Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

The effects of copper on photosynthetic electron transfer systemsin isolated spinach chloroplasts were studied. Two differentinhibitions were observed. First, copper markedly inhibitedferredoxin-catalyzed reactions such as NADP⁺ photoreduction.The concentration required for 50% inhibition was about 2 µMof cupric sulfate. However, electron flow from reduced 2,6-dichloroindophenol(DCIP) to methyl viologen was not affected. The dissociationconstant between ferredoxin and ferredoxin-NADP⁺ reductase wasunchanged in the presence of 2.5 µM of cupric sulfate.In enzymic reaction systems, the ferredoxin-dependent electronflow from NADPH to cytochrome c was also strongly inhibitedin the presence of cupric sulfate, while DCIP reduction withNADPH as the electron donor was not affected. Second, DCIP photoreductionwas weakly blocked by copper and the lost activity could notbe recovered by adding 1,5-diphenylcarbazide (DPC). It can be concluded that copper directly interacted with ferredoxincausing inhibition of ferredoxin-dependent reactions. Further,copper caused weak inactivation between the oxidizing side ofthe reaction center of photosystem II and the electron donatingsite of DPC. (Received August 8, 1977; )     

Sulfide-induced oxygen uptake by isolated spinach chloroplasts catalyzed by photosynthetic electron transport

In addition to an inhibitory effect on the photoreduction of NADP⁺ by isolated spinach chloroplasts ( Spinacea oleracea L. cv. Melody Hybrid), sulfide initiated oxygen uptake by chloroplasts upon illumination, both in presence and absence of an electron acceptor. Sulfide-induced oxygen uptake was sensitive to DCMU demonstrating the involvement of photosynthetic electron transport. Addition of superoxide dismutase to the chloroplast suspension prevented the sulfide-induced oxygen uptake, which indicated that sulfide may be oxidized by the chloroplast, its oxidation being initiated by superoxide formed upon illumination (at the reducing side of PSI). Tris-induced inhibition of NADP⁺ photo-reduction could not be abolished by sulfide, which indicated that sulfide could not act as an electron donor for PSI.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine-washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a₆₈₀, and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides.     

Characteristics of Cu deficiency-induced inhibition of photosynthetic electron transport in spinach chloroplasts

In the present work we studied the effect of Cu deficiency on spinach chloroplasts. We found that in spinach the electron transport was inhibited as reported previously for sugar beet (Droppa, M., Terry, N. and Horváth, G. (1984) Proc. Natl. Acad. Sci. USA (1984) 81, 2369–2373). The breakpoint of the Arrhenius plot of the whole electron-transport activity was shifted from +6°C to +12°C in Cu-deficient chloroplasts. A similar effect could be observed with a spin-labelled probe, when the rotational correlation time was plotted vs. the reciprocal temperatures. This indicates that the membrane fluidity might be changed by Cu deficiency. The lipid/protein ratios were similar in both control and deficient chloroplasts. On the other hand, the saturated/unsaturated ratio of phosphatidylcholine (PC), phosphatidylglycerol (PG) and sulpholipids (SL) was increased but that of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) decreased. We conclude that Cu deficiency does not change the entire membrane fluidity but rather the lipid composition of the microenvironment of some electron-transport components. The inhibition of Photosystem II electron transport in Cu-deficient chloroplasts was characterized by thermoluminescence and 2-dimensional gel electrophoresis. It was found that Cu deficiency shifted the main peak of the glow curve from +18°C to +8°C, similar to that of DCMU-poisoned chloroplasts. Two apoproteins of the 29 kDa polypeptide disappeared in Cu-deficient chloroplasts which indicates that this polypeptide has a regulatory role in ensuring the normal electron flow between Q_A and Q_B.     

Effects of long-chained aliphatic amines on photosynthetic reactions in isolated spinach chloroplasts

The effect of six long-chained aliphatic amines on ¹⁴CO₂-reduction, electron transport and the 515 nm absorbance change and shrinkage in isolated intact and broken chloroplasts from spinach ( Spinacia oleracea L. cv. Weibulls Medania) was investigated. Five of the six investigated amines affected photosynthesis in intact chloroplasts by inhibiting ¹⁴CO₂-reduction. In broken chloroplasts the same amines uncoupled electron transport. When added to intact chloroplasts the five amines induced a light-dependent oxygen uptake leading to (he formation of hydrogen peroxide. The oxygen uptake was not due to the amines acting as Mehler reactants. The mode of action, different from that of simple aliphatic amines, was an effect on membrane integrity, first affecting the membrane potential. At higher amine concentrations a more general effect on the ion conditions in the thylakoids was evident.     

The inhibition of photosynthetic electron transport in spinach chloroplasts by low osmolarity.

