Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans. The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C.The Arrhenius plot of the phosphorylation mediated by N-methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C.The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.     

PHOTOSYNTHETIC ELECTRON TRANSPORT IN CELL-FREE EXTRACTS OF DIVERSE PHYTOPLANKTON1,2

A simple and rapid procedure for preparing thylakoid membranes that are active in photosynthetic electron transport from diverse phytoplankton species is described. The method requires disruption of algal cells with glass beads, exposure to mild hypotonic stress, and subsequent enrichment of the thylakoid membranes by differential centrifugation. Isolated thylakoid membranes were assayed for photosynthetic electron transport activity by measuring rates of oxygen consumption and oxygen production, using a variety of electron donors and acceptors. In the dinoflagellate Gonyaulax polyedra Stein, a relatively broad pH optimum between 7.0 and 8.0 was determined for the whole chain electron transport from water to methyl viologen. The preparation maintained maximum activity for 45 min following the preparation. The assay for photosystem I activity in G. polyedra, determined as electron flow from ascorbate/2,6-dichlorophenolindophenol to methyl viologen, had a somewhat narrower pH optimum around 8.0. Rates of whole chain photosynthetic electron transport on a per cell and on a per chlorophyll a basis were shown to decrease dramatically with cell age in batch cultures of G. polyedra. Using the procedures optimized for G. polyedra, reproducible rates of electron transport on a per cell chlorophyll a basis were also measured in cultures of the dinoflagellate Glenodinium sp., the diatom Nitzschia closterium (Ehrenberg 1839) Wm. Smith 1853 and the chrysophyte Monochrysis lutheri Droop {= Pavlova lutheri (Droop) Green}. Other electron transport assays applied to G. polyedra, and that resulted in comparable rates to those found in other algal groups, include the photosystem II assay from water to diaminodurene/ferricyanide and the photosystem I assay from durohydroquinone to methyl viologen.     

The effect on photosynthetic electron transport of temperature-dependent changes in the fluidity of the thylakoid membrane in a thermophilic blue-green alga

Various electron transport reactions in cell or isolated thylakoid membranes of the thermophilic blue-green alga, Synechococcus sp. were measured at different temperatures between 72 and 3 degrees C. They are classified into two groups with respect to their temperature dependency. The first group involves cytochrome 553 photooxidation, methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol as electron donor and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea-resistant ferricyanide photoreduction determined in the presence or absence of silicomolybdate. The Arrhenius plot of these reactions showed a single straight line with the activation energy of about 10 kcal/mol throughout wide temperature ranges studied. Methyl viologen photoreduction with water as electron donor, reduction of flash-oxidized cytochrome 553, ferricyanide photoreduction and photosynthetic O2 evolution form the second group. Their arrhenius plots are characterized by discontinuities or breaks at about 30 and 10 degrees C, which respectively correspond to the upper and lower boundaries of the lateral phase separation of the membrane lipids. The first group reactions represent short spans of electron transport which are mediated either by Photosystem I or Photosystem II alone and not related to plastoquinone, whereas all the reactions of the second group involve plastoquinone. It is concluded therefore that the membrane fluidity affect electron transport specifically at the region of plastoquinone. It is proposed that the reaction center chlorophyll-protein complexes of both Photosystems I and II are closely associated with related electron carrier proteins to form functional supramolecular assemblies so that electron transfer within such a cluster of proteins proceeds independently of the phase changes in the membrane lipids. On the other hand, the role of plastoquinone as a mobile electron carrier mediating electron transfer from the protein assembly of Photosystem II to that of Photosystem I through the fluid hydrophobic matrix of the membranes is highly sensitive to the physical state of the membrane lipids.     

Adenylate regulation of photosynthetic electron transport and the coupling sites of phosphorylation in spinach chloroplasts

The regulation by adenylates of activities of various partial electron transport systems in spinach chloroplasts was studied using systems from H₂O to 2,5-dimethyl-p-benzoquinone, H₂O to 2,6-dichlorophenolindophenol, reduced 2,6-dichlorophenolindophenol to methyl viologen, and H₂O to methyl viologen or ferricyanide. Adenylates regulated all of them. The ratio of the amount of esterified Pi (P) to that of electrons transported (e) in coupling with phosphorylation manifested that there are two phosphorylation sites: one between H₂O and 2,5-dimethyl-p-benzoquinone or 2,6-dichlorophenolindophenol and another between reduced 2,6-dichlorophenolindophenol and methyl viologen, under the proposed stoichiometries,i.e., P/H⁺=0.5 and H⁺/e=1, where H⁺ is the amount of protons pumped by electron transport (= those translocated during phosphorylation), when the basal electron transport (the part not regulated by adenylates) was excluded. The effects of pH, phlorizin, and methylamine on the adenylate regulation of electron transport, and the stimulation profile of electron transport coupled with quasiarsenylation suggested no distinction between the two phosphorylation sites.     

