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1.
Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made 1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators. 2. The light- and dithioerythritol-dependent Mg2+-ATPase was also inhibited by the lipophilic chelators. 3. Electron transport through either partial reaction. Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors. The electron transport inhibition site is localized in the region of platoquinone leads to cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near + 290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f. just where our data suggests there to be a functional metalloprotein. 4. Some of the lipophilic chelators induce H+ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1-3 mM nonanedione does not induce significant H+ leakiness, while inhibiting ATP formation and the Mg2+-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H+ uptake. 5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.  相似文献   

2.
Valinomycin inhibits the coupled portion of whole-chain noncyclic electron transport and phosphorylation and also inhibits the anaerobic menadione-dependent cyclic phosphorylation. Both of these electron transport systems are thought to involve plastoquinone. Electron transport and phosphorylation in various photosystem I and II partial reactions not involving plastoquinone or phosphorylation using other cyclic cofactors that are not dependent on plastoquinone were relatively insensitive to valinomycin. Single-beam ultraviolet spectrophotometric measurements showed that valinomycin decreased the apparent first-order rate constant for plastohydroquinone oxidation by more than twofold and had no observable effect on the reduction rate constant. The valinomycin inhibitions did not require K+ addition to the media. However, it was shown that extensive washing or dialysis did not reduce the chloroplast endogenous K+ content below 0.1 μmol K+/mg of chloroplasts.  相似文献   

3.
(1) Using asolectin (mixed soybean phospholipids) liposomes, extra lipid, with or without additional plastoquinone, has been introduced into isolated thylakoid membranes of pea chloroplasts. (2) Evidence for this lipid enrichment was obtained from freeze-fracture which indicated that a decrease in the numbers of EF and PF particles per unit area of membrane occurred with increasing lipid incorporation. The decrease was not due to loss of integral membrane polypeptides as judged by assay of cytochrome present or SDS-polyacrylamide gel electrophoresis of lipid-enriched membrane fractions. Moreover, the enrichment procedure did not lead to extraction of low molecular weight lipophilic membrane components or of thylakoid membrane lipids. (3) The introduction of phospholipids into the membrane affected steady-state electron transport. Inhibition of electron transport was observed when either water (Photosystem (PS) II + PS I) or duroquinol (PS I) was used as electron donor with methyl viologen as electron acceptor, and the degree of inhibition increased with higher enrichment levels. Introduction of exogenous plastoquinone with the additional lipid had little effect on whole-chain electron transport, but caused an increase in the 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-sensitive rate of PS I electron transport. The inhibition was also detected by flash-induced oxidation-reduction changes of cytochrome f.  相似文献   

4.
Barr R  Crane FL 《Plant physiology》1976,57(3):450-453
The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H2O→methyl viologen, H2O→silicomolybdic acid H2O→ferricyanide, and H2O→dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H2O→silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition.  相似文献   

5.
《BBA》1985,809(2):167-172
Uncouplers have been previously observed to relieve appreciably the inhibition of photosynthetic electron transport from water to NADP+ by the plastoquinone analogues, dibromothymoquinone (DBMIB) and dinitrophenyl ether of iodonitrothymol (DNP-INT). These results were now extended by demonstrating that the reversal by uncouplers of DBMIB and DNP-INT inhibition occurred under conditions when the uncouplers did not stimulate or inhibit NADP+ reduction in control treatments without the plastoquinone analogues. Since effects of uncouplers on photosynthetic electron transport depend on external pH, we determined for each of the four uncouplers, gramicidin, nigericin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and SF 6847 (a ditertiary phenol derivative) its effect on oxygenic electron transport (H2O to NADP+) over a range of external pH from 6.7 to 8.7. The effect of each uncoupler on counteracting the inhibition of DBMIB and DNP-INT was then measured at its crossover external pH at which the uncoupler had little or no effect on electron transport in the uninhibited controls. Under these controlled conditions, uncouplers increased the rate of plastoquinone-inhibited electron transport, in some cases by almost 300%. To explain these results, a role for plastoquinone in processing protons released by the oxidation of water is postulated.  相似文献   

6.
Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP+. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the Pe2 ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.  相似文献   

7.
Wolfgang Haehnel 《BBA》1982,682(2):245-257
Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl2, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700+ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700+ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b6 - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700+. The P-700 kinetics indicate an electron transfer from the cytochrome b6 - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.  相似文献   

8.
Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl2 (less than 1 μmole Hg2+/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl2 concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl2 to these sites.  相似文献   

9.
David B. Knaff 《BBA》1973,325(2):284-296
1. Cytochrome f (λmax = 554 nm, Em = +0.35 V) and cytochrome b558 (λmax = 558 nm, Em = +0.35 V) were photooxidized by Photosystem I and photoreduced by Photosystem II in a cell