Effect of phloridzin on photosynthetic electron transport

Phloridzin (2',4',6',4-tetraoxyhydrochalcon-2'-glucoside) was used to study the localization of synthesis of ATP in the electrontransporting chain of photosynthesis. It was shown that phloridzin inhibits the rate of photoreduction of NADP+ by isolated pea chloroplasts by 40%, electron transport via cytochrome f by 100% and via plastocyanin--by 50%. The "crossover" experiments demonstrated that phloridzin inhibits ADP-induced photoreduction of cytochrome f, having no effect on plastocyanin under identical conditions. It is assumed that the site of ATP synthesis is localized on the reduced site of cytochrome f, while the carrier itself is located in the electron transporting chain coupled to phosphorylation. It is possible that only part of the plastocyanin molecules are located in the phosphorylating pathway of electron transport.     

The site of inhibition of the chloroplast electron-transport system by 2,3-dithiopropan-1-ol (BAL)

BAL (2,3-dithiopropan-1-ol) treatment of chloroplasts has previously been reported to induce a block in electron transport from water to NADP+ at a site preceding plastocyanin [Belkin et al. (1980) Biochim. Biophys. Acta 766, 563-569]. In the present work the block was further characterized. The following properties of BAL treatment are described. Inhibition of electron transport from water to lipophilic acceptors but not to silicomolybdate. Inhibition of the slow, sigmoidal phase of chlorophyll a fluorescence induction. Inability of N,N,N',N',-tetramethyl-p-phenylenediamine to bypass the inhibition of NADP+ photoreduction with water as the electron donor. Inhibition of electron transport from externally added quinols to NADP+. Inhibition of cytochrome f reduction by photosystem II, but not its oxidation by photosystem I. Inhibition of cytochrome b6 turnover and cytochrome f rereduction after single-turnover flash illumination under cyclic electron-flow conditions. The BAL-induced block is therefore located between the secondary quinone acceptor (QB) and the cytochrome b6f complex. It was further found that (a) the isolated cytochrome complex is not inhibited after BAL treatment; (b) BAL-reacted plastoquinone-1 inhibits electron transport in chloroplasts; (c) BAL does not inhibit electron transport in chromatophores of Rhodospirilum rubrum or Rhodopseudomonas capsulata. It is suggested that the inhibition of electron transport in chloroplasts results from specific reaction of BAL with the endogenous plastoquinone.     

Participation of ferredoxin in oxygen reduction in a photosynthetic electron-transport chain

Oxygen reduction in a photosynthetic electron-transport chain (PETC) was studied in isolated pea thylakoids in the presence of either ferredoxin, or ferredoxin + NADP+, or cytochrome c. The contribution of the electron flow through ferredoxin to the total oxygen reduction was evaluated by comparing the rate of oxygen reduction and the rate of oxidation of reduced ferredoxin in the light. It was found that at ferredoxin concentrations optimal for NADP+ reduction, 30-50% of electrons transferred to oxygen went through ferredoxin both in the absence and presence of NADP+. However, the absolute rate of oxygen reduction by membrane components of PETC in the presence of NADP+ was 3-4 times less than that in the presence of ferredoxin alone and close to the rate of oxygen reduction in the presence of cytochrome c. It was assumed that a Photosystem I component, whose role in this process depends on the rate of electron outflow from terminal acceptors of this photosystem, participates in oxygen reduction, and this component is phylloquinone.     

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.     

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(+).     

Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium.

A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit. Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 microM plastocyanin included), using 100 nM ferredoxin:NADP(+) reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP(+) photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type. This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase. In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant. A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP(+), and test for possible causes for the observed change in affinity for FNR. The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant [Barth, P., et al., (1998) Biochemistry 37, 16233-16241], do not significantly influence the rate of linear electron transport to NADP(+). Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR. We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport. Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins.     

Mechanism of photoinactivation in photosynthetic systems II. The occurrence and properties of two different types of photoinactivation

Photoinactivation of the activity of NADP photoreduction withreduced DPIP or with reduced TMPD as the electron donor wasinhibited by the absence of oxygen in the atmosphere or by thepresence of photosynthetic inhibitors (CMU, DCMU, o-phenanthroline)in the preillumination mixture. Photoinactivation of the photoreductionof NADP or DPIP with water as the electron donor was not affected,or even accelerated, by these conditions of preillumination.The concentrations of inhibitors required for maximum inhibitionin the former case corresponded to those required for inhibitionof photosynthetic electron transport. The results indicatedthe occurrence of 2 different types of photoinactivation, eachspecifically affecting photosystems I and II, and differingin behaviours; including their requirement for oxygen in theatmosphere and their responses toward the presence of photosyntheticinhibitors during the preillumination period. (Received July 30, 1969; )     

