Inhibition Of Photophosphorylation Research Articles

Metabolism of Oat Leaves during Senescence

The ATP content of 7-day-old Avena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. l-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, i.e. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling.

Reversible uncoupling of photophosphorylation by a new bifunctional maleimide.

A new bifunctional maleimide that contains a disulfide bond has been synthesized. This maleimide, dithiobis-N-ethylmaleimide (DTEM), like another bifunctional maleimide, o-phenylenebismaleimide, is about 500-fold more effective as an inhibitor of photophosphorylation than N-ethylmaleimide. Thylakoids must be illuminated in the presence of DTEM before the assay of phosphorylation for the inhibition to occur. Phosphoryalation in thylakoids treated with DTEM in the light is uncoupled and proton permeability of the treated thylakoids is enhanced. This uncoupling of photophosphorylation in thylakoids treated with DTEM can be reversed by thiol compounds. The addition of 50 mM dithiothreitol restores H+ uptake in thylakoids treated with DTEM in the light to control levels and partially reverses the inhibition of phosphorylation. Evidence is provided to show that DTEM cross-links groups within the gamma subunit of the coupling factor 1, and that the cross-link is broken by high concentrations of thiols. These results suggest that cross-linking is the cause for the increased proton permeability in thylakoids treated with bifunctional maleimides in the light.

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF 1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: 1. (a) the disappearance of three out of four accessible thiol groups, two from γ and one from a β subunit as a consequence of CF 1 modification by o-iodosobenzoate; 2. (b) the total free sulphydryl groups of CF 1 were reduced from 8 to 4 after modification of CF 1 by o-iodosobenzoate. Two groups disappeared from β and two from γ subunits; 3. (c) a second heating step of CF 1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF 1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF 1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from β and γ subunits.

Photooxidative Damage in Photosynthetic Activities of Chromatium vinosum

The capacity of photosynthetic CO(2) fixation in the anaerobic purple-sulfur bacterium, Chromatium vinosum is markedly impaired by strong illumination (9 x 10(4) lux) in the presence of 100% O(2). In the absence of HCO(3) (-), decline in activity occurred gradually, with about 40% of the initial activity remaining after a 1-hour incubation. The addition of 50 millimolar HCO(3) (-) to the incubation medium resulted in a measurable delay (about 30 minutes) of the inactivation process. Ribulose-1,5-bisphosphate carboxylase activity and light-dependent O(2) uptake (electron flow) or crude extracts prepared after pretreatment of the bacterial cells with O(2) and light were not affected but the photophosphorylation capacity of either bacterial cells or chromatophores was drastically reduced. The inhibition of photophos-phorylation in the chromatophore preparations was significantly reduced by the addition of either an O(2) (-) scavenger, Tiron, or an (1)O(2) scavenger, alpha-tocopherol. These results suggest that the active O(2) species, O(2) (-) or (1)O(2), might take part in the observed inactivation.The pretreatment of the bacteria with O(2) and light inhibited CO(2) assimilation through the Calvin-Benson cycle, while relatively stimulating the formation of aspartate and glutamate. It also inhibited the conversion of glycolate to glycine, resulting in a sustained extracellular excretion of glycolate. The inactivation of photosynthetic CO(2) fixation by intact cells was enhanced by low temperature, KCN, or methylviologen addition during the pretreatment with O(2) and light. The mechanism(s) of O(2)-dependent photoinactivation of photosynthetic activities in Chromatium are discussed in relation to the possible role of photorespiration as a means of producing CO(2) in the photosynthetic system.

Analysis of the Prompt Fluorescence Induction by Means of Computer Simulation of the Primary Photosynthetic Reactions

Abstract The induction phenomenon of prompt fluorescence of the photosynthetic system in green plants reflects the adaptation of the electron transport system from a dark adapted state to a steady state under light condition. The appearance of the P-peak in the prompt fluorescence induction curve is explained by the decay of the electrochemical gradient across the thylakoid membrane in the dark. The decay of the electrochemical gradient in the dark is calculated using measured fluorescence induction curves with different times of dark adaptation. The influence of photophosphorylation inhibitors is discussed and the threshold effect of ATP-formation depending on the electrochemical gradient is explained by the coupling of the ATP-formation with the electron transport in the thylakoid membrane.

