The effect of norflurazon on protein composition and chlorophyll organization in pigment–protein complex of Photosystem II

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides in the 29.5–21 kDa region. The Chl a forms at 668, 676, and 690 nm that belong to LHC and antenna part of PS I disappear completely after treatment. The intensity of the Chl b form at 648 nm is sharply decreased in treated seedlings grown under 30 or 100 lx light intensity. The bands of carotenoid absorption at 421, 448 (Chl a), 452, 480, 492, 496 (β-carotene), and 508 nm also disappear. The band shift from 740 to 720 nm and decrease in its intensity relative to the 687 nm emission peak in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chla form at 710–712 nm.     

Normal-phase HPLC quantitation of chlorophyll a′ and phylloquinone in Photosystem I particles

Fractionated Photosystem (PS) I particles consisting of six, five or two core proteins were analyzed by HPLC for chlorophyll (Chl) a and phylloquinone (PhQ). Each particle had a Chl a/P700 molar ratio of 50–55 and contained ca. 2 molecules of Chl a per P700. Deliberate control of eluent composition led to isolated elution of PhQ and -carotene in the normal-phase chromatogram. Based on these a simple HPLC procedure has been established to determine the PhQ/P700 molar ratio, which was ca. 2 for the larger two PS I particles and ca. 1 for the smallest particle, in line with previous reports.     

Room temperature photooxidation of β-carotene and peripheral chlorophyll in photosystem II reaction centre

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.     

Similarity between electron donor side reactions in the solubilized Photosystem II–LHC II supercomplex and Photosystem-II-containing membranes

The PS II–LHC II supercomplex is a novel type of oxygen evolving Photosystem II (PS II) core particle that contains the light harvesting complex proteins Lhcb1/2/4/5 in addition to the PS II reaction centre, oxygen evolving complex (OEC) and inner antennae [Hankamer et al. (1997) Eur J Biochem 243: 422–429]. The 33 and 23 kDa extrinsic proteins in these particles have been localised by image analysis of electron micrographs and averaging techniques [Boekema et al. (1998) Eur J Biochem 252: 268–276]. To assay the functionality of the water splitting complex, we compared the single flash P680⁺ reduction kinetics in these supercomplexes with those of PS II-rich granal stack membranes (BBYs). We found that the P680⁺ reduction kinetics in PS II–LHC II supercomplexes were indistinguishable from those in BBYs. We also examined a number of PS II core particles lacking the Lhcb components. All of these had different P680⁺ reduction kinetics, which we attributed to partial loss of OEC function before and during the measurements.     

Changes in the antenna size of Photosystem I and Photosystem II in Synechococcus sp. strain PCC 7942 grown in the presence of SANDOZ 9785 — a Photosystem II inhibitor

SANDOZ 9785, also known as BASF 13.338, is a pyridazinone derivative that inhibits Photosystem II (PS II) activity leading to an imbalance in the rate of electron transport through the photosystems. Synechococcus sp. strain PCC 7942 cells grown in the presence of sublethal concentration of SANDOZ 9785 (SAN 9785) for 48 hours exhibited a 20% decrease in Chl a per cell. However, no changes were observed in the content of phycocyanin per cell, the size of the phycobilisomes or in the PS II:PS I ratio. From an estimate of PS II electron transport rate under varying light intensities and spectral qualities and analysis of room temperature Chl a fluorescence induction, it was deduced that growth of Synechococcus PCC 7942 in the presence of SAN 9785 leads to a redistribution of excitation energy in favour of PS II. Though the redistribution appears to be primarily caused by changes affecting the Chl a antenna of PS II, the extent of energetic coupling between phycobilisomes and PS II is also enhanced in SAN 9785 grown Synechococcus PCC 7942 cells. There was a reduction in the effective size of PS I antenna based on measurement of P700 photooxidation kinetics. These results indicate that when PS II is partially inhibited, the structure of photosynthetic apparatus alters to redistribute the excitation energy in favour of PS II so that the efficiency of utilization of light energy by the two photosystems is optimized. Our results suggest that under the conditions used, drastic structural changes are not essential for redistribution of excitation energy between the photosystems.Abbreviations APC Allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophyenyl)-1,1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - F_o fluorescence when all the reaction centres are open - f_m fluorescence yield when all the reaction centres are closed - F_v variable chlorophyll fluorescence - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic Acid - I₅₀ concentration that causes 50% inhibition in activity - MV methyl viologen - pBQ para benzoquinone - PBS phycobilisome - PC phycocyanin - PS I, PS II Photosystem I, Photosystem II - P700 reaction centre Chl a of PS I - SAN 9785 SANDOZ 9785 i.e. 4-chloro-5-dimethylamino-2-phenyl-3 (2H) pyridazinone, also known as BASF 13.338     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review

