Basal Electron Flow Research Articles

Inhibition and Uncoupling of Photosynthetic Electron Transport by Diterpene Lactone Amide Derivatives

Nine diterpene lactone amide derivatives 1-9 were synthesized from 6-oxovouacapan-7beta,17beta-lactone, which was obtained from 6alpha,7beta-dihydroxyvouacapan-17beta-oic acid isolated from Pterodon polygalaeflorus Benth., and tested for their activity on photosynthetic electron transport. Amide derivatives 3-5 behaved as electron transport chain inhibitors; they inhibited the photophosphorylation and uncoupled non-cyclic electron transport from water to methylviologen (MV). Furthermore, 4 and 5 enhanced the basal electron rate acting as uncouplers. Compound 6 behaved as an uncoupler; it enhanced the light-activated Mg2+-ATPase and basal electron flow, without affecting the uncoupled non-cyclic electron transport. Compounds 1-2 and 7-9 were less active or inactive. Compounds 3-5 did not affect photosystem I (PSI); they inhibited photosystem II (PSII) from water to 2,6-dichlorophenol indophenol (DCPIP). Compound 4 inhibited PSII from water to silicomolybdate (SiMo), but it had no effect on the reaction from diphenylcarbazide (DPC) to DCPIP indicating that its inhibition site was at the water splitting enzyme complex (OEC). Compounds 3 and 5 inhibited PSII from water to DCPIP without any effect from water to SiMo, therefore they inhibited the acceptor site of PSII. Chlorophyll a fluorescence kinetics confirmed the behaviour of 3-5.

Synthesis of Photosynthesis-Inhibiting Nostoclide Analogues

A series of 34 3-benzyl-5-(arylmethylene)furan-2(5H)-ones, designed using the naturally occurring toxins nostoclides as a lead structure, was synthesized as potential inhibitors of the photosynthetic electron transport. All compounds were fully characterized by IR, NMR (1H and 13C), and MS spectrometry. HMBC and HSQC bidimensional experiments allowed 13C and 1H assignments. Their biological activities were evaluated in vitro as the ability to interfere with light-driven reduction of ferricyanide by isolated spinach chloroplasts. About two-thirds of the compounds exhibited inhibitory properties in the micromolar range against the basal electron flow from water to K3[Fe(CN)6]. The inhibitory potential of these 3-benzyl-5-(arylmethylene)furan-2(5H)-one lactones is higher than that of other nostoclide analogues previously synthesized in the same laboratories.

Alternaria alternataCrofton-Weed Toxin: a Natural Inhibitor of Photosystem II inChlamydomonas reinhardtiiThylakoids

The action site of Alternaria alternata Crofton-weed toxin (AAC-toxin), isolated first from Alternaria alternata (Fr.) Keissler, was investigated in Chlamydomonas reinhardtii thylakoids. The results revealed that AAC-toxin inhibited photophosphorylation in a concentration-dependent pattern. Similarly, toxin inhibited uncoupled, basal electron flow and photosystem II (PSII) electron transport as well. However, toxin did not affect photosystem I (PSI) activity or the partial reaction of electron transport from H2O to silicomolybdic acid (SiMo). Therefore, the action site of toxin was located at QB level. In addition, the toxin may behave as an energy-transfer inhibitor at high concentrations by inhibiting phosphorylating electron transport and Mg2+ATPase activity. Chlorophyll a fluorescence induction and JIP test corroborated the inhibition at QB level. Through observations of the different sensitivity of toxin on D1 mutants of C. reinhardtii, evidence further confirmed that AAC-toxin inhibited electron transport by displacing the QB on the D1 protein, and the mode of action was similar to phenol-type PSII inhibitors.

Annonaceous acetogenins: Naturally occurring inhibitors of ATP synthesis and photosystem II in spinach chloroplasts

The effects of squamocin (1), bullatacin (2) and motrilin (3), 3 bis‐tetrahydrofuran Annonaceous acetogenins, isolated from Annona purpurea (Annonaceae), were investigated on several photosynthetic activities in spinach thylakoids. The results indicated that compounds 1–3 significantly inhibited both ATP synthesis and uncoupled electron transport. In addition, they enhanced light‐activated Mg2+‐ATPase, and basal electron flow. Therefore, acetogenins 1–3 behave as uncouplers and Hill reaction inhibitors. Natural products 1–3 did not affect photosystem I (PSI) activity but they inhibited photosystem II (PSII) electron flow. The study of the partial PSII reactions from H2O to DCPIPox, H2O to SiMo and diphenylcarbazide to DCPIP established that the site of inhibition was at the oxygen‐evolving complex (OEC). Chlorophyll a fluorescence measurements confirmed the behavior of the Annonaceous acetogenins as water‐splitting enzyme inhibitors.

