Hill Reaction Inhibitors Research Articles

Biflavonoids Isolated from Selaginella lepidophylla Inhibit Photosynthesis in Spinach Chloroplasts

Through bioactivity-guided chemical analysis of Selaginella lepidophylla, biflavonoids robustaflavone (1), 2,3-dihydrorobustaflavone (2), and 2,3-dihydrorobustaflavone-5-methyl ether (3) were isolated and their structures confirmed by spectroscopic and spectrometric analyses. Their NMR resonances were unambiguously assigned from HMBC, NOESY, and NOESY-1D experiments, and absolute configurations of 2 and 3 were established. Compound 3 has not been reported, and although structure of 2 was described before, the (13)C NMR assignment does not correlate with the structure reported. Therefore, this is the first report of 2. All compounds inhibited ATP production. Compounds 1 and 2 behaved as Hill reaction inhibitors. 1 interacted with photosystem II, transforming the reaction centers to silent centers at 300 and 600 microM. The interaction and inhibition target of 2 was located on Cyt b6f to PC. The three compounds also behaved as energy transfer inhibitors, 3 being the most active.

Sesquiterpenes from Celastrus vulcanicola as Photosynthetic Inhibitors

Three new sesquiterpenoids (1- 3) with a dihydro-beta-agarofuran skeleton were isolated from Celastrus vulcanicola. Their structures were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC, and HMBC), and the absolute configurations were determined by circular dichroism and chemical correlations. Their effects on photosynthesis were tested. Sesquiterpene 1 (50 microM) inhibits both light-dependent synthesis of ATP and the electron flow in chloroplasts, whereas at high concentrations the electron flow inhibition was partially reversed. Therefore, 1 behaves as a Hill reaction inhibitor and a weak energy transfer inhibitor and has two targets of interaction: one located at the oxygen-evolving complex, and the other located at the light-activated Mg (2+)-ATPase. Compound 2 was inactive, whereas 3 acts with the same mechanisms as 1 but was less active. Celastrus vulcanicola J. Donnell Smith (Celastraceae) is a subtropical woody vine distributed in Central America and the Caribbean. Its chemical constituents and biological activity have not yet been investigated.

Siderin from Toona ciliata (Meliaceae) as photosystem II inhibitor on spinach thylakoids

Four natural products were isolated from plants of the Rutaceae and Meliaceae families and their effect on photosynthesis was tested. Siderin ( 1) inhibited both ATP synthesis and electron flow (basal, phosphorylating, and uncoupled) from water to methylviologen (MV); therefore, it acts as Hill reaction inhibitor in freshly lysed spinach thylakoids. Natural products 2– 4 were inactive. Secondary metabolite 1 did not inhibit PSI electron transport. It inhibits partial reactions of PSII electron flow from water to 2,6-dichlorophenol indophenol (DCPIP), from water to sodium silicomolybdate, and partially inhibits electron flow from diphenylcarbazid (DPC) to DCPIP. These results established that the site of inhibition of 1 was at the donor and acceptor sides of PSII, between P 680 and Q A. Chlorophyll a fluorescence measurements confirmed the behavior of the Toona ciliate coumarin 1 as P 680 to Q A inhibitor by the creation of silent centers. May be this is the mechanisms of action of 1 and is the way in which it develops a phytotoxic activity against photosynthesis.

Inhibition of Photophosphorylation and Electron Transport Chain in Thylakoids by Lasiodiplodin, a Natural Product fromBotryosphaeria rhodina

Four natural products were isolated from the fungus Botryosphaeria rhodina, and their effects on photosynthesis were tested. Only lasiodiplodin (1) inhibited ATP synthesis and electron flow from water to methylviologen; therefore, it acts as a Hill reaction inhibitor in freshly lysed spinach thylakoids. Photosystem I and II and partial reactions as well as ATPase were measured in the presence of 1. Three new different sites of 1 interaction and inhibition were found: one at CF1, the second in the water-splitting enzyme, and the third at the electron-transfer path between P680 and QA; these targets are different from that of the synthetic herbicides present. Electron transport chain inhibition by 1 was corroborated by fluorescence induction kinetics studies.