The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at approximately 730 nm (Frese et al., Biochemistry 42:9205-9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84-91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of Q(A) photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841-851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (B(A) and B(B)) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of B(A) molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P(870)* increased up to approximately 200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the B(A) appears to be lost and B(B) is replaced by bacteriopheophytin Phi(B) with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of approximately 2 ps. These results are discussed in terms of obligatory role of B(A) and Phi(B) molecules located near P for efficient electron transfer from P*.     

Role of the oxidized secondary acceptor Q B of Photosystem II in the delayed ‘afterglow’ chlorophyll luminescence

Leaf discs of dark-adapted tobacco plants were excited by 2 flashes and kept in darkness at 20 °C for various time periods, then thermoluminescence emission was recorded without freezing the sample. The B band at 30 °C decreased with a half-time t_1/2~1 min and the AG band at 45 °C with a t_1/2~5 min. This corresponds to the decay kinetics of S_2/3 in PS II centres in the state S_2/3 Q_B^- (B band) or S_2/3 Q_B. Assuming that the 45 °C band is an ‘afterglow’ emission originating from those centres with an oxidized Q_B on which an electron is back-transferred from stroma reductants through a pathway induced by warming, the theoretical ratio of the B and AG band was compared to that measured experimentally. After 2 or 3 flashes producing mainly S₃, the intensity of AG band encompassed several fold that of the B band, because recombining S₃ recreated S₂ Q_B AG-emitting centres. In order to confirm that the AG band is governed by the heat-induced activation of a dark Q_B-reducing pathway rather than by PS II charge recombination, the AG emission was characterized in triazine-resistant Chenopodium album weed biotypes. In these mutants where the Q_B pocket is altered, the B band is strongly downshifted to 18 °C, compared to 32 °C in the wild type, whereas the AG band is only downshifted by 3 or 4 °C, demonstrating that S_2/3 Q_B^- is not the limiting step of the AG emission.     

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading α-ketoglutarate dioxygenase

 The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the α-ketoglutarate (α-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and α-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g–tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of α-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding. Received: 3 July 1998 / Accepted: 13 October 1998     

A thermoluminescence study of Photosystem II back electron transfer reactions in rice leaves – effects of salt stress

The recombination reactions of Photosystem II have been investigated in vivo in rice leaves by using the thermoluminescence (TL) emission technique. Excitation of dark-adapted leaf segments at 0 °C with different number of single turn-over flashes induced the appearance of complex TL glow curves. The mathematical analysis of these curves showed the existence of four TL components: B1-band (temperature maximum, t_max, at 24 °C, originating from S₃Q_B⁻ recombination), B2-band (t_max at 35 °C, from S₂Q_B⁻), AG-band (t_max at 46 °C) and C-band (t_max at 55 °C, from Tyr_D⁺Q_A⁻). Their contributions to the total TL signal were different depending on the number of flashes given. AG-band seems to reflect a special electron transfer from some unknown stroma donor to PS II. Q-band (t_max at 19 °C), originating from S₂Q_A⁻ recombination, was recorded after flashing samples incubated in the presence of DCMU. The recombination halftimes (t_1/2) at 20 °C of S₂Q_A⁻, S₃Q_B⁻, S₂Q_B⁻ and Tyr_D⁺Q_A⁻ were, respectively, 0.8 s, 48 s, 74 s and about 1 h. A sharp AG-band (t_max at 50 °C and t_1/2 of 210 s) could be also observed after illumination of leaves with far-red light and after a dark incubation period of whole plants. Incubation of leaf segments with 0.5 M NaCl abolished the inductions of AG-band by darkness and far-red illumination, significantly decreased Q-band intensity, whereas induced a strong increase in C-band intensity. The possible inhibition of S₂/S₃ formation and quinone oxidation by saline stress are discussed.     