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl₂ at a molar ratio of HgCl₂ to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ? was similarly sensitive towards HgCl₂, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ? and light-induced increase in fluorescence yield were enhanced by the HgCl₂ treatment of chloroplasts.     

Light Activation of Purified Aconitase by Washed Thylakoid Membranes of Pea (Pisum sativum L.)

Purified aconitase, an iron-sulfur protein, from either beef heart mitochondria or pig heart can be activated fully by light when combined with washed thylakoid membranes from pea (Pisum sativum L.) chloroplasts. The light activation of the enzyme does not require any other additive or cofactor and is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,6-dichlorophenol-indophenol, ferricyanide, and methyl viologen, indicating that the photoelectron transport system of the thylakoid membranes, and in particular, photosystem I, is involved in the process of activation. Light activation of the enzyme is also markedly inhibited when the thylakoid membranes are treated with sulfite or arsenite, and abolished totally when the membranes are treated with Zwittergent, suggesting that the light effect mediator involved in the light modulation of chloroplastic enzymes mediates the activation of purified aconitase also.     

Light-dependent inhibition of photosynthetic electron transport by zinc

The effects of zinc concentrations up to 400 μ M were examined on three photosynthetic electron transport reactions of thylakoids isolated from Pisum sativum L. cv. Meteor. Zinc (400 μ M ) had no effect on photosystem I mediated electron transport from reduced N,N,N',N'-tetramethyl- p -phenylenediamine to methyl viologen, but inhibited uncoupled electron flow from water to methyl viologen by ca 50% and to 2,6-dichlorophenol-indophenol (DCPIP) by ca 30% at saturating light levels. Zinc inhibition of DCPIP photoreduction was independent of the light intensity to which thylakoids were exposed. Decreasing the photon flux density below 400 μmol m⁻² s⁻¹ produced a logarithmic reduction in the zinc-induced inhibition of methyl viologen photoceduction; a stimulation of this reaction was observed below 80 μmol photons m⁻² s⁻¹. Increasing light intensity decreased the amount of zinc tightly bound to the thylakoid membranes, but increased the weakly associated zinc which could be removed by washing the membranes with buffer containing Mg². The results suggest that zinc acts on the photosynthetic electron transport system at two sites. Site 1 is on the oxidizing side of photosystem 2 and the inhibition by zinc is independent of the light intensity. Site 2 is between photosystems 1 and 2 and the electron flow can be positively or negatively affected by zinc depending on the light intensity.     

Photosynthetic phosphorylation by a membrane preparation of the cyanobacterium Spirulina Maxima

A new method for the isolation of photosynthetic membranes from the cyanobacterium Spirulina maxima has been developed. When illuminated, these membranes evolve oxygen in the presence of ferricyanide (Hill reaction) and consume oxygen in the presence of methyl viologen (Mehler reaction). When the membranes are left to stand at 4°C for 30 min, they develop the ability to consume oxygen in the light without an added, artificial electron acceptor. The Hill and Mehler reactions are not affected by the presence of ADP or uncouplers, but are inhibited by triphenyltin chloride. We have detected a cryptic ATPase activity stimulated by trypsin in the 2000×g supernatant fraction of the membrane preparation. In addition, the membrane vesicles contain an ATPase activity which is enhanced by treatment with dithiothreitol in the presence of light. These observations of ATPase led us to try a careful titration of the membrane vesicles with both triphenyltin chloride and N,N′-dicyclohexylcarbodiimide. When the vesicles were sealed with these reagents, we could observe both cyclic and stoichiometric photosynthetic phosphorylation.     

