll a molecule lacking a central Mg(2+) ion, as determined from NMR data. Intake of pheophytin a and chlorophyll a significantly increased IL-2 and IFN-γ production, and the percentage of IL-2- and IFN-γ-producing CD4+ T-cells in PP. Taken together, our data suggest that ECPs produce a higher content of pheophytin a than CCPs, and pheophytin a and chlorophyll a are immune-modulating components in green vegetables.

Topics: Agriculture; Animals; Capsicum; CD4-Positive T-Lymphocytes; Cells, Cultured; Chlorophyll; Chlorophyll A; Interferon-gamma; Interleukin-2; Male; Mice; Mice, Inbred C57BL; Peyer's Patches; Pheophytins; Plant Extracts

In view of the folklore use of green leaves to treat inflammation, the anti-inflammatory property of chlorophylls and their degradation products were studied. Chlorophyll a and pheophytin a (magnesium-free chlorophyll a) from fresh leaves showed potent anti-inflammatory activity against carrageenan-induced paw edema in mice and formalin-induced paw edema in rats. Chlorophyll a inhibited bacterial lipopolysaccharide-induced TNF-α (a pro-inflammatory cytokine) gene expression in HEK293 cells, but it did not influence the expression of inducible nitric acid synthase and cyclooxygenase-2 genes. Chlorophyll b only marginally inhibited both inflammation and TNF-α gene expression. But both chlorophyll a and chlorophyll b showed the same level of marginal inhibition on 12-O-tetradecanoyl-phorbol-13-acetate-induced NF-κB activation. Chlorophylls and pheophytins showed in vitro anti-oxidant activity. The study shows that chlorophyll a and its degradation products are valuable and abundantly available anti-inflammatory agents and promising for the development of phytomedicine or conventional medicine to treat inflammation and related diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Chlorophyll; Chlorophyll A; Chromolaena; Cyclooxygenase 2; Edema; Eupatorium; Formaldehyde; HEK293 Cells; Humans; Inflammation; Lipopolysaccharides; Mice; Moraceae; NF-kappa B; Nitric Oxide Synthase Type II; Pheophytins; Plant Extracts; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

We present a set of force field (FF) parameters compatible with the AMBER03 FF to describe five cofactors in photosystem II (PSII) of oxygenic photosynthetic organisms: plastoquinone-9 (three redox forms), chlorophyll-a, pheophytin-a, heme-b, and β-carotene. The development of a reliable FF for these cofactors is an essential step for performing molecular dynamics simulations of PSII. Such simulations are important for the calculation of absorption spectrum and the further investigation of the electron and energy transfer processes. We have derived parameters for partial charges, bonds, angles, and dihedral-angles from solid theoretical models using systematic quantum mechanics (QM) calculations. We have shown that the developed FF parameters are in good agreement with both ab initio QM and experimental structural data in small molecule crystals as well as protein complexes.

Topics: beta Carotene; Chlorophyll; Chlorophyll A; Heme; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Pheophytins; Photosystem II Protein Complex; Plastoquinone; Quantum Theory

A mathematical model has been developed that describes the changes of pyropheophytin a (pyphya) in virgin olive oil (VOO). The model has been created using multivariate statistical procedures and is used in the prediction of the stability and loss of freshness of VOO. An earlier thermokinetic study (Aparicio-Ruiz, R.; Mı́nguez-Mosquera, M. I.; Gandul-Rojas, B. Thermal degradation kinetics of chlorophyll pigments in virgin olive oils. 1. Compounds of series a. J. Agric. Food Chem.2010, 58, 6200-6208) that looked at the characterization of the degradation of pheophytin a (phya), the main chlorophyll compound in VOO and a precursor of pyphya, allowed the authors to obtain the kinetic parameters necessary for mathematically expressing the percentage of pyphya, according to the time and temperature of storage using the Arrhenius model. Data regarding the percentage of pyphya obtained during the actual degradation of VOO in darkness, at room temperature and with a limited supply of oxygen, has allowed the mathematical prediction model to be validated. Using average monthly temperatures in the calculation of kinetic constants, theoretical data are obtained that are generally found to be within 95% confidence levels of experimental data.