Phytotoxic constituents from Nimbya alternantherae

A phytotoxic compound identified as 6-(3,3-dimethylallyloxy)-4-methoxy-5-methylphthalide ( 1), was isolated from the phytopathogenic fungus Nimbya alternantherae, cultivated on solid media. Catalytic hydrogenation of 1 resulted in the quantitative formation of second compound ( 2). Both compounds ( 1, 2) act by inhibiting the ATP synthesis in spinach thylakoids. A detailed investigation on the mechanism of action indicates that compound 1 acts as a Hill reaction inhibitor and uncoupler of photosynthesis.

Uncoupling and inhibition properties of 3,4-seco-friedelan-3-oic acid isolated from Maytenus imbricata

3,4-Seco-friedelan-3-oic acid was isolated from Maytenus imbricata (Celastraceae). At low concentrations it inhibited non-cyclic electron transport and ATP synthesis in spinach chloroplasts, i.e., it behaved as a Hill reaction inhibitor, and at high concentrations it acts as an uncoupler by enhancing uncoupled electron transport and Mg 2+–ATPase activity. 3,4-Seco-friedelan-3-oic acid did not inhibit PSII electron transport from DPC to DCPIP ox and photosystem I activity, but it enhanced from TMQH 2 to MV, corroborating its action as uncoupler. It inhibits electron flow through PSII from water to sodium silicomolybdate. The whole results indicate that the 3,4-seco-friedelan-3-oic acid target is at the OEC complex enzyme, the donor side of PSII. The fluorescence decay data shows the formation of the K-band, which match this result, acting as inhibitor at the donor side of PSII and it as an uncoupler.

A Diterpene γ-Lactone Derivative from Pterodon polygalaeflorus Benth. as a Photosystem II Inhibitor and Uncoupler of Photosynthesis

6alpha,7beta-Dihydroxyvouacapan-17beta-oic acid (1) was isolated from Pterodon polygalaeflorus Benth. Modification of 1 yielded 6alpha-hydroxyvouacapan-7beta,17beta-lactone (2) and then 6-oxovouacapan-7beta,17beta-lactone (3). Photosynthesis inhibition by 3 was evaluated in spinach chloroplasts. The uncoupled non-cyclic electron transport rate and ATP synthesis were inhibited by 3, which behaved as a Hill reaction inhibitor. Furthermore, 3 acted as an uncoupler because it enhanced the basal and phosphorylating electron transport rate on thylakoids. This last property of 3 was corroborated when it was observed that it enhances the Mg2+-ATPase activity. In contrast, 3 did not affect photosystem I (PSI) activity. Analysis of the partial photosystem II (PSII) reactions from water to DCPIPOX and water to silicomolybdate allowed to locate the inhibition sites at the redox components of PSII. The OJIP test of the chlorophyll a fluorescence transient confirmed that the inhibition sites were 1.) the oxygen-evolving complex (OEC) and 2.) by the formation of silent centers in the non-QA reducing centers.

Phytogrowth‐ and photosynthesis‐inhibiting properties of nostoclide analogues

Six nostoclide analogues were synthesised from 3-benzyl-2(5H)-furanone in one step, with yields ranging from 10 to 71%, and subjected to several biological assays. The two most active of these, 5d and 5e, were shown to be phytogrowth inhibitors of the radicle of Lolium multiflorum Lam, while enhancing the root growth of Physalis ixocarpa Brot. Both compounds inhibited electron flow (basal, phosphorylating and uncoupled) from water to methylviologen (MV); both acted as Hill reaction inhibitors, since the synthesis of ATP was prevented. The uncoupled electron transport from photosystem II (PSII) (water to 2,6-dichlorophenol-indophenol (DPIP)) and photosystem I (PSI) (2,6-dichlorophenol-indophenol reduced (DPIPred) to MV) was inhibited with 500 microM of 5d by 22 and 14% respectively. In addition, 400 microM of 5d inhibited PSI (from tetramethyl-p-benzohydroquinone (TMQH(2)) to MV) by 40%. Thus 5d inhibited electron transport at the b(6)f complex. Finally, 500 microM of 5e inhibited electron flow (basal and phosphorylating) by 25%, and 300 microM of 5e enhanced light-activated membrane-bound Mg(2+)-ATPase by 66%. Thus 5e behaved as a weak Hill reaction inhibitor and an uncoupler. In general, the phytotoxicity of the synthetic lactones was only weakly related to inhibition of photosynthesis.