Comparative studies on the properties of spinach chloroplasts prepared by media containing choline chloride or sucrose

Spinach chloroplasts were isolated and stored in a medium containing0.5 M choline chloride. The properties of these chloroplastswere compared with those of chloroplasts prepared in an ordinarysucrose medium. In marked contrast to sucrose-prepared chloroplasts, choline-preparedchloroplasts did not show "conventional uncoupling" (stimulationof ferricyanide reduction paralleling the inactivation of phosphorylation)after transient warming in the temperature range between 20?and 55?C. The thermal stability of the oxygen evolving system, the vulnerabilityof the lipophilic structure in the thylakoids to alcohol duringthe warming treatment, and the effects of amine on photophosphorylationwere similar in both chloroplast preparations. After the warming treatment, the degree of swelling in chloroplastswas larger and the intermediate electron transport systme (fromreduced 2,6-dichlorophenolindophenol to methyl viologen) wasmore stable against uncoupling, in choline-prepared chloroplaststhan in sucrose-prepared ones. The presence and absence of "conventional uncoupling" in twochloroplast preparations were ascribed to differences in thermalstability. In choline-prepared chloroplasts the uncoupling temperatureof the electron transport system was higher than the inactivationtemperature of the oxygen evolving system, but it was lowerin the sucrose-prepared ones. (Received February 13, 1974; )     

N,N-diethylhydroxylamine: a new electron donor to photosystem II

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. Diethylhydroxylamine supports dichlorophenolindophenol photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II before the herbicide binding site. The stimulation of the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed that the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamine. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations with diethylhydroxylamine either in light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that diethylhydroxylamine is a useful electron donor to the photosystem II.     

Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase

The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO₂ (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously.     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O₂ evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O₂ uptake with methyl viologen as acceptor.

Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H₂O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.

The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O₂ evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O₂ evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

     

Time-dependent changes in the in vitro effects of sodium chloride on photosynthetic electron transport of isolated chloroplasts

The effects of a wide concentration range of NaCl and sorbitol on three photosynthetic electron transport reactions of Pisum sativum L. cv. Feltham First chloroplasts were examined as a function of time from thylakoid membrane isolation. Rates of electron flow from water to diaminodurene (DAD) and ferricyanide were determined polarographically, whilst photoreduction of 2,6-dichlorophenolindophenol (DCPIP) was monitored spectrophotometrically. Assay of thylakoids immediately after isolation showed that the rate of photoreduction of all three electron acceptors decreased with increasing salt concentration. However, 100 min after leaf homogenisation the response pattern of ferricyanide and DCPIP photoreduction to increasing NaCl, but not increasing sorbitol concentration, became significantly modified. This was not the case for DAD photoreduction. The results are discussed in relation to the assessment of the possible effect of salinity on photosynthetic electron transport in vivo.     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

Photosynthetic properties of permaplasts of anacystis

A treatment procedure using lysozyme and ethylenediaminetetracetic acid gave intact but permeable cells (permeaplasts) of Anacystis nidulans. Rates of electron transport from water to carbon dioxide, ferricyanide, 2,6-dichlorophenol indophenol, benzoquinone, and methyl viologen, and from reduced indophenol to methyl viologen were measured as a function of treatment time. Rates of oxygen evolution in complete photosynthesis and electron flow from water to methyl viologen showed rapid and parallel decline with treatment time. Electron flow from water to ferricyanide and from reduced indophenol to methyl viologen increased during the first half hour of treatment (phase 1) to 60 to 80% of the original photosynthetic rate. Longer treatment (phase 2) resulted in decreased rate of ferricyanide reduction but not in rate of methyl viologen reduction from indophenol. Electron flow from water to quinone was two to three times higher than for complete photosynthesis in intact cells. It remained high during phase 1 and declined during phase 2. Phase 1 permeaplasts apparently retain high activity for photosystems 1 and 2 photoreactions.     

Oxygenic photoreduction of methyl viologen and nicotinamide adenine dinucleotide phosphate without the involvement of photosystem I during plastid development