Phytotoxic compounds from Esenbeckia yaxhoob

Investigation of the aerial parts of Esenbeckia yaxhoob Lundell (Rutaceae) led to the isolation of a new dammarane-type of triterpene which was characterized by spectral means as (24S)-24-methyl-dammara-20,25-diene-3β-yl-acetate. In addition, 2-tridecanone, asarinin, imperatorin, lupeol and hesperidin were obtained. (24S)-24-Methyl-dammara-20,25-diene-3β-yl-acetate, 2-tridecanone, asarinin and imperatorin caused significant inhibition of the radicle growth and\\or germination of seedlings of Amaranthus hypochondriacus, Echinochloa crusgalli, Lactuca sativa and Lycopersicum esculentum. Also, it has been found that imperatorin inhibited ATP synthesis and both phosphorylating and uncoupled electron flow from H2O to K3[Fe(CN)6]. On the other hand, this coumarin stimulated the basal electron flow from H2O to K3[Fe(CN)6] and the activity of the light-activated Mg2-ATPase. These effects were measured in freshly lysed illuminated spinach chloroplasts and allowed to determine that imperatorin acts as an uncoupler and as a Hill reaction inhibitor.

Uncoupling Behavior of the 4-Phenylcoumarins in Spinach Chloroplasts: Structure−Activity Relationships

4-Phenylcoumarins isolated from Exostema caribaeum and Hintonia latiflora (Rubiaceae) and some semisynthetic derivatives acted as uncouplers in spinach chloroplasts. The glycoside 5-O-β-d-glucopyranosyl-7-methoxy-3‘,4‘-dihydroxy-4-phenylcoumarin (3), 5,7,3‘,4‘-tetrahydroxy-4-phenylcoumarin (1a), and 7-methoxy-5,3‘,4‘-trihydroxy-4-phenylcoumarin (2a) inhibited ATP synthesis and proton uptake. On the other hand, basal and phosphorylating electron transport were enhanced by these compounds. The light-activated Mg2+-ATPase was slightly stimulated by coumarins 1a and 2a. In addition, at alkaline pH compound 1a stimulated the basal electron flow from water to methylviologen, but at the pH range from 6.0 to 7.5 the coumarin did not have any enhancing effect. Compound 1a, which possesses four free phenolic hydroxyl groups, was the most active uncoupler agent. Methylation (2b, 4), acetylation (2a), or glycosylation (1−3) of the phenolic groups at C-3‘, C-4‘, and C-5 resulted in a reduction or loss of the uncoupling activity. Therefore, the phenolate anions may be the active form responsible for the uncoupling behavior of 4-phenylcoumarins. Keywords: Exostema caribaeum; Hintonia latiflora; Rubiaceae; phenylcoumarin; photosynthesis; uncouplers

Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction.

Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.

Formate as an Inhibitor of Photosynthetic Electron Flow

The effects of formate on the Hill reaction in isolated broken pea chloroplasts were in­vestigated. Addition of formate to chloroplasts has two distinct effects: I. basal electron flow can be stimulated 3-fold; 2. uncoupled electron flow is inhibited. The stimulating effect is due to uncoupling by formate and appears instantaneous. Maximal inhibition by form ate is only observed after prolonged illumination. The inhibitory action of form ate on electron flow can be relieved by bicarbonate *.

Quantitative relationships between phosphorylation, electron flow, and internal hydrogen ion concentrations in spinach chloroplasts.