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O 2. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K 4Fe(CN) 6, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c 2 from the heavy chromatophores caused a decrease in the light-induced O 2 uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c 2 from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c 2 level which faces the outside medium. However, cytochrome c 2 is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c 2 in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O 2 whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O 2 consumed is 4, which means that the end product of O 2 reduction is H 2O.

Glycolate synthesis by intact chloroplasts: Studies with inhibitors of photophosphorylation

Intact spinach chloroplasts, capable of evolving O 2 in response to CO 2 at rates greater than 70 μmol/h · mg of chlorophyll, synthesize glycolate from added dihydroxyacetone phosphate, ribose 5-phosphate, or xylulose 5-phosphate, when illuminated in the presence of O 2. The synthesis of glycolate from these compounds is dependent upon photophosphorylation and is inhibited by each of the three classes of photophosphorylation inhibitors [Izawa, S., and Good, N. E. (1972) in Methods in Enzymology, Vol. 24, Part B, pp. 355–377)]: an uncoupler, carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP), an energy transfer inhibitor, Dio-9, and a phosphate analog, arsenate. Neither arsenate nor Dio-9 causes the collapse of the light-generated proton gradient between thylakoid and stroma compartments of the chloroplasts, so that the inhibition of glycolate synthesis by these compounds cannot be ascribed to an inactivation of Calvin cycle enzymes thought to be associated with the collapse of such a proton gradient. The dependency of glycolate synthesis upon photophosphorylation indicates that an ATP-consuming reaction(s) is involved in the conversion of the substrates (triose and pentose monophosphates) to glycolate. The formation of dihydroxyethylthiamine pyrophosphate, the “active glycolaldehyde” intermediate of the transketolase reaction, from triose and pentose monophosphates has no known requirements for ATP. On the other hand, the conversion of both triose and pentose monophosphates to ribulose 1,5-bisphosphate, the substrate for the ribulose 1,5-bisphosphate oxygenase reaction, requires ATP. It is concluded that glycolate synthesis by intact isolated chloroplasts is primarily the result of ribulose 1,5-bisphosphate oxygenase activity. No substantial role in glycolate synthesis can be attributed to the oxidation of dihydroxyethylthiamine pyrophosphate, the intermediate of the transketolase reaction.

The Effect of Electron Donors and Acceptors on Light‐Induced Absorbance Changes and Photophosphorylation in Rhodospirillum rubrum Chromatophores

Light-induced difference spectra between 400 and 640 nm of Rhodospirillum rubrum chromatophores were performed in the presence and absence of exogenous electron donor/acceptor systems and compared with the chemical oxidation spectrum. The results indicate that the component previously defined as P430 is not a unique entity but rather represents different species, or a mixture of species, under various conditions. Under all conditions in which the reaction center bacteriochlorophyll is reversibly photooxidized, as indicated by the bleaching around 600 nm, it is also contributing to the absorbance increase around 430 nm. In one case, in presence of reduced dichloroindophenol and in the absence of oxygen, the photooxidation of reaction center bacteriochlorophyll is fully supressed. Under these conditions an irreversible change around 430 nm is still observed and seems to be due to the Soret band of b-type cytochrome. In the presence of reduced dichloroindophenol and absence of oxygen there is a marked inhibition of photophosphorylation. This inhibition is apparently due to the complete reduction of the cyclic electron carriers. Addition of the low potential dye benzyl viologen facilitates an almost complete recovery of the reversible photooxidation of reaction center bacteriochlorophyll as well as of photophosphorylation. These results indicate that the apparent mid-point potential of the primary electron acceptor in Rhodospirillum rubrum chromatophores is probably in the range of that of benzyl viologen (E'o = - 340 mV).

Peptide alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

Peptide alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

Membrane-bound ATPase in chloroplasts of Euglena gracilis

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca 2+- and Mg 2+-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. K m values of Ca 2+ and Mg 2+ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca 2+- or Mg 2+-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca 2+ or Mg 2+ or from two different enzymes are discussed.