Pachypodol from Croton ciliatoglanduliferus Ort. as Water-Splitting Enzyme Inhibitor on Thylakoids

A bioactivity-guided chemical study of aerial parts of Croton ciliatoglanduliferus Ort. led to the isolation for the first time of the flavonoids retusin (5-hydroxy-3,7,3',4'-tetramethoxyflavone) (1) and pachypodol (5,4'-dihydroxy-3,7,3'-trimethoxyflavone) (2) from the n-hexane extract. Compounds 1 and 2 were separated by preparative thin-layer chromatography. Compound 2 was the most active compound on ATP synthesis inhibition. The I50 value was 51 microM. Pachypodol behaves as a Hill reaction inhibitor. It inhibited the uncoupled electron flow on photosystem II partial reaction from water to dichlorophenol indophenol (DCPIP) and from water to sodium silicomolybdate. However, the uncoupled partial reaction from diphenylcarbazide to DCPIP and the uncoupled photosystem I from DCPIPred to MV were not inhibited by 2. These results were corroborated by fluorescence decay data. Therefore, pachypodol inhibits the water-splitting enzyme activity. Compound 1 with a 4'-methoxy group was a weak inhibitor, indicating that the 4' free -OH group is important for strong inhibition.

Natural diterpene β-lactone derivative as photosystem II inhibitor on spinach chloroplasts

α,7β-Dihydroxyvouacapan-17β-oic acid ( 1) was isolated from Pterodon polygalaeflorus Benth. Then, ( 1) was modified to obtain 6α-hydroxyvouacapan-7β,17β-lactone ( 2). Inhibition properties of both ( 1) and ( 2) were evaluated in spinach chloroplasts. Only compound ( 2) inhibited non-cyclic electron transport and ATP synthesis, i.e., ( 2) behaved as a Hill reaction inhibitor. Compound ( 2) did not affect photosystem I (PSI) activity but it did inhibit electron flow through PSII. Analysis of the partial PSII reactions from water to DCPIP ox, water to silicomolybdate, and diphenylcarbazide to DCPIP ox allowed locating inhibition sites at (i) the oxygen-evolving complex (OEC) and (ii) the redox enzymes of the electron transport chain, in the span of P 680 to Q A. Chlorophyll a fluorescence measurements confirmed the action site for ( 2).

Cobalt(II) Coordination Compounds of Ethyl 4‐Methyl‐5‐Imidazolecarboxylate: Chemical and Biochemical Characterization on Photosynthesis and Seed Germination

In this work we present the synthesis, structural and spectroscopic characterization of Co2+ coordination compounds with ethyl 4-methyl-5-imidazolecarboxylate (emizco). The effects of emizco, the metal salts CoCl2.6H2O, CoBr2, Co(NO3)2.6H2O and their metal coordination compounds [Co(emizco)2Cl2], [Co(emizco)2 Br2].H2O, [Co(emizco)2 (H2O)2(NO2)2.2H2O were evaluated on photosynthesis in spinach chloroplasts. Seed germination and seedling growth of the monocotyledonous species Lolium multiflorum and Triticum aestivum and the dicotyledonous species Trifolium alexandrinum and Physalis ixocarpa were also assayed under the effect of the compounds and salts. The results showed that cobalt(II) salts and their emizco coordination compounds inhibit photosynthetic electron flow and ATP-synthesis, behaving as Hill reaction inhibitors. Coordination compounds are more potent inhibitors than the salts. It was found that the salts target is at the b6f level while the complexes targets are at QB(D1)-protein and b6f level. The QB inhibition site was confirmed by variable chlorophyll a fluorescence yield. On the other hand, emizco inhibits seed germination, root and shoot development, in both weed and crop species. Cobalt(II) coordination compounds are the most effective photosynthesis inhibitors, but they are less potent than emizco in germination and seedling growth, while the metal salts are the least active of all.