Studies on the appearance of various electron transport functions were followed during greening of etiolated cucumber cotyledons. Appearance of dichlorodimethoxy-p-benzoquinone, dimethyl quinone, tetramethyl-p-phenylenediamine, dichlorophenol indophenol and ferricyanide Hill reactions were observed after 8h of greening. However, photoreduction of methyl viologen (MV) and nicotinamide adenine dinucleotide phosphate (NADP) was observed from 2h of greening. Variable fluorescence, which is a direct indication of water-splitting function, was observed from 2h of greening in cotyledons, thylakoid membranes and photosystem II (PSII) particles. The decrease in variable fluorescence in the presence of MV (due to rapid reoxidation of Q-) observed from early stages of greening confirmed the photoreduction of MV by PSII. The early development of water-splitting function was further confirmed by the abolition of variable fluorescence in thylakoid membranes and PSII particles by heat treatment and concomittant loss of light dependent oxygen uptake in the presence of MV in heat treated chloroplasts. However, the photoreduction of MV and NADP was insensitive to intersystem electron transport inhibitors, dichlorophenyl dimethylurea or dibromomethyl isopropyl-p-benzoquinone till 8h of greening. Though the oxidation of intersystem electron carrier cytochrome f was observed from early stages of greening, the reduction of cytochrome f was not observed till 8h of greening. All these observations confirm that during early stages of greening MV and NADP are photoreduced by PSII without the involvement of intersystem electron carriers or the collaboration of PSI. Since these observations are at variance with the currently prevalent concept (Z-Scheme) of the photosynthetic generation of reducing power, which requires definite collaboration of the two photosystems, an alternate electron flow pathway is proposed.     

Electron flow from hydrogen peroxide in photosystem II-catalyzed oxidation-reduction reactions of spinach chloroplast fragments

Oxidation-reduction reactions of photosystem II were investigatedin spinach chloroplast fragments. Chloroplast fragments treatedwith 8-hydroxyquinoline sulfate showed only a low activity forthe 2,6-dichlorophenolindophenol (DPIP) Hill reaction, as wasobserved in chloroplast fragments treated with a high-concentrationof Tris buffer. Hydrogen peroxide could donate electrons tophotoreaction center II in chloroplast fragments treated with8-hydroxyquinoline, high-concentration Tris, or ethylene glycol,but water could not serve as an electron donor in these preparations.Electrons from hydrogen peroxide were transferred to DPIP, ferricyanide,and p-benzoquinone viaphotosystem II. (Received May 12, 1971; )     

Reconstitution of energy transfer and electron transfer between solubilised pigment-protein complexes from thylakoid membranes. The role of acyl lipids

Solubilisation of thylakoid membranes from young leaves of Pisum sativum in the presence of Triton X-100 resulted in an almost complete loss of quenching of light-harvesting chlorophyll-protein (LHCP) fluorescence, as measured at 77°K. There were concomitant changes in the kinetics of light-saturation curves of electron transport from 2,6-dichlorophenolindophenol/ascorbate to methyl viologen. These effects were accompenied by a physical dissociation of LHCP polypeptides from photosystem I (PSI) and photosystem II (PSII) polypeptides, as determined by polyacrylamide gel-electrophoresis. Detergent-dialysis in the presence of exogenous purified galactolipids, about 80% of which were linoleoyl molecular species, only partially reversed these effects. However, detergent-dialysis using the phospholipids, phosphatidylglycerol and phosphatidylcholine, resulted in the substantial restoration of 77°K fluorescence quenching and the restoration of both emission spectra and electron transport kinetics of both Photosystems I and II that were typical of native membranes.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DGD digalactosyldiacylglycerol - LHCP light-harvesting chlorophyll-protein - MGD monogalactosyldiacylglycerol - PCi phosphatidylcholine — Sigma grade NS - PCii -oleoyl, -palmitoyl phosphalidylcholine - PG phosphatidylglycerol - PSI photosystem I - PSII photosystem II     

Reversibility of the cyanide inhibition of electron transport in spinach chloroplast thylakoids

The prior treatment of thylakoids with cyanide (30 mM) was shown to inhibit plastocyanin-dependent electron transport reactions. We find that cyanide inhibition of electron flow from either water or diaminodurene to methyl viologen, but not from water to ferricyanide, is partially reversed when the thylakoids are collected by centrifugation and resuspended in a cyanide-free medium. However, methyl viologen reduction in thylakoids pretreated with cyanide is sensitive to cyanide (～1 mM) added to the reaction mixtures, whereas that in control thylakoids is unaffected. The cyanide must be added in the dark. Electron transport to methyl viologen in chloroplasts pretreated with cyanide is also sensitive to inhibition by EDTA and bathocuproine sulfonate. Thus, KCN inhibition of electron transport in thylakoids is partially reversible. Moreover, the accessibility of plastocyanin to various reagents is probably altered by the KCN treatment.