Topics: Chlorophyll; Darkness; Drug Stability; Food Preservation; Kinetics; Models, Theoretical; Olive Oil; Pheophytins; Plant Oils; Temperature; Thermodynamics; Time Factors

In a previous study, we measured the redox potential of the primary electron acceptor pheophytin (Phe) a of photosystem (PS) II in the chlorophyll d-dominated cyanobacterium Acaryochloris marina and a chlorophyll a-containing cyanobacterium, Synechocystis. We obtained the midpoint redox potential (E(m)) values of -478 mV for A. marina and -536 mV for Synechocystis. In this study, we measured the redox potentials of the primary electron acceptor quinone molecule (Q(A)), i.e., E(m)(Q(A)/Q(A)(-)), of PS II and the energy difference between [P680·Phe a(-)·Q(A)] and [P680·Phe a·Q(A)(-)], i.e., ΔG(PhQ). The E(m)(Q(A)/Q(A)(-)) of A. marina was determined to be +64 mV without the Mn cluster and was estimated to be -66 to -86 mV with a Mn-depletion shift (130-150 mV), as observed with other organisms. The E(m)(Phe a/Phe a(-)) in Synechocystis was measured to be -525 mV with the Mn cluster, which is consistent with our previous report. The Mn-depleted downshift of the potential was measured to be approximately -77 mV in Synechocystis, and this value was applied to A. marina (-478 mV); the E(m)(Phe a/Phe a(-)) was estimated to be approximately -401 mV. These values gave rise to a ΔG(PhQ) of -325 mV for A. marina and -383 mV for Synechocystis. In the two cyanobacteria, the energetics in PS II were conserved, even though the potentials of Q(A)(-) and Phe a(-) were relatively shifted depending on the special pair, indicating a common strategy for electron transfer in oxygenic photosynthetic organisms.

Topics: Benzoquinones; Chlorophyll; Chlorophyll A; Cyanobacteria; Electron Transport; Energy Metabolism; Oxidation-Reduction; Pheophytins; Photosystem II Protein Complex; Spinacia oleracea; Synechocystis

Water oxidation by photosystem (PS) II in oxygenic photosynthetic organisms is a major source of energy on the earth, leading to the production of a stable reductant. Mechanisms generating a high oxidation potential for water oxidation have been a major focus of photosynthesis research. This potential has not been estimated directly but has been measured by the redox potential of the primary electron acceptor, pheophytin (Phe) a. However, the reported values for Phe a are still controversial. Here, we measured the redox potential of Phe a under physiological conditions (pH 7.0; 25 degrees C) in two cyanobacteria with different special pair chlorophylls (Chls): Synechocystis sp. PCC 6803, whose special pair for PS II consists of Chl a, and Acaryochloris marina MBIC 11017, whose special pair for PS II consists of Chl d. We obtained redox potentials of -536 +/- 8 mV for Synechocystis sp. PCC 6803 and -478 +/- 24 mV for A. marina on PS II complexes in the presence of 1.0 M betaine. The difference in the redox potential of Phe a between the two species closely corresponded with the difference in the light energy absorbed by Chl a versus Chl d. We estimated the potentials of the special pair of PS II to be 1.20 V and 1.18 V for Synechocystis sp. PCC 6803 (P680) and A. marina (P713), respectively. This clearly indicates conservation in the properties of water-oxidation systems in oxygenic photosynthetic organisms, irrespective of the special-pair chlorophylls.

Topics: Chlorophyll; Chlorophyll A; Cyanobacteria; Oxidation-Reduction; Pheophytins; Photosystem II Protein Complex; Synechocystis; Water

(-)-N-Formylanonaine (1), (-)-oliveroline (2), (+)-nornuciferine (3), lysicamine (4), (+)-cyperone (5), (+)-epi-yangambin (6), ficaprenol-10 (7), pheophytin a (8), aristophyll C (9) and michephyll A (10) were isolated from the leaves of Michelia alba DC (Magnoliaceae). Among them, 10 is a new compound. The structures of these compounds were characterised and identified by spectral analyses. We have also presented the antioxidation activity of 10.

Topics: Alkaloids; Antioxidants; Aporphines; Benzothiazoles; Chlorophyll; Magnoliaceae; Nuclear Magnetic Resonance, Biomolecular; Pheophytins; Plant Extracts; Plant Leaves; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Sulfonic Acids; Terpenes

We report a new device for the estimation of the content of chlorophyll a pigment in water samples as an indicator of water quality. The extraction of the pigment from water was also optimized. 10 mL of water was filtered through a nylon filter (45 microm pore size and 13 mm of diameter), after the chlorophylls were dissolved by immersing the filter in 1 mL of a low non-hazardous solvent as ethanol. An in-valve in-tube SPME device coupled to capillary liquid chromatography with diode array detection was designed. A capillary column of 70 cm in length (0.32 mm i.d. coated with 5% diphenyl-95% polydimethylsiloxane, 3 microm coating thickness) was used as the loop of the injection valve for preconcentration and a Zorbax SB C(18) (SiO(2)-based) 150 mm x 0.5 mm i.d., 5 microm column (Agilent) was used as analytical column. The achieved detection limit was 0.05 microg L(-1) and the working range of concentrations was 0.1-1 microg L(-1). % RSD values between 2 and 11 were obtained. Chlorophyll a in several water matrices was determined with good results in presence of other pigments such as chlorophyll b, pheophytin a and pheophytin b.