Biosynthesis of non-fluorescent chlorophyll of Photosystem II core in greening plant leaves. Effect of etiolated plants growing under heat shock conditions (38 °C)

Preliminary dark incubation of etiolated pea and maize plants at 38 °C allowed to observe a new dark reaction of Chl biosynthesis occuring after photoconversion of protochlorophyllide Pchld 655/650 into chlorophyllide Chld 684/676. This reaction was accompanied by chlorophyllide esterification and by the bathochromic shift of pigment spectra: Chld 684/676 Chl 688/680. After completion of the reaction, a rapid (20–30 s at 26 °C) quenching of Chl 688/680 low-temperature fluorescence was observed. The reaction Chld 684/676 Chl 688/680 was inhibited under anaerobic conditions as well as in the presence of KCN; the reaction accompanied by Chl fluorescence quenching was inhibited in the leaves of pea mutants with impaired function of Photosystem II reaction centers. The spectra position of newly formed Chl, effects of Chl fluorescence quenching allowed to assume that the new dark reaction is responsible for biosynthesis of P–680, the key pigment of Photosystem II reaction centres.     

Biosorption of Cd(II) and Pb(II) onto brown seaweed, Lobophora variegata (Lamouroux): kinetic and equilibrium studies

The present work deals with the biosorption performance of raw and chemically modified biomass of the brown seaweed Lobophora variegata for removal of Cd(II) and Pb(II) from aqueous solution. The biosorption capacity was significantly altered by pH of the solution delineating that the higher the pH, the higher the Cd(II) and Pb(II) removal. Kinetic and isotherm experiments were carried out at the optimal pH 5.0. The metal removal rates were conspicuously rapid wherein 90% of the total sorption occurred within 90 min. Biomass treated with CaCl₂ demonstrated the highest potential for the sorption of the metal ions with the maximum uptake capacities i.e. 1.71 and 1.79 mmol g⁻¹ for Cd(II) and Pb(II), respectively. Kinetic data were satisfactorily manifested by a pseudo-second order chemical sorption process. The process mechanism consisting of both surface adsorption and pore diffusion was found to be complex. The sorption data have been analyzed and fitted to sorption isotherm of the Freundlich, Langmuir, and Redlich–Peterson models. The regression coefficient for both Langmuir and Redlich–Peterson isotherms were higher than those secured for Freundlich isotherm implying that the biosorption system is possibly monolayer coverage of the L. variegata surface by the cadmium and lead ions. FT-IR studies revealed that Cd(II) and Pb(II) binding to L. variegata occurred primarily through biomass carboxyl groups accompanied by momentous interactions of the biomass amino and amide groups. In this study, we have observed that L. variegata had maximum biosorption capacity for Cd(II) and Pb(II) reported so far for any marine algae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of plastoquinone and β-carotene in the primary reaction of plant Photosystem II

Extraction of Triton Photosystem II chloroplast fragments with 0.2% methanol in hexane for 3 h results in the removal of 90 to 95% of the plastoquinone in the original preparation. The extracted fragments (chlorophyll : plastoquinone ratio, 900 : 1) showed no P-680 photooxidation at 15 K after a single laser flash. The extracted fragments also showed no light-induced C-550 absorbance change at 77 K. Reconstitution of the primary reaction of Photosystem II, as evidenced by restoration of low-temperature photooxidation of P-680, could be obtained by the addition of plastoquinone A but not by the addition of β-carotene. The addition of β-carotene plus plastoquinone A restored the C-550 absorbance change. These results indicate that plastoquinone functions as the primary electron acceptor of Photosystem II and that β-carotene does not play a direct role in the primary photochemistry but is required for the C-550 absorbance change.     