Interference of ruthenium red analogues at photosystem II of spinach thylakoids

The effect of ruthenium red analogues on several thylakoid photosynthetic activities has been investigated. RR, RV, RRPh1, RRPh2 and Ph inhibit ATP synthesis and electron flow from water to MV (basal, phosphorylating and uncoupled) as their concentration increases, thus, they act as a Hill reaction inhibitor. They inhibit uncoupled electron transport through PSII from water to DCPIP and partially from DPC to DCPIP. However, these compounds do not affect uncoupled PSI electron transport from DCPIP to MV. Therefore, the target of interaction is at the level of OEC and the span P680 to QA for RR, RRPh1 and RRPh2. Chlorophyll a fluorescence studies corroborate the already found interference sites and may affect the disconnection between chlorophyll molecules within the LHCII and/or between antennae and RCs, or decreases the exciton to reach the RC and inhibition of PSII occurs. RRPh2 is six times more active than RR. Finally, Ph inhibits electron flow interacting at the level of QB.

Interference of ruthenium red analogues at photosystem II of spinach thylakoids

The effect of ruthenium red analogues on several thylakoid photosynthetic activities has been investigated. RR, RV, RRPh1, RRPh2 and Ph inhibit ATP synthesis and electron flow from water to MV (basal, phosphorylating and uncoupled) as their concentration increases, thus, they act as a Hill reaction inhibitor. They inhibit uncoupled electron transport through PSII from water to DCPIP and partially from DPC to DCPIP. However, these compounds do not affect uncoupled PSI electron transport from DCPIP to MV. Therefore, the target of interaction is at the level of OEC and the span P 680 to Q A for RR, RRPh1 and RRPh2. Chlorophyll a fluorescence studies corroborate the already found interference sites and may affect the disconnection between chlorophyll molecules within the LHCII and/or between antennae and RCs, or decreases the exciton to reach the RC and inhibition of PSII occurs. RRPh2 is six times more active than RR. Finally, Ph inhibits electron flow interacting at the level of Q B.

Inhibition of photosystem II in spinach chloroplasts by trachyloban-19-oic acid

The effect of trachyloban-19-oic acid isolated from Iostephane heterophylla (Asteraceae), was investigated on several photosynthetic activities in spinach thylakoids. The results indicated that this compound inhibited ATP synthesis and uncoupled electron transport, as well as basal and phosphorylating electron flow. Therefore, trachyloban-19-oic acid acts as Hill reaction inhibitor. This compound did not affect photosystem I activity but inhibited uncoupled photosystem II electron flow from H 2O to DCPIP, and has not effect on electron flow from H 2O to SiMo, indicating that the site of inhibition of this compound is at the level of Q A–Q B. Chlorophyll a fluorescence measurements confirm the behavior of this diterpene as Q A–Q B inhibitor, and in the other hand, this results indicate that a perturbation in the thylakoid membranes at the level of LHC II occurs.

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.

Inhibition of Photophosphorylation and Electron Transport by Flavonoids and Biflavonoids from Endemic Tephrosia spp. of Mexico

Abstract The biflavonoids tepicanol 1 and crassifolin 2 and the flavonoids elongatin 3, tephrobotin 4, tephroleocarpin 5, glabranin 6, and methylglabranin 7 were isolated from Tephrosia tepicana, T. crassifolia, T. viridiflora, T. abbottiae, T. leiocarpa, and T. madrensis, respectively, Leguminoseae-Papilionidae, a large tropical and subtropical genus, and their in vitro effects on different photosynthetic activities were investigated. All the compounds isolated, except 1 and 3, induced a concentration-dependent inhibition of photophosphorylation. Compounds 2 and 4-7 interfered with the energetic metabolism of plants at the level of photosynthesis with different mechanisms of action. Compounds 2, 4, and 6 behave as Hill reaction inhibitors, and compounds 5 and 7 act as energy transfer inhibitors (H+-ATPase inhibitor).