Topics: Chlorophyll; Filtration; Limit of Detection; Pheophytins; Quality Control; Solid Phase Extraction; Solvents; Water Pollutants, Chemical

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll --> pheophytin --> pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Cellular Senescence; Chlorophyll; Chloroplasts; Hydrolases; Membrane Proteins; Molecular Sequence Data; Mutation; Phenotype; Pheophytins; Photosynthesis; Phylogeny; Phytol; Plant Leaves; Recombinant Fusion Proteins; Sequence Alignment

Chlorophyll degradation is an important phenomenon in the senescence process. It is necessary for the degradation of certain chlorophyll-protein complexes and thylakoid membranes during leaf senescence. Mutants retaining greenness during leaf senescence are known as 'stay-green' mutants. Non-functional type stay-green mutants, which possess defects in chlorophyll degradation, retain greenness but not leaf functionality during senescence. Here, we report a new stay-green mutant in rice, nyc3. nyc3 retained a higher chlorophyll a and chlorophyll b content than the wild-type but showed a decrease in other senescence parameters during dark incubation, suggesting that it is a non-functional stay-green mutant. In addition, a small amount of pheophytin a, a chlorophyll a-derivative without Mg(2+) ions in its tetrapyrrole ring, accumulated in the senescent leaves of nyc3. nyc3 shows a similar but weaker phenotype to stay green (sgr), another non-functional stay-green mutant in rice. The chlorophyll content of nyc3 sgr double mutants at the late stage of leaf senescence was also similar to that of sgr. Linkage analysis revealed that NYC3 is located near the centromere region of chromosome 6. Map-based cloning of genes near the centromere is very difficult because of the low recombination rate; however, we overcame this problem by using ionizing radiation-induced mutant alleles harboring deletions of hundreds of kilobases. Thus, it was revealed that NYC3 encodes a plastid-localizing alpha/beta hydrolase-fold family protein with an esterase/lipase motif. The possible function of NYC3 in the regulation of chlorophyll degradation is discussed.

Topics: Amino Acid Sequence; Chlorophyll; Chloroplasts; Cloning, Molecular; Gene Expression Regulation, Plant; Genetic Linkage; Hydrolases; Microscopy, Electron, Transmission; Molecular Sequence Data; Mutation; Oryza; Phenotype; Pheophytins; Phylogeny; Plant Leaves; Plant Proteins; Protein Stability; RNA, Plant

Chlorella, when heterotrophically cultivated in the dark, is able to grow with Zn2+ at 10-40 mM, which is 10 times the concentration lethal to autotrophically grown cells. However, the lag phase is prolonged with increasing concentrations of Zn2+; for example, in this study, 1 d of the control lag phase was prolonged to about 16 d with Zn2+ at 16.7 mM (x2,000 of the control). Once the cells started to grow, the log phase was finished within 4-6 d regardless of Zn concentration, which was almost the same as that of the control. The photosysystem I reaction center chlorophyll, P700, and the far-red fluorescence were detected only after the late log phase of the growth curve, suggesting that chlorophyll-protein complexes can be organized after cell division has ceased. Interestingly, at more than 16.7 mM of Zn2+, Zn-chlorophyll a was accumulated and finally accounted for about 25% of the total chlorophyll a in the late stationary phase. We found that the Zn-chlorophyll a was present in the thylakoid membranes and not in the soluble fractions of the cells. The rather low fluorescence yield at around 680 nm in the stationary phase suggests that Zn-chlorophyll a can transfer its excitation energy to other chlorophylls. Before accumulation of Zn-chlorophyll a, a marked amount of pheophytin a was temporally accumulated, suggesting that Zn-chlorophyll a could be chemically synthesized via pheophytin a.