Nano-Anatase Relieves the Inhibition of Electron Transport Caused by Linolenic Acid in Chloroplasts of Spinach

Linolenic acid is an inhibitor of electron transport in chloroplasts of higher plants. It has obvious effects on the structure and function of chloroplasts. In the present paper, we investigated the nano-anatase relieving the inhibition of photoreduction activity and oxygen evolution caused by linolenic acid in spinach chloroplasts. The results showed that linolenic acid in various concentrations could obviously reduce the whole chain electron transport and the photoreduction activity of two photosystems, especially on the oxidative reside and reduce reside of photosystem II (PS II). After adding nano-anatase to chloroplasts treated by linolenic acid, the whole chain electron transport rate, the photoreduction activity of two photosystems, and the oxygen evolution rate were increased significantly, indicating that nano-anatase could obviously decrease the inhibition of linolenic acid on the electron transport, photoreduction activity, and oxygen evolution of spinach chloroplasts.     

Porton uptake by isolated chloroplasts during cyclic and non-cyclic electron transport catalyzed by photosystem I in the presence of ferredoxin]

Proton uptake by isolated chloroplasts during cyclic electron transport in the presence of ferredoxin and under NADP+ reduction from the ascorbate--TMPD donor pair under anaerobic conditions was studied. It was found that during cyclic transport the proton uptake is less intensive than under NADP+ reduction. In the presence of ATP the proton uptake is increased in the first case and is decreased in the second one. During cyclic transport in the presence of gramicidin D the proton uptake is completely suppressed and under NADP+ reduction is decreased down to 0,08--0,09 mk equiv H+ per mg of chlorophyll, irrespective of ferredoxin or NADP+ concentrations. The role of ferredoxin NADP+ reductase in a proton uptake by thylakoids is discussed.     

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.     

Effects of Optically Active 1-(α-Methylbenzyl)-3-(3,4-dichlorophenyl)urea on Reactions of Mitochondria and Chloroplasts

Effects of the R- and S-isomers and racemate of 1-(alpha-methylbenzyl)-3-(3,4-dichlorophenyl)urea (MBPU) were measured on phosphorylation and electron transport in mung bean (Phaseolus aureus L.) mitochondria and spinach (Spinacia oleracea L.) chloroplasts.In chloroplasts, S-MBPU inhibited basal and methylamine-uncoupled electron transport with ferricyanide as the oxidant, both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide and nicotinamide adenine dinucleotide phosphate (NADP) as oxidants, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. With ascorbate 2,6-dichloro-phenolindophenol as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but the reduction of NADP was not inhibited. The R-isomer of MBPU, like the S-isomer, inhibited all of the photophosphorylation reactions studied. However, unlike the S-isomer, the R-isomer either did not inhibit or was a very weak inhibitor of all photoreduction reactions. The effects of the MBPUs on the chloroplast reactions can be explained by action at two different sites: an optically specific site near photosystem II and the oxygen evolution pathway, and a second optically nonspecific site associated with the generation of ATP.In mitochondria, both the R- and S-isomers stimulated state 4 respiration, inhibited state 3 respiration, and released oligomycin-inhibited respiration with malate, succinate, and NADH as substrates. Both enantiomers were equally active in all studies with malate and succinate as substrates. However, with NADH as substrate, R-MBPU was a stronger inhibitor of state 3 respiration and a weaker stimulator of state 4 respiration than S-MBPU.     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO2-fixation was initiated by oxygen concentrations as low as about 10 microM. Once CO2 fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO2 reduction.     

Transgenic tobacco plants overexpressing chloroplastic ferredoxin-NADP(H) reductase display normal rates of photosynthesis and increased tolerance to oxidative stress

Ferredoxin-NADP(H) reductase (FNR) catalyzes the last step of photosynthetic electron transport in chloroplasts, driving electrons from reduced ferredoxin to NADP+. This reaction is rate limiting for photosynthesis under a wide range of illumination conditions, as revealed by analysis of plants transformed with an antisense version of the FNR gene. To investigate whether accumulation of this flavoprotein over wild-type levels could improve photosynthetic efficiency and growth, we generated transgenic tobacco (Nicotiana tabacum) plants expressing a pea (Pisum sativum) FNR targeted to chloroplasts. The alien product distributed between the thylakoid membranes and the chloroplast stroma. Transformants grown at 150 or 700 micromol quanta m(-2) s(-1) displayed wild-type phenotypes regardless of FNR content. Thylakoids isolated from plants with a 5-fold FNR increase over the wild type displayed only moderate stimulation (approximately 20%) in the rates of electron transport from water to NADP+. In contrast, when donors of photosystem I were used to drive NADP+ photoreduction, the activity was 3- to 4-fold higher than the wild-type controls. Plants expressing various levels of FNR (from 1- to 3.6-fold over the wild type) failed to show significant differences in CO2 assimilation rates when assayed over a range of light intensities and CO2 concentrations. Transgenic lines exhibited enhanced tolerance to photooxidative damage and redox-cycling herbicides that propagate reactive oxygen species. The results suggest that photosynthetic electron transport has several rate-limiting steps, with FNR catalyzing just one of them.     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO₂-fixation was initiated by oxygen concentrations as low as about 10 μM. Once CO₂ fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO₂ reduction.     