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Stoichiometries of electron transport complexes in spinach chloroplasts

The stoichiometric relationship among photosystem II complexes, photosystem I complexes, cytochrome b/f complexes, high-potential cytochrome b-559, and chlorophyll in spinach chloroplasts has been determined. Two features of this data stand out, in contrast to currently proposed stoichiometries in which the ratio of photosystem II to photosystem I is reported to be 2:1 and the chlorophyll to reaction center ratio to be as low as 260:1. Using a variety of techniques it was found that the stoichiometry of photosystem II:photosystem I:cytochrome b/f complex was 1:1:1, within 10%, and that the ratio of total chlorophyll to these components was 600:1, also within 10%. A ratio of two high-potential cytochrome b-559 molecules per 640 chlorophyll, or two molecules per photosystem II reaction center, was found. These ratios were remarkably constant regardless of the time of year or the source of the spinach. The concentration of photosystem II complexes was determined using a pH electrode to measure the flash-induced proton release resulting from water oxidation. The photosystem I reaction center concentration was measured by two different techniques that compared favorably. In the first method a pH electrode was used to measure the amount of flash-induced proton consumption associated with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive oxidation of N,N,N',N'- tetramethylphenylenediamine , resulting in the production of hydrogen peroxide. In the second method the amount of P700 oxidized by far-red light was determined using dual-wavelength spectroscopy. The concentration of the cytochrome b/f complex was determined assuming 1 mol of cytochrome f per complex. The concentration of cytochrome f was measured spectroscopically by its light-induced turnover and by chemical difference spectra. The concentration of high-potential cytochrome b-559 was determined by chemical difference spectra. In addition to these studies, the light-induced absorbance change exhibiting a peak at 323 nm that has been attributed to the reduction of the primary quinone acceptor of photosystem II has been investigated. This measurement frequently has been used to quantitate the photosystem II to chlorophyll ratio. However, in view of these results it is argued that this technique significantly overestimates the photosystem II concentration.     

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.     

The cupric ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts

Strong inhibition of uncoupled photosynthetic electron transport by Cu²⁺ in isolated spinach chloroplasts was observed by measuring changes in O₂ concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu²⁺ to the chloroplast membranes resulted in removal of Cu²⁺ from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu²⁺ binding to inhibitory sites. Preincubation in the dark resulted in Cu²⁺ binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.     

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.     

The effects of high concentrations of salts on photosynthetic electron transport in spinach (Spinacia oleracea) chloroplasts.

1. Photosynthetic electron transport from water to lipophilic Photosystem II acceptors was stimulated 3--5-fold by high concentrations (greater than or equal to 1 M) of salts containing anions such as citrate, succinate and phosphate that are high in the Hofmeister series. 2. In trypsin-treated chloroplasts, K3Fe(CN)6 reduction insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea was strongly stimulated by high concentrations of potassium citrate, but there was much less stimulation of 2,6-dichloroindophenol reduction in Tris-treated chloroplasts supplied with 1,5-diphenylcarbazide as artificial donor. The results suggest that the main site of action of citrate was the O2-evolving complex of Photosystem II. 3. Photosystem I partial reactions were also stimulated by intermediate concentrations of citrate (up to 2-fold stimulation by 0.6--0.8 M-citrate), but were inhibited at the highest concentrations. The observed stimulation may have been caused by stabilizaton of plastocyanin that was complexed with the Photosystem I reaction centre, 4. At 1 M, potassium citrate protected O2 evolution against denaturation by heat or by the chaotropic agent NaNO3. 5. It is suggested that anions high in the Hofmeister series stimulated and stabilized electron transport by enhancing water structure around the protein complexes in the thylakoid membrane.     

Inhibition of the photosynthetic activities of isolated spinach chloroplasts by phosphonate compounds

The effects of three closely related phosphonate compounds on several photosynthetic activities of isolated chloroplasts were investigated. Phosphonoformic and phosphonopropionic acid were found to inhibit both CO₂ fixation and the reduction of 3-phosphoglyceric acid, with CO₂ fixation being more sensitive. In contrast, phosphonoacetic acid was only slightly inhibitory. The lack of inhibition appeared to be due to its inability to enter the stroma via the phosphate translocator. Measurements of changes in stromal metabolite levels following the inhibition of CO₂ fixation by either phosphonoformic or phosphonopropionic acid indicated that the activity of ribulose bisphosphate carboxylase/oxygenase was reduced. Studies with the isolated enzyme confirmed that both of these compounds were effective competitive inhibitors of the carboxylase activity of the enzyme.     

Effects of ethanol on the interaction of photosynthetic processes in spinach chloroplasts

The interaction and coupling between photosynthetic processeswere studied in ethanol-treated chloroplasts (in the absenceof ethanol) or in the presence of ethanol. Light-induced H⁺uptake and photophosphorylation were suppressed and electrontransfer was enhanced by ethanol treatment or in the presenceof ethanol (10–25%). Dark recoveries of the H⁺ uptakeand the 515-nm absorbance change were accelerated by ethanol.The half-effective concentrations of ethanol for these processeswere higher in the ethanol-treated chloroplasts than when ethanolwas present in the reaction mixture. The maximum rate of electrontransfer in the ethanol-treated chloroplasts, which was at thesame level as that of the control with an uncoupler, was notaffected by uncouplers. The marked acceleration of recoveryof the 515-nm absorbance change by SCN- or valinomycin plusK⁺ in the untreated chloroplasts was much smaller in the ethanol-treatedchloroplasts or in the presence of ethanol. The ethanol-treatedpreparation had the same characteristics as those of the control,in chlorophyll fluorescence, light-intensity dependence of electrontransfer (compared with the control with an uncoupler), andsensitivity to sucrose osmolarity except for a slight increaseof the packed volume. Under the present conditions, the ethanoltreatment mainly induced an increase of permeability of thylakoidmembranes to ions. In chloroplasts treated with ethanol at various concentrations,the relationships of the uncoupler-stimulated part of electrontransfer with the dark half-recovery time of H⁺ uptake and withphotophosphorylation were linear. Logarithms of the photophosphorylationand the extent of H⁺ uptake had a linear relationship with aslope of about 3. This slope may indicate the stoichiometryof H⁺/ATP. (Received June 17, 1978; )