Topics: Cell Division; Chlorella; Chlorophyll; Chlorophyll A; Darkness; Dose-Response Relationship, Drug; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Thylakoids; Zinc

Chlorophylls, owing to their adjustable pi-electron system and intense, well-separated electronic transitions, can serve as convenient intrinsic spectroscopic probes of ligand-metal center interactions. They are also interesting for their photosensitizing properties. In order to examine the heavy-atom effects on the chlorophyll triplet state, a key intermediate in chlorophyll-photosensitized reactions, the synthesis of a novel Pt(II)-substituted chlorophyll a was carried out, and the effects of the substitution on steady-state and transient photophysical properties of chlorophyll were studied by absorption and fluorescence spectroscopies, and by laser flash photolysis. The presence of highly electronegative platinum as the central ion increases the energies of the chlorophyll main absorption transitions. As laser flash photolysis experiments show, in air-equilibrated solutions, chlorophyll triplets are efficiently quenched by molecular oxygen. Interestingly, this quenching by oxygen is more effective with metal-containing pigments, in spite of the increased spin-orbit coupling, introduced with the central metals. This points to occurrence of nonspecific interactions of molecular oxygen with metallochlorophylls. The differences in the effects exerted on the pigment triplet by the central metal become distinct after the removal of oxygen. The lifetime of a Pt-chlorophyll triplet remains very short, in the range of only a few microseconds, unlike in the free-base and Mg- and Zn-substituted chlorophylls. Such drastic shortening of the triplet lifetime can be attributed to a large heavy-atom effect, implying that strong interactions must occur between the central Pt(II) ion and the chlorophyll macrocycle, which lead to a more efficient spin-orbit coupling in Pt-chlorophyll than in Pt-porphyrins.

Topics: Chlorophyll; Chlorophyll A; Energy Transfer; Magnesium; Metals, Heavy; Pheophytins; Platinum; Spectrometry, Fluorescence; Zinc

A 'metal-free' chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.

Topics: Chlorophyll; Chlorophyll A; Electron Transport; Energy Transfer; Eukaryota; Molecular Structure; Petroselinum; Pheophytins; Photosystem II Protein Complex

Photosystem II (PSII) electron transfer (ET) in the chlorophyll d-containing cyanobacterium Acaryochloris marina (A. marina) was studied by time-resolved electron paramagnetic resonance (EPR) spectroscopy at room temperature, chlorophyll fluorescence, and low-temperature optical spectroscopy. To maximize the ability to measure PSII ET in the intact cells of this organism, growth conditions were optimized to provide the highest specific O(2) activity and the instrumental parameters for the EPR measurements of tyrosine Z (Y(Z)) reduction were adjusted to give the best signal-to-noise over spectral resolution. Analysis of the Y(Z)(*) reduction kinetics revealed that ET to the oxygen-evolving complex on the donor side of PSII in A. marina is indistinguishable from that in higher plants and other cyanobacteria. Likewise, the charge recombination kinetics between the first plastoquinone acceptor Q(A) and the donor side of PSII monitored by the chlorophyll fluorescence decay on the seconds time scale are not significantly different between A. marina and non-chlorophyll d organisms, while low-temperature optical absorption spectroscopy identified the primary electron acceptor in A. marina as pheophytin a. The results indicate that, if the PSII primary electron donor in A. marina is made up of chlorophyll d instead of chlorophyll a, then there must be very different interactions with the protein environment to account for the ET properties, which are similar to higher plants and other cyanobacteria. Nevertheless, the water oxidation mechanism in A. marina is kinetically unaltered.

Topics: Bacterial Proteins; Chlorophyll; Cyanobacteria; Electron Spin Resonance Spectroscopy; Electron Transport; Oxidation-Reduction; Oxygen; Pheophytins; Photosystem II Protein Complex; Plastoquinone; Spectrometry, Fluorescence

Pigment-protein complexes enriched in photosystem II (PS II) have been isolated from the chlorophyll (Chl) d containing cyanobacterium, Acaryochloris marina. A small PS II-enriched particle, we call 'crude reaction centre', contained 20 Chl d, 0.5 Chl a and 1 redox active cytochrome b-559 per 2 pheophytin a, plus the D1 and D2 proteins. A larger PS II-enriched particle, we call 'core', additionally bound the antenna complexes, CP47 and CP43, and had a higher chlorophyll per pheophytin ratio. Pheophytin a could be photoreduced in the presence of a strong reductant, indicating that it is the primary electron acceptor in photosystem II of A. marina. A substoichiometric amount of Chl a (less than one chlorophyll a per 2 pheophytin a) strongly suggests that Chl a does not have an essential role in the photochemistry of PS II in this organism. We conclude that PS II, in A. marina, utilizes Chl d and not Chl a as primary electron donor and that the primary electron acceptor is one of two molecules of pheophytin a.

