The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II

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

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa II. Reduction kinetics and electric field increase in the 10 ms range

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC⁺ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern.The reduction of PC⁺ and cytochrome f⁺ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267–284). Both need the presence of a compound R′ in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R′ as terminal reducant, PC⁺ and cytochrome f⁺ as terminal oxidants, would account for this field generation.Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.     

The function of diphenylamines as modifiers of Photosystem II electron transport in isolated spinach chloroplasts

Diphenylamines with highly electronegative substituents are effective inhibitors of photosynthetic electron transport and photophosphorylation. They inhibit only Photosystem II- and not Photosystem I-dependent photoreductions. As judged from the missing tetramethylphenylenediamine-bypass, displacement experiments with [¹⁴C]metribuzin, and measurements of oxygen evolution in trypsinated chloroplasts, diphenylamines act neither as dibromomethylisopropylbenzoquinone- nor as dichlorophenyldimethylurea-type inhibitors. All of the diphenylamines tested were found to function as ADRY-type reagents, (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439) which modify the stability of redox equivalents stored within the water-splitting enzyme system Y.The site of inhibition of diphenylamines is assumed to be located at the reducing side of Photosystem II or the reaction center itself. The inhibitory effect could involve a modification of cytochrome b-559 or its surrounding. In an assay for herbicidal activity, diphenylamines showed more pronouncing effect on mono- than on dicotyledonous plants.     

Protective effect of bicarbonate against extraction of the extrinsic proteins of the water-oxidizing complex from Photosystem II membrane fragments

A protective effect of bicarbonate (BC) against extraction of the extrinsic proteins, predominantly the Mn-stabilizing protein (PsbO protein), during treatment of Photosystem II (PS II) membrane fragment from pea with 2 M urea, and at low pH (using incubation in 0.2 M glycine-HCl buffer, pH 3.5 or 0.5 M citrate buffer, pH 4.0-4.5) was detected. It was shown that the extraction of the proteins with Mw 24 kDa (PsbP protein) and 18 kDa (PsbQ protein) by the use of highly concentrated solutions of NaCl does not depend on the presence of BC in the medium. An optimal concentration of BC at which it produces the maximum protecting effect was shown to be between 1 mM and 10 mM. The addition of formate did not influence the protein extraction but it reduced the stabilizing effect of BC. Independence of the stabilizing effect on the presence of the functionally active Mn within the water-oxidizing complex indicates that the protecting effect of BC is not related to its interaction with Mn ions. The fact that there is a preferable sensitivity of the PsbO protein to the absence of BC in the medium during all the treatments makes it possible to suggest that either BC interacts directly with the PsbO protein or it binds to some other sites within PS II and this binding facilitates the preservation of the native structure of this protein.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q_z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Effects of DDT on photosynthetic electron flow in Secale species

Following a survey of a range of varieties of rye, mainly Secale cereale, for reaction to DDT, the mode of action of the pesticide in a susceptible variety was studied. Two sites of interaction of DDT with the photosynthetic electron transport chain were demonstrated. The first site of inhibition was on the oxidizing side of photosystem 2, between the sites of electron donation from diphenylcarbazide at pH 6.0 and pH 8.0 in Tris-washed chloroplasts. The second site of DDT inhibition was in the intermediate electron transport chain, and was demonstrated by using dichlorophenol-indophenol and phenyldiamines as electron donors in chloroplasts where electron flow from photosystem 2 was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The sites are distinct from those characteristic of herbicides which affect photosynthetic electron flow.     

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Optimization of ferredoxin-mediated cyclic photophosphorylation and electric potential generation

Optimal conditions for cyclic photophosphorylation and electric potential generation have been established in well coupled Photosystem (PS)I-enriched subchloroplast vesicles supplemented with ferredoxin. Using NADPH and oxygen as redox-poising agents, it is shown that accurate redox poising of the cyclic system is required for optimal electron transfer. The molar ratio of NADPH to oxygen, rather than their concentrations, regulates the rate of cyclic photophosphorylation. In the present experimental system, the actual redox potential of ferredoxin is of crucial importance for optimal cyclic electron transfer and energy transduction. Under conditions for optimal redox poising of the cyclic system, a relatively strong expression of the flash-induced slow electric potential component was found, as monitored by the absorption changes of carotenoids and of oxonol VI. The function and regulation of cyclic electron transfer in stroma lamellae membranes in vivo are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.     

Redox titration of electron acceptor Q and the plastoquinone pool in Photosystem II

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve.The titration of Q, as indicated by the dark level of F_i, occurs in two discrete steps. The high-potential component (Q_h) has a midpoint potential of +68 mV (pH 7.2) and accounts for ～67% of Q. The pH sensitivity of the midpoint potential is ?60 mV, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The low-potential component (Q₁) accounts for the remaining 33% of Q and shows a midpoint potential near?300 mV (pH 7.2).The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30–40 mV more oxidizing than that of Q_h, i.e., at 106 mV (pH 7.2). The E_m shows a pH sensitivity of ?60 mV/pH unit, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The observation that all 12–14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property.Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.     