Chloroplast coupling factor 1: A species-specific receptor for tentoxin

Tentoxin, a cyclic tetrapeptide, induces chlorosis in certain plant species. It inactivated photophosphorylation and coupling factor 1 (CF(1)) ATPase in lettuce, a sensitive species. This effect was due to binding of tentoxin with CF(1) at a single site (affinity constant 1.3 to 20 x 10(7) M(-1)). Neither AMP nor adenyl-5'-yl imidodiphosphate appeared to bind to this site. In radish, an insensitive species, 20 times more tentoxin was required for 50% inhibition of photophosphorylation. In this species CF(1) ATPase was unaffected by tentoxin, and its CF(1) bound tentoxin only weakly (affinity constant less than 1 x 10(4) M(-1)). Sensitivity of photophosphorylation to tentoxin was correlated with chlorosis sensitivity in six other species examined.

Inhibition of electron transport on the oxygen-evolving side of photosystem II by an antiserum to a polypeptide isolated from the thylakoid membrane.

A polypeptide fraction with the apparent molecular weight 11 000 was isolated from stroma-freed chloroplasts from Anthirrhinum majus. An antiserum to this polypeptide fraction inhibits photosynthetic electron transport in chloroplasts from Nicotiana tabacum. The relative degree of inhibition is pH dependent and has its maximum at pH 7.4. The maximal inhibition observed was 93%. The dependence of the inhibition on the amount of antiserum yields a sigmoidal curve which hints at a cooperative effect. A calculation of the Hill interaction coefficient gave the value of 10. The inhibition occurs on the water splitting side of photosystem II between the sites of electron donation of tetramethyl benzidine and diphenylcarbazide. Tetramethyl benzidine donates its electrons before the site where diphenylcarbazide feeds in its electrons. Analysis of the steady state level of the variable fluorescence also indicates that the inhibition site is on the water splitting side of photosystem II. Tris-washed chloroplasts are equally inhibited by the antiserum and the inhibition is also observed in the presence of an inhibitor of photophosphorylation like dicyclohexyl carbodiimide and in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) which means that the inhibitory action is directed towards the electron transport chain. Valinomycin which is supposed to affect the cation permeability of the thylakoid membrane has no influence on the inhibitory action of the antiserum. The same is valid for gramicidin. Methylamine on the other hand can induce a state in the thylakoids in which the antiserum is not effective. If the antibodies are already adsorbed prior to the methylamine addition then the high inhibitory effect by the antiserum remains unchanged upon addition of methylamine. From the experiments it follows that a component from the vicinity of photosystem II is accessible to antibodies that is, the component is located in the outer surface of the thylakoid membrane. It appears that the inhibitory effect is produced in the course of the light reaction.

Sulphydryl groups in photosynthetic energy conservation III. Inhibition of photophosphorylation in spinach chloroplasts by CdCl 2

Low concentrations of CdCl 2 inhibited cyclic and non-cyclic photophosphorylation in spinach chloroplasts and depressed coupled electron transport to the basal level. The basal electron transport and the electron flow stimulated by several uncouplers, except arsenate, were not affected. The inhibitory effects of cadmium were reversed by dithiols like dithioerythritol or 2,3-dimercaptopropanol. The light-induced pH rise of chloroplast suspensions and the trypsin-activated Ca-ATPase were not affected by cadmium at the concentrations that inhibited ATP synthesis but the latter was affected by higher concentrations.

Suppression of the stomatal opening by morphactins in isolated epidermal strips

The effects of three morphactins, chlorflurenol, flurenol and EMD 7301 W, were examined on the stomatal aperture using isolated epidermal strips of Commelina benghalensis. Morphactins produced, a striking decrease in the stomatal opening in light but had no effect on stomatal closure in darkness. Various catalysts and inhibitors of photophosphorylation had no influence on the morphactin-induced stomatal closure. The stimulatory effects of ATP, pyruvate and KC1 on stomatal opening were suppressed by the morphactins. The cytokinin, benzyladenine stimulated the stomatal opening even in the presence of a morphactin. The influence of morphactins on the stomatal aperture closely resembled the effect of abscisic acid.