Topics: Chlorophyll; Cyanobacteria; Cytochromes b; Oxidation-Reduction; Pheophytins; Photochemistry; Photosystem II Protein Complex; Protein Binding; Spectrum Analysis; Temperature

The effects of acid and alkali treatment on the light absorption, energy transfer and protein secondary structure of the photosystem II core antenna CP43 and CP47 of spinach were investigated by the absorption spectra, fluorescence emission spectra and circular dichroism spectra. It has been found that acid treatment caused the appearance of absorption characteristic of pheophytin a (Pheo a), whereas alkali treatment induced a new absorption peak at 642 nm. The energy transfer between beta-carotene and chlorophyll a (Chl a) in CP43 was easily disturbed by alkali, whereas in CP47 was readily affected by acid. As to the effects on the secondary structure of proteins in CP43 and CP47, effects of acid were far less than those of alkali. Both acid and alkali disturbed the microenvironment of Chl a and interfered exciton interaction between Chl a molecules. It was suggested that acid and alkali affect the light absorption, energy transfer and protein secondary structure of CP43 and CP47 in a different way. H+ can permeate into the internal space of alpha-helix, change Chl a into Pheo a and disturb the microenvironment of pigments without damaging the secondary structure of protein, whereas OH- can induce the protein unfolding at first, then saponify Chl a to chlorophyllide and disturb the microenvironment of pigments.

Topics: Acids; Alkalies; beta Carotene; Chlorophyll; Chlorophyll A; Circular Dichroism; Energy Transfer; Hydrogen-Ion Concentration; Light; Light-Harvesting Protein Complexes; Pheophytins; Photosensitizing Agents; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Protein Structure, Secondary; Spectrometry, Fluorescence; Spinacia oleracea

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.

Topics: Chlorophyll; Cyanobacteria; Electron Transport; Energy Metabolism; Kinetics; Light-Harvesting Protein Complexes; Luminescent Measurements; Oxidation-Reduction; Oxygen; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Proteins; Spectrometry, Fluorescence; Temperature

The resolution of quaternary mixtures of chlorophylls a and b and pheophytins a and b has been accomplished by partial least-squares (PLS) multivariate calibration, applied to the fluorescence signals of these pigments. The total luminescence information of the compounds has been used to optimize the spectral data set to perform the calibration. After preliminary studies, a method is described in acetone media, to avoid emulsions with the olive oil samples. Different scanning paths have been selected for each method. For the simultaneous determination of the pigments in olive oil samples, a comparative study of the results found by using excitation, emission, and synchronous spectral data, as analytical signal, was performed. The excitation spectra were selected as the better analytical signals for the determination of the pigments in olive oil samples. The optimum wavelength range to record the excitation spectra (lambda(em) = 662 nm) was selected to minimize the contribution of pheophytin a and to maximize the contribution of the other pigments, which are the minor constituents in olive oil. Determination of these pigments in olive oil samples was effected from the excitation spectra of dissolutions o suitable aliquots in acetone. Recovery values from olive oil, spiked with chlorophylls a and b and pheophytins a and b, were in the ranges of 70-112, 71-111, 76-105, and 82-109%, respectively.

Topics: Chlorophyll; Chlorophyll A; Olive Oil; Pheophytins; Plant Oils; Spectrometry, Fluorescence

Pigment-depleted Photosystem II reaction centers (PS II-RCs) from a higher plant (pea) containing five chlorophyll a (Chl) per two pheophytin a (Phe), were treated with Chl and several derivatives under exchange conditions [FEBS Lett. 434 (1998) 88]. The resulting reconstituted complexes were compared to those obtained by pigment exchange of "conventional" PS II-RCs containing six Chl per two Phe. (1) The extraction of one Chl is fully reversible. (2) The site of extraction is the same as the one into which previously extraneous pigments have been exchanged, most likely the peripheral D1-H118. (3) Introducing an efficient quencher (Ni-Chl) into this site results in only 25% reduction of fluorescence, indicating incomplete energy equilibration among the "core" and peripheral chlorophylls.