Photosynlhetic Inhibitory Activity of Dihydro-β-agarofurans Sesquiterpenes from Maytenus dislicha and Maytenus boaria (Celastraceae)

Abstract The effects of 9β-benzoyloxy-la, 2α, 6β, 8α, 15-pentaacetoxy-dihydro-β-agarofuran and 9β furoyloxy-1α, 6β, 8α-triacetoxy-dihydro-β-agarofuran, major phytogrowth inhibitors isolated from the aerial parts of Maytenus disticha (Celastraceae) and seeds of Maytenus boaria (Celastaraceae), respectively, on different photosynthetic activities of isolated spinach chloroplasts have been investigated. Photophosphorylation and electron transport (basal, phosphorylating and uncoupled) were inhibited in a concentration dependent manner by both compounds, therefore acting as Hill reaction inhibitors. The site of action of these natural compounds was located in the span from P680 to QA. 9β-benzoyloxy-1,2,6,8,15-pentaacetoxydihydro-β-agarofuran was one order of magnitude more potent (I50 = 2.6 μм) than 9β-furoyloxy-1,6,8,-triacetoxydihydro-β-agarofuran, suggesting that the substitution at C-9 and the acetoxy groups at carbons 2 and 15 are important structural requirements for the displayed inhibitory activity.

Two Inhibition Targets by [Cr(2gb)3]3+ and [Co(2gb)3]3+ on Redox Enzymes of Spinach Thylakoids

[Cr(2gb)(3)]Cl(ZnCl(4)), [Cr(2gb)(3)]Cl(3), and [Co(2gb)(3)]Cl(3) were synthesized and characterized. Their chemical structures and the oxidation states of their metal centers remained unchanged in solution. The effects of these compounds, CrCl(3) and [Co(NH(3))(6)]Cl(3), on photosynthesis were investigated. The coordination compounds inhibit ATP synthesis and electron flow (basal, phosphorylating, and uncoupled), behaving as Hill reaction inhibitors. The target for [Cr(2gb)(3)]Cl(ZnCl(4)) is located at the Q(B) level. In contrast, the interaction sites of [Cr(2gb)(3)]Cl(3) and [Co(2gb)(3)]Cl(3) are located in the span from P(680) to Q(A) and at the b(6)f complex. Neither CrCl(3) nor [Co(NH(3))(6)]Cl(3) inhibited photosynthesis. The 100% inhibition on PS II of [Cr(2gb)(3)]Cl(ZnCl(4)) is explained in terms of a synergystic effect between the 2gb-chromium(III) coordination compound and the ZnCl(4)(2)(-) anion.

Effect of selected coumarins on spinach chloroplast photosynthesis.

Xanthyletin (1), 3-(1',1'-dimethylallyl)xanthyletin (2), and chalepensin (3), the major coumarins isolated from Stauranthus perforatus, inhibit ATP synthesis from water to methylviologen in spinach thylakoids in a concentration-dependent manner. At low concentration chalepensin (3) inhibits basal and phosphorylating electron flow from water to K(3)[Fe(CN)(6)] without affecting uncoupled electron flow but accelerating Mg(2+)-ATPase activity. Thus, at low concentration the compound behaves as an energy transfer inhibitor. However, at higher concentrations this coumarin acts as an uncoupler because it enhances basal and phosphorylating electron transfer. On the other hand, coumarins 1 and 2 act as Hill reaction inhibitors, although 2 exhibited also uncoupler properties because it induces stimulation of basal and phosphorylating electron flow from water to ferricyanide. The site of interference of xanthyletin was located at the b(6)f-PC level of the electron transport chain.

Interference by Nickel(II) Salts and Their 5-Methylimidazole-4-carboxylate Coordination Compounds on the Chloroplast Redox Chain

Abstract Nickel(II) salts and their coordination com pounds with ethyl 5-m ethylim idazole-4-carboxylate (emizco), [Ni(emizco)2Cl2], [Ni(emizco)2Br2], [Ni(emizco)2(H2O)2] (NO3)2 H2O · Ni(NO3)2, inhibit photosynthetic electron flow (basal, phosphorylating and uncoupled) and ATP-synthesis, therefore behave as Hill reaction inhibitors. Coordination compounds are more potent inhibitors than the salts. It was found that the target for NiCl2; NiBr2 and Ni(NO3)2 is at the b6f level. On the other hand, the complexes [Ni(Emizco)2Cl2], [Ni(Emizco)2Br2] and [Ni(emizco)2(H2O )2] (NO3)2H2O binding sites are located at QB(D1)-protein and b6f level. Therefore, they have a common inhibition site located at b6f avoiding the PQH2 oxidation. The QB inhibition site was corroborated by variable chlorophyll a fluorescence yield [V(j)]. The emizco ligand has no activity on photosynthetic electron flow.