Crassulacean Acid Metabolism and Photochemical Efficiency of Photosystem II in the Adaxial and Abaxial Parts of the Succulent Leaves of Kalanchoë daigremontiana Grown at Four Photon Flux Densities

Kalanchoë daigremontiana, a species possessing crassulacean acid metabolism, was grown at four photon flux densities (1300, 400, 60, and 25 micromole photons per square meter per second). In leaves which had developed at 1300 and 400 micromole photons per square meter per second, CO₂ was mainly incorporated through the lower, shaded leaf surfaces, and the chlorenchyma adjacent to the lower surfaces showed a higher degree of nocturnal acid synthesis than the chlorenchyma adjacent to the upper surfaces. In leaves acclimated to 60 and 25 micromole photons per square meter per second, the gradient in CAM activity was reversed, i.e. more CO₂ was taken up through the upper than through the lower surfaces and nocturnal acidification was higher in the tissue next to the upper surfaces. Total net carbon gain and total nocturnal acid synthesis were highest in leaves which had developed at 400 micromole photons per square meter per second. Chlorophyll content was markedly reduced in leaves which had developed at 1300 micromole photons per square meter per second, especially in the exposed adaxial parts. There was also a sustained reduction in photosystem II photochemical efficiency as indicated by measurements of the ratio of variable over maximum chlorophyll a fluorescence. These findings suggest that, at high growth photon flux densities, the reduced activity of the exposed portions of these succulent leaves is caused by (a) the adverse effects of excess light, (b) together with a genotypic component which favors CO₂ uptake and acid synthesis in the abaxial (lower) leaf parts even when light is not or only marginally excessive. This latter component is predominant at medium photon flux densities, e.g. at 400 micromole photons per square meter per second. It becomes overridden, however, under conditions of deep shade when strongly reduced light levels in the abaxial parts of the leaf chlorenchyma severely limit photosynthesis.     

Synthesis, structure and magnetic properties of two μ-oxo and thiocyanato-bridged manganese(II)-mercury(II) coordination polymers

Mixed-metal thiocyanate complexes [MnHg(SCN)₄(NOP)] (1) and [MnHg(SCN)₄(DMSO)] (2) (NOP = 3-methyl-4-nitropyridine-N-oxide, DMSO = dimethylsulfoxide) have been synthesized and structurally characterized by single crystal X-ray analysis. Complex 1 and 2 both contain a [Mn₂(μ₂-O)₂] lozenge, which is bridged to Hg(II) ions by end-to-end thiocyanate groups to form a 2-D and 3-D polymeric network, respectively. Magnetic studies indicate that both complexes are anti-ferromagnets, with 1 showing anti-ferrimagnetic ordering below 8.0 K.     

Ligand substitution reactions of bis(μ-acetato)dichlorodicarbonyldiiridium(II)

Seven diiridium(II) complexes were synthesized by ligand substitution reactions of [Ir₂(μ-O₂CMe)₂Cl₂(CO)₂] (1) and [Ir₂(μ-O₂CMe)₂Cl₂(CO)₂(py)₂] (2).The reaction of 2 with the silver salt of a less coordinating anion, AgSbF₆, gave a cationic complex [Ir₂(μ-O₂CMe)₂Cl(CO)₂(py)₃]SbF₆ (3).A tricarbonyl cationic complex [Ir₂(μ-O₂CMe)₂(CO)₃Cl(py)₂]SbF₆ (4) was obtained under a CO atmosphere.Complex 2 reacted with AgO₂CCF₃ to give [Ir₂(μ-O₂CMe)₂Cl(O₂CCF₃)(CO)₂(py)₂] (5) in toluene.[Ir₂(μ-hiq)₂(CO)₂Cl₂] (Hhiq = 1-hydroxyisoquinoline, 6) was synthesized by the bridging-ligand substitution of 1 with Hhiq.Its axial adducts [Ir₂(μ-hiq)₂Cl₂(CO)₂L₂] (L = Mepy (4-methylpyridine), 7 or PPh₃, 8) were synthesized by addition of the ligands to a suspension of 6.In the structures of 7 and 8, two iridium atoms are bridged by two hiq ligands in a head-to-tail arrangement.The reaction of 1 with Hmhp (2-hydroxy-4-methylpyridine) led to triply bridged [Ir₂(μ-mhp)₃(CO)₂Cl(Hmhp)] (9).In complex 9, all the mhp ligands bridge between the Ir atoms in a head-to-head manner.The Ir-Ir distances of 3, 4, 5, 7 and 8 are 2.6047(7), 2.6216(9), 2.5899(9), 2.5933(5) and 2.634(2) Å, respectively, which are similar to those observed in[Ir₂(μ-O₂CMe)₂Cl₂(CO)₂L₂]. The Ir-Ir distance of 2.5512(4) Å in 9 is shorter than in the other complexes.     