Topics: Binding Sites; Chlorophyll; Chromatography, High Pressure Liquid; Circular Dichroism; Energy Transfer; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pigments, Biological; Pisum sativum; Plant Leaves; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

The absorption and fluorescence properties of pheophorbide-a, Sodium salt of pheophorbide-a and its long chain (C20H39) ester (Pheophytine) were investigated in air-saturated micellar aqueous solutions before and after ultrasound irradiation (48 kHz, 10 min). The absorption spectra changed depending on the surfactant; cetyltrimethyl ammonium bromide (CTAB) or sodium dodecyl sulfate concentrations. The formation of different molecular species in various micellar solutions was estimated from the analysis of the absorption spectra. The absorption bands resulted from an aggregated form of the chromophore present in 50 mM phosphate buffer and in pre-micellar solutions. The specific bands of the aggregate disappeared with a simultaneous increase of the bands of monomer in normal micellar solution. The fluorescence spectra, the lifetimes and the fraction of each component (with a characteristic lifetime) of the chromophore in the micellar solutions changed significantly before and after ultrasound irradiation although the changes in absorption spectra were small. The fluorescence emission band at 710 nm due to the aggregate almost disappeared in the pre-micellar solution after ultrasound irradiation. The fraction of the short-lifetime component estimated for the aggregates decreased 55% in H2O or 85% in 2 mM CTAB, however the long-lifetime components increased after the ultrasound treatment. From these fluorescence properties, it was concluded that the aggregated molecules were converted to a stable monomeric form by ultrasound. Extrapolation of these data to in vivo situations suggests that pretreatment of certain photosensitizers with ultrasound in micellar solutions may lead to increased efficiency of photodynamic therapy since only the monomers are photodynamically active.

Topics: Cetrimonium; Cetrimonium Compounds; Chlorophyll; Micelles; Molecular Conformation; Pheophytins; Photosensitizing Agents; Radiation-Sensitizing Agents; Sodium Dodecyl Sulfate; Solutions; Spectrometry, Fluorescence; Spectrophotometry; Surface-Active Agents; Ultrasonics; Water

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.

Topics: Chlorophyll; Chlorophyll A; Chromatography, High Pressure Liquid; Light; Light-Harvesting Protein Complexes; Pheophytins; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Plant Proteins; Spinacia oleracea; Thylakoids; Time Factors; Vitamin K 1

Normal-phase HPLC conditions have been developed for separating the C17(3) isoprenoid isomers, which are expected to be formed as biosynthetic intermediates of chlorophyll (Chl) a, Chl a' (C13(2)-epimer of Chl a), pheophytin (Pheo) a and protochlorophyll (PChl). The application of these conditions to pigment composition analysis of greening etiolated barley leaves allowed us to detect, for the first time, the C17(3) isomers of Chl a', a possible constituent of the primary electron donor of photosystem (PS) I, P700, and those of Pheo a, the primary electron acceptor of PS II, in the very early stage of greening. The C17(3) isomer distribution patterns were approximately the same between Chl a and Chl a', but significantly different between Pheo a and Chl a', probably reflecting the similarity and difference, respectively, in the biosynthetic pathways of these pigment pairs.

Topics: Carbohydrate Sequence; Chlorophyll; Chlorophyll A; Chromatography, High Pressure Liquid; Hordeum; Isomerism; Pheophytins; Plant Leaves; Plant Proteins

Topics: Chlorophyll; Chromatography, High Pressure Liquid; Eukaryota; Pheophytins; Photosynthesis; Pigments, Biological

A series of modified chlorophylls (chlorophyll a, pyrochlorophyll a, Zn-pheophytin a and Zn-pheophorbide a) have been inserted into lamellar phases of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT). The role played by the different functional groups in affecting the bilayer formation and organisation has been investigated by means of the NMR quadrupolar splitting technique. Evidence is reported for the first time on the capacity of the phytyl chain of the chlorophylls to anchor the tetrapyrroles into the bilayer, favouring at the same time the regular formation of the lamellae.

Topics: Chlorophyll; Lipid Bilayers; Magnetic Resonance Spectroscopy; Pheophytins; Phytic Acid; Spinacia oleracea; Time Factors

The reduction of chlorophyllide b and its analogue zinc pheophorbide b in etioplasts of barley (Hordeum vulgare L.) was investigated in detail. In intact etioplasts, the reduction proceeds to chlorophyllide a and zinc pheophorbide a or, if incubated together with phytyldiphosphate, to chlorophyll a and zinc pheophytin a, respectively. In lysed etioplasts supplied with NADPH, the reduction stops at the intermediate step of 7(1)-OH-chlorophyll(ide) and Zn-7(1)-OH-pheophorbide or Zn-7(1)-OH-pheophytin. However, the final reduction is achieved when reduced ferredoxin is added to the lysed etioplasts, suggesting that ferredoxin is the natural cofactor for reduction of chlorophyll b to chlorophyll a. The reduction to chlorophyll a requires ATP in intact etioplasts but not in lysed etioplasts when reduced ferredoxin is supplied. The role of ATP and the significance of two cofactors for the two steps of reduction are discussed.