黄瓜叶片光合电子传递对水分胁迫的响应

黄瓜叶片在水分胁迫下叶片相对含水量减少,类囊体室温吸收光谱的吸收峰降低,同时其NADP光还原活性、Ca^2 -ATPase活性也相应降低,全链电子传递明显受阻。类囊体膜蛋白电泳分析结果显示：类囊体膜色素蛋白复合体含量有不同程度的降低,其中PSⅡ色素蛋白复合体含量下降较多,试验结果表明水分胁迫通过限制光能的吸收,传递双及转换效率,抑制了光合电子传递过程。     

Photophosphorylation capacity of stable spheroplast preparations of anabaena

Spheroplasts from Anabaena 7119 (formerly designated Nostoc muscorum) were prepared in the presence of serum albumin in 0.5 molar sucrose. Electron transport and photophosphorylation were preserved (> 70% of the maximum rate for 1 week). The pH profile of electron transport and photophosphorylation in the reactions H₂O → NADP, H₂O → methyl viologen, and H₂O → ferricyanide shows that uncoupling by ammonia is small throughout and increases slightly with higher pH. ADP + Pi increased NADP reduction from H₂O by 2.5-fold. The ratios of ATP formed per electron pair transported ranged from 0.9 to 1.5. Effects of catalase and superoxide dismutase on the overall O₂ balance implicate pseudocyclic electron transport and phosphorylation. The quenching of 9-aminoacridine fluorescence indicates the formation of a Δ pH from 2 to 2.6 during illumination. This pH gradient is abolished by uncouplers; however, complete uncoupling is achieved only by 3-chlorocarbonyl cyanide phenylhydrazone or valinomycin + NH₄⁺. In the presence of NH₄⁺ alone, the membrane potential may act as the driving force for photophosphorylation.     

EFFECT OF PLASTOCYANIN ON PHOTOPHOSPHORYLATION IN ISOLATED CHLOROPLASTS

The effect of addition of plastocyanin on photophosphorylationin isolated chloroplasts was studied in comparison with itseffect on photoreduction. Catalytic amounts of added plastocyaninstimulated the HILL reaction with NADP as oxidant, but the photophosphorylationcoupled to this reaction was not influenced by the additionof plastocyanin. The addition of NH₄+ or the complete phosphorylatingsystem did not affect the plastocyanin-induced increase in rateof the photoreduction. The rate of photoreduction of NADP, FMN,or indigo carmine in the presence of DPIP and ascorbate wasmuch accelerated by added plastocyanin, but these reactionswere not coupled to ATP formation. As reported earlier, isolated chloroplasts can utilize plastocyaninas oxidant in the HILL reaction. This reaction was found tobe accompanied by ATP formation, but the efficiency in thiscase was somewhat lower than in photophosphorylation coupledto the HILL reaction with the usual oxidants. The possible mechanism of these reactions is described brieflyand a scheme for reactions of plastocyanin in the electron transferin chloroplasts is presented. (Received July 20, 1965; )     

Overexpression of petH Gene of Cyanobacterium synechococcus sp. PCC 7002 in Escherichia coll and Purification of the Expressed Product

Fd:NADP+ oxidoreductase (FNR) is one of the key enzymes in photosynthetic electron transport. The gene petH encoding FNR of Synechococcus sp. PCC 7002 was cloned into the expressing vector pET-3 d' and overexpressed in E. coli. The amount of recombinant FNR (rFNR) was over 50% of the total cellular proteins. There were two forms of FNR activity, one is soluble and the other one was in the form of inclusion bodies. The soluble rFNR was purified through ion exchange chromatography and gel chromatography. The rFNR in the form of inclusion bodies was first solubilized with 6.7 mol/L urea, and then refolded into the active form in the presence of flavin adenine dinucleotide (FAD). Further purification was performed by ion exchange chromatography. The rFNR pmified from either form of the expressed product had the maximum absorption spectrum as that of the natural FNR from cyanobacteria, whose maximum absorption was at 273, 385 and 456 ran respectively. N-tenninal sequencing showed that rFNR was indeed a product of petH gene expression, rFNR could catalyze the electron transport from P700 to NADP+ in the presence of ferredoxin. The optimal pH for diaphorase activity of rFNR was 8.0 and the optimal temperature was 30 ℃.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.