DFT study of (17)O, (1)H and (13)C NMR chemical shifts in two forms of native cellulose, I(α) and I(β)

This computational study is intended to shed light on the crystalline and molecular structure, together with the hydrogen bonding (H-bonding) differences between two forms of native cellulose. DFT calculations were carried out to characterize the ¹⁷O, ¹H and ¹³C nuclear magnetic resonance (NMR) parameters in cellulose I_α and I_β with the B3LYP functional employing the 6–311++G7 and 6–31+G1 basis sets. Geometry optimization revealed that the average HB length is shortened by 0.01–0.08 Å when the chains are aligned, whereas the average bond angle increases by about 4–8° exhibiting the enhancement of HB strength. For the isolated cellotetramer chains, the isotropic ¹⁷O–H chemical shifts were plotted as a function of HB length. Our results indicated that as the HB length in cellotetramer I_α increases, the ¹⁷O–H chemical shift isotropy increases, but this parameter changes in the opposite direction for the other structure. Moreover, B3LYP/6–311++G7 calculations reveal that there is an acceptable correlation between the calculated ¹³C chemical shifts of the two structures and their experimental values.     

The efficiency of electron transfer from QA − to the donor side of Photosystem II decreases during induction of photosynthesis: Evidences from chlorophyll fluorescence and photoacoustic techniques

The amplitudes ratio of the fast and slow phases (A_fast/A_slow) in the kinetics of the dark relaxation of variable chlorophyll fluorescence (F_V) was studied after various periods of illumination of dark-adapted primary barley leaves. Simultaneously, photosynthetic activity was monitored using the photoacoustic technique and the photochemical and non-photochemical fluorescence quenching parameters. The ratio A_fast/A_slow changed with the preceding illumination time in a two-step manner. During the first stage of photosynthetic induction (0–20 s of illumination), characterized by a drop in O₂-dependent photoacoustic signal following an initial spike and by a relatively stable small value of photochemical F_V quenching, the ratio A_fast/A_slow remained practically unaltered. During the second stage (20–60 s of illumination), when both the rate of O₂ evolution and the photochemical F_V quenching were found to be sharply developed, a marked increase in the above ratio was also observed. A linear correlation was found between the value of the photochemical quenching and the ratio A_fast/A_slow during the second phase of photosynthetic induction. It is concluded that the slow phase appearing in the kinetics of F_V dark relaxation is not due to the existence of Photosystem II reaction centres lacking the ability to reduce P700⁺ with high rates, but is instead related to the limitation of electron release from Photosystem I during the initial stage of the induction period of photosynthesis. This limitation keeps the intersystem electron carriers in the reduced state and thus increases the probability of back electron transfer from Q_A ^– to the donor side of Photosystem II.Abbreviations A_fast/A_slow the ratio of magnitudes between the fast and slow phases of dark relaxation of variable fluorescence - F_O initial level of chlorophyll fluorescence - F_V variable chlorophyll fluorescence (F-F_O) - (F_V)_S the yield of variable chlorophyll fluorescence under saturating pulse in illuminated leaves - (F_V)_M the yield of variable chlorophyll fluorescence under saturating pulse in dark-adapted leaves - PA photoacoustic - PSI Photosystem I - PS II Photosystem II - qN non-photochemical quenching - qQ photochemical quenching