Topics: Adenosine Triphosphate; Alcohol Oxidoreductases; Chlorophyll; Chlorophyll A; Ferredoxin-NADP Reductase; Ferredoxins; Hordeum; Intracellular Membranes; Oxidation-Reduction; Pheophytins; Plastids; Subcellular Fractions; Zinc

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Topics: Chlorophyll; Chlorophyll A; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plants; Zinc

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH4-). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH4- in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13(1)-deoxo-13(1)-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo- and P680+. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Qy and Qx transitions of this molecule were determined to be located at 5 degrees C at 679.5-680 nm and 542 nm, respectively.

Topics: Borohydrides; Chenopodiaceae; Chlorophyll; Chlorophyll A; Cytochrome b Group; Dithionite; Light; Light-Harvesting Protein Complexes; Paraquat; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Proteins; Spectrophotometry

In this study a model for decomposition and pigment assignment of the low-temperature (10 K) absorption spectrum of the photosystem II reaction center (D1-D2-cytochrome b559 complex, PSII-RC) is developed. It is based on theoretical calculations of the line shapes of the inhomogeneously broadened pigment spectra, taking into account electron-phonon coupling. The analysis is performed under the hypothesis that exciton coupling is weak, except for the P680 special pair. In this way a detailed decomposition of the absorption spectrum is obtained. Within the model the temperature dependence of the spectrum can be well explained. It is mainly caused by the temperature-dependent changes of the homogeneous absorption spectra of the individual pigments in the PSII-RC. In addition, slight changes in the inhomogeneous distribution functions have to be taken into account. Two slightly different parameter sets are found. We prefer one of these parameter sets which indicates that an accessory chlorophyll (Chl) is the lowest energy pigment in the RC core and that the two antenna Chls have their spectral maxima at 667.7 and 677.9 nm, respectively. The relationship between the shape of the absorption spectrum and the pigment stoichiometry of the sample (ratio of chlorophyll a:pheophytin a), which was noticed by comparison of a variety of different independently prepared samples, can be explained by the presence of "additional" Chl molecules which are nonstoichiometrically bound to part of the PSII-RCs. These Chls can be grouped into three spectrally distinguishable pools. One of them has its absorption maximum at about 683 nm and is responsible for the prominent shoulder that is present in the 10 K absorption spectra of most PSII-RC preparations. Our results suggest that the Chl content of the samples has been underestimated in many spectroscopic studies on the PSII-RC.

Topics: Chlorophyll; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spectrophotometry; Temperature

Photosystem II, the photosynthetic water-oxidizing complex, can be isolated from both plants and cyanobacteria. A variety of methods have been developed for purification of this enzyme, which can be isolated in several functional and structural forms. Knowledge of the pigment content of photosystem II preparations is important for precise spectroscopic, biochemical, and functional analysis. We have determined pigment stoichiometries in oxygen-evolving photosystem II preparations from plants and cyanobacteria. We have employed a solvent system for the isocratic elution of a reverse phase HPLC column in which we have determined the extinction coefficients of the relevant pigments. Pigments were extracted from four photosystem II preparations. These preparations included spinach photosystem II membranes [Berthold, D. A., Babcock, G. T., & Yocum, C. F. (1981) FEBS Lett. 134, 231-234], spinach photosystem II reaction center complexes [Ghanotakis, D. F., & Yocum, C. F. (1986) FEBS Lett. 197, 244-248], spinach photosystem II complexes [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856], and photosystem II particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803 [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Pigment stoichiometries were determined using two different methods of data analysis and were based on the assumption that there are two pheophytin a molecules per photosystem II reaction center. The pigment stoichiometries obtained were comparable for the two methods of data analysis and agreed with previous biophysical and biochemical characterizations of the preparations. The average pigment stoichiometries (chlorophyll:plastoquinone-9 per 2 pheophytin a) determined using the two data analysis methods were as follows: photosystem II membranes, 274:3.2; photosystem II reaction center complexes, 78:2.5; Synechocystis PS II particles, 55:2.4; photosystem II complexes, 121:2.0.