Effects of alcohol/water mixtures on the structure and reactivity of cytochrome c

The oxidation reaction of ferrocytochrome c (produced in situ by pulse radiolysis) by Fe(CN)^3?₆, was used to probe the effect of alcohol/water mixtures on the reactivity of the protein. Reduced cytochrome c is oxidized in a biphasic process. The relative contribution of each phase depended on: pH, alcohol concentration and temperature. pK_a values were derived from the kinetic data. These pK_a values were identical with the spectroscopic pK_a values determined under similar conditions by monitoring the 695 nm absorption band of the oxidized protein. The two phases of oxidation were therefore related to the oxidation of a relaxed and a nonrelaxed conformer of reduced cytochrome c produced in situ. A shift in the pK_a of ferricytochrome c and a retardation of the redox reactions of both the reduced and the oxidized protein were observed at low alcohol concentrations (up to 5 mol %). These low alcohol concentrations are known to affect the structure of water (Yaacobi, M. and Ben-Naim, A. (1973) J. Sol. Chem. 5, 425?443; Ben-Naim, A. (1967) J. Phys. Chem. 71, 4002?4007 and Ben-Naim, A. and Baer, S. (1964) Trans. Faraday Soc. 60, 1736?1741) but have only minor effects on the protein. Accordingly, the kinetic results are interpreted on the basis of involvement of water molecules in the reaction complex of cytochrome c with its redox substrates.     

Isolation and Structural Determination of the Antifouling Diketopiperazines from Marine-Derived Streptomyces praecox 291-11

Marine derived actinomycetes constituting 185 strains were screened for their antifouling activity against the marine seaweed, Ulva pertusa, and fouling diatom, Navicula annexa. Strain 291-11 isolated from the seaweed, Undaria pinnatifida, rhizosphere showed the highest antifouling activity and was identified as Streptomyces praecox based on a 16S rDNA sequence analysis. Strain 291-11 was therefore named S. praecox 291-11. The antifouling compounds from S. praecox 291-11 were isolated, and their structures were analyzed. The chemical constituents representing the antifouling activity were identified as (6S,3S)-6-benzyl-3-methyl-2,5-diketopiperazine (bmDKP) and (6S,3S)-6-isobutyl-3-methyl-2,5-diketopiperazine (imDKP) by interpreting the nuclear magnetic resonance and high-resolution mass spectroscopy data. Approximately 4.8 mg of bmDKP and 3.1 mg of imDKP were isolated from 1.2 g of the S. praecox 291-11 crude extract. Eight different compositions of culture media were investigated for culture, the TBFeC medium being best for bmDKP and TCGC being the optimum for imDKP production. Two compounds respectively showed a 17.7 and 21 therapeutic ratio (LC₅₀/EC₅₀) to inhibit zoospores, and two compounds respectively showed a 263 and 120.2 therapeutic ratio to inhibit diatoms.     

On the interaction of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with bound electron carriers in spinach chloroplasts

2,5-Dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), when added to chloroplasts as the sole electron donor, is an effective reducing agent. Low concentrations of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone reduce cytochrome f, plastocyanin, and P700 in the dark but do not reduce the high-potential form of cytochrome b₅₅₉. 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone appears to interact at or near the site of function of the “Rieske” iron-sulfur center, as evidenced by a shift in the g value of the electron paramagnetic resonance signal of the reduced center.     

Characterization of wave phenomena in the relaxation of flash-induced chlorophyll fluorescence yield in cyanobacteria

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

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Absence of the psbH gene product destabilizes the Photosystem II complex and prevents association of the Photosystem II-X protein in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

PS II-H is a small hydrophobic protein that is universally present in the PS II core complex of cyanobacteria and plants. The role of PS II-H was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The psbH disruptant could grow photoautotrophically; however, its growth was much slower than that of the wild type cell. Chromatography enabled the isolation of active oxygen-evolving PS II complexes from both the mutant and the wild type. The mutant yielded a relatively large amount of inactive PS II complex that lacked the following extrinsic proteins: the 33-kDa protein, the 12-kDa protein, and cytochrome c ₅₅₀ . There were differences between the psbH disruptant and the wild type in terms of the oxygen evolution activities of the cells, thylakoids, and PS II complexes. At high concentrations of 2,6-DCBQ, the activity was much lower in the mutant than in the wild type. Gel filtration chromatography of the PS II complexes showed that both active and inactive PS II complexes isolated from the mutant were mostly in the monomeric form, while the active PS II complex from the wild type was in the dimeric form. The polypeptide composition of both active and inactive PS II complexes from the mutant showed the absence of another small polypeptide, PS II-X. These results suggest that the PS II-H protein is essential for stable assembly of native dimeric PS II complex containing PS II-X.