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation

ATP and pyrophosphate at high concentration (> 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl 2, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg · ADP −) and magnesium-phosphate (Mg · P i) complexes. It is highly probable that Mg · ADP − and Mg · P i, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg 2+ which is available for the formation of the complex with ADP and phosphate.

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D- threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO 2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO 2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO 2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO 2 fixation without affecting phosophoglycerate reduction. This inhibition of CO 2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO 2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO 2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO 2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO 2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO 2 fixation in the light. This demonstrates that CO 2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8. 7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO 2 fixation from zero to maximal activity. This offers a mechanism for light control of CO 2 fixation, avoiding wasteful CO 2 fixation in the dark.

Synergistic uncoupling of spinach chloroplasts by combinations of photophosphorylation inhibitors and carbonyl cyanide p-trifluoromethoxyphenylhydrazone

Combinations of low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhy-drazone (FCCP) with suboptimal concentrations of Dio-9, phloridzin, ajmaline, and dihydrodiscarine B synergistically inhibited cyclic and noncyclic photophosphorylation in spinach chloroplasts but their effects on the light-triggered ATPase were additive rather than synergistic. The effect was reversed by washing and prevented by dithioerythritol and by cistein. Carbonyl cyanide m-chlorophenylhydrazone (CCP) could replace FCCP but uncouplers of other types like atebrin did not substitute for FCCP. Combinations of FCCP with the four inhibitors synergistically uncoupled ferricyanide reduction in the presence of ADP and P i but not in their absence. The synergistic uncoupling was not observed on the light-dependent pH rise of chloroplast suspensions. Association of FCCP with any of the inhibitors completely abolished the stimulation of proton uptake or the inhibition of electron transport induced by low concentrations of ATP. This synergistic and peculiar uncoupling can not be ascribed to a modification of membrane permeability. One possible explanation is that the effect requires a conformational state of the membrane-bound coupling factor 1 (CF 1) induced by phosphorylating conditions which would facilitate the interaction of inhibitors and FCCP with the membrane.

Effects of tentoxin on enzymic activities in cucumber and cabbage cotyledons.

Tentoxin, produced by Alternaria alternata (Fr.) Keissl. causes severe variegated chlorosis in germinating seedlings of certain dicotyledonous species. However, it does not impair radicle and hypocotyl elongation or cotyledon expansion. Effects of the toxin on the activity of selected enzymes from both chloroplasts and cytoplasm were determined. Cucumber (Cucumis sativus L.), which is highly sensitive to tentoxin and cabbage (Brassica oleracea L.), which is resistant, were used as test plants. The activities of chloroplastic, but not cytoplasmic, enzymes were decreased by treatment of cucumber cotyledons with tentoxin. Neither group of enzymes was affected by the toxin in cabbage cotyledons. The decreased enzymic activities are probably related to reported inhibition of photophosphorylation by tentoxin.

Bioenergetic aspects of bacteriophage replication in the photosynthetic bacterium Rhodopseudomonas capsulata

The energy requirements for replication of the virulent bacteriophage RC1 in the nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata were investigated. The host bacterium can grow either with energy obtained by photophosphorylation (anaerobically) or aerobic (dark) oxidative phosphorylation, and the conditions of growth influence the relative capacities of the alternative energy-converting systems. These capacities are, in turn, reflected in the ability of infected cells to support virus growth under photosynthetic or aerobic, dark circumstances. In photosynthetically grown host cells, phage replication is supported by both kinds of energy conversion; anaerobically, virus growth in illuminated infected cells is markedly suppressed by inhibitors of photophosphorylation (or by transfer of the infected cells to darkness before the end of the latent period). When the cells are cultivated aerobically in darkness, synthesis of the photosynthetic pigment system is greatly diminished; in such organisms, the energy for growth of phage RC1 can be supplied by oxidative phosphorylation, but the anaerobic photophosphorylation capacity of the cells does not suffice. Other results obtained indicate that the energy requirement for phage RC1 multiplication is more stringent than that for (uninfected) host cell growth. We suggest that nonsulfur purple bacteria-phage systems may offer unique advantages for further study of the bioenergetics of phage growth, and may also be useful for investigation of the in vivo development of energy-converting membranes.