Topics: Chlorophyll; Chlorophyll A; Chromatography, High Pressure Liquid; Cyanobacteria; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plastoquinone; Spectrophotometry; Spinacia oleracea

The pigment composition of the isolated photosystem II reaction center complex in its most stable and pure form currently is a matter of considerable debate. In this contribution, we present a new method based on a combination of gel filtration chromatography and diode array detection to analyze the composition of photosystem II reaction center preparations. We show that the method is very sensitive for the detection of contaminants such as the core antenna protein CP47, pigment-free and denatured reaction center proteins, and unbound chlorophyll and pheophytin molecules. We also present a method by which the photosystem II reaction center complex is highly purified without using Triton X-100, and we show that in this preparation the contamination with CP47 is less than 0.1%. The results strongly indicate that the photosystem II reaction center complex in its most stable and pure form binds six chlorophyll a, two pheophytin a, and two beta-carotene molecules and that the main effect of Triton X-100 is the extraction of beta-carotene from the complex. Analysis of 4 K absorption and emission spectra indicates that the spectroscopic properties of this preparation are similar to those obtained by a short Triton X-100 treatment. In contrast, preparations obtained by long Triton X-100 treatment show decreased absorption of the shoulder at 684 nm in the 4 K absorption spectrum and an increased number of pigments that trap excitation energy at very low temperatures. We conclude that the 684 nm shoulder in the 4 K absorption spectrum should at least in part be attributed to the primary electron donor of photosystem II.

Topics: beta Carotene; Chlorophyll; Chlorophyll A; Chromatography, Gel; Chromatography, High Pressure Liquid; Detergents; Light-Harvesting Protein Complexes; Octoxynol; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spectrometry, Fluorescence; Spectrophotometry; Spinacia oleracea; Temperature

Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Zn-pheophytin a. Zn-pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90-300 pmol of Zn-pheophytin a or of about 400-600 pmol of chlorophyll a/4.2 x 10(7) etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-pheophytin a, but was unaffected by higher concentrations of chlorophyll a.

Topics: Apoproteins; Chlorophyll; Chlorophyll A; Light-Harvesting Protein Complexes; Magnesium; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Zinc

A D1-D2-cyt b-559 complex containing 4 chlorophyll alpha, 1 beta-carotene and 1 cytochrome b-559 per 2 pheophytin a has been isolated from spinach with 30% yield using a Q-Sepharose Fast-Flow anion-exchange column equilibrated with 0.1% Triton X-100, 10 mM MgSO4 and 50 mM Tris-HCl (pH 7.2). The preparation was then stabilized with 0.1% dodecyl-beta-D-maltoside. This method gave a yield 10 times higher than that using a Fractogel TSK-DEAE 650(S) column equilibrated with 0.1% Triton X-100, 30 mM NaCl and 50 mM Tris-HCl (pH 7.2). The PS II RC complex was characterized using absorption and fluorescence spectroscopy at 277 and 77 K. A selective reversible bleaching under reducing conditions with maximum at 682 nm, associated with pheophytin a reduction, and light-induced absorption differences with a lifetime of 1.0 ms, ascribed to the triplet state of P680 were measured and indicated that the isolated D1-D2-cyt b-559 complex is active in charge separation. The results are compared with the data obtained for a PS II RC preparation containing 6 chlorophyll alpha, 2 beta-carotene and 1 cyt b-559 per 2 pheophytin a.

Topics: Chlorophyll; Chlorophyll A; Cytochrome b Group; Glucosides; Hydrogen-Ion Concentration; Light-Harvesting Protein Complexes; Macromolecular Substances; Magnesium Sulfate; Molecular Weight; Octoxynol; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plants; Spectrometry, Fluorescence; Spectrophotometry

The EPR and ENDOR characteristics of the intermediate electron acceptor radical anion I-. in Photosystem II (PS II) are shown to be identical in membrane particles and in the D1D2 cytochrome b-559 complex (Nanba, O. and Satoh, K. (1987) Proc. Natl. Acad. Sci. USA 84, 109-112). These findings provide further evidence that the D1D2 complex is the reaction center of PS II and show that the pheophytin binding site is intact. A hydrogen bond between I-. and the protein (GLU D1-130) is postulated on the basis of D2O exchange experiments. The ENDOR data of I-. and of the pheophytin a radical anion in different organic solvents are compared and the observed differences are related to structural changes of the molecule on the basis of molecular orbital calculations (RHF-INDO/SP). The importance of the orientation of the vinyl group (attached to ring I) on electron transfer is discussed.

Topics: Anions; Chlorophyll; Chloroplasts; Cytochrome b Group; Electron Transport; Electrons; Free Radicals; Hydrogen Bonding; Light-Harvesting Protein Complexes; Molecular Structure; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plant Proteins; Protons; Spectrum Analysis

Topics: Chemistry Techniques, Analytical; Chlorophyll; Chlorophyll A; Pheophytins; Research; Water

Topics: Chlorophyll; Fermentation; Humans; Nursing Care; Pheophytins; Photosynthesis