1. Further evidence that the uptake of [14C]hexylamine, determined by centrifugal filtration of spinach chloroplast thylakoids through silicone fluid layers, gives precise estimations of light-induced H+ concentration gradients (deltapH) is presented. DeltapH was independent of the amount of thylakoids used or of the concentration of hexylamine. Moreover, hexylamine uptake was sensitive to the osmolarity of the suspending medium. 2. Internal H+ concentration ([H+]in) is proportional to the rate of electron flow when light intensity was used to vary these parameters. Proportionality was still observed in the presence of 0.1 and 1.0 muM gramicidin D. When, however, [H+]in and electron flow were varied by increasing the concentration of gramicidin D, at constant light intensity the rate of electron flow was approximately proportional to 1/[H]in. 3. The phosphorylation efficiency (P/e2 ratio) falls with decreasing light intensity or increasing concentrations of the phosphorylation inhibitor, 4'-deoxyphlorizin. The proportionality between the rate of electron flow and [H+]in allows the calculation of the rate of nonphosphorylating (basal) electron flow if [H+]in under phosphorylating conditions is known. The contribution of basal electron flow, a consequence of passive efflux of H+ from the thylakoids, to the overall rate of electron flow increases as the rate of phosphorylation decreases. P/e2 ratios calculated using rates of electron flow from which the basal component has been subtracted are constant. A calculated P/e2 ratio of about 1.3 is obtained. 4. It is shown that the reciprocal of the phosphorylation efficiency should be proportional to 1/[H+]in2 when these parameters are varied using light intensity. This relationship was verified and provided an estimate of the P/e2 at infinite [H+]in. This value was 1.3. These results provide further evidence that a H+ electrochemical gradient serves to couple photophosphorylation to electron flow and that the rate of phosphorylation is proportional to [H+]in3. That is, three H+ are translocated out of thylakoids for each adenosine triphosphate formed.

The interaction of peroxyacetyl nitrate (PAN) with the electron flow of isolated chloroplasts

Peroxyacetyl nitrate (PAN), a component of photochemical smog, inhibits the electron flow in both photosystems of isolated chloroplasts with no uncoupling (no increase in basal electron flow rate). Furthermore, PAN is relatively stable in solution (with a half-life of ca. 3 min under the assay condition), and thus the Hill reaction rate continues to decline slowly after gassing. Dithiothreitol decomposes the PAN and eliminates this decline after-gassing effect, but it cannot reverse the inhibition of the Hill reaction. Light stimulates the inhibition produced by PAN, but only in aged chloroplasts.

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP 2 e ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 1 1 ratio, was about 4 both at pH 7.6 and at 7.0. ATP 2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2. Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO 2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP 2 e ratios were calculated to be between 1.1 and 1.4. Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg −1 chlorophyll · h −1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO 2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP 2 e ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials [ ATP] [ ADP] [ P i ] . Flexible coupling implies, that ATP 2 e ratios should decrease with increasing phosphorylation potentials inside the chloroplasts.

Inhibition of Coupling Factor Activity of Chloroplast Membranes by Diazonium Compounds

Abstract 1. Chemical modification of chloroplast membranes by diazonium benzenesulfonic acid (DABS) resulted in marked inhibition of noncyclic and cyclic photophosphorylation. Accompanying the inhibition of ATP synthesis there was an increased rate of basal electron flow and loss of the ability of ADP to stimulate the basal electron transport rate, both characteristic of uncoupling. 2. The calcium-dependent ATPase activity of the coupling factor was also severely inhibited after treatment with the diazonium compound. This, in conjunction with the binding of [35S]DABS to the coupling factor subunits as revealed by sodium dodecyl sulfate disc gel electrophoresis, suggests that the inhibition of phosphorylation results, in part at least, from inactivation of the coupling factor complex by the chemical modifier. 3. Treatment of chloroplast membranes (from which coupling factor was largely removed by two EDTA washes) with [35S]DABS, followed by digitonin fractionation of the membranes into Photosystem I and Photosystem II fractions revealed that the Photosystem I fraction incorporated 10-fold more [35S]DABS than the Photosystem II fraction. This is consistent with previous findings (Dillley, R. A., Peters, G. A., and Shaw, E. R. (1972) J. Membrane Biol. 8, 163–180) using intact chloroplasts (with coupling factor present) that the intrinsic proteins associated with Photosystem I are localized at the external surface of the grana membrane.

Inhibition of photophosphorylation by tentoxin, a cyclic tetrapeptide

Tentoxin, a fungal toxin which is known to cause seedling chlorosis, was found to inhibit cyclic photophosphorylation but not reversible proton accumulation by isolated chloroplasts. The toxin, at low concentrations, also inhibited coupled electron flow (in the presence of ADP and phosphate) but did not effect basal electron flow (in the absence of ADP and phosphate) or uncoupled electron transport. It is suggested that tentoxin is an energy transfer inhibitor which acts at the terminal steps of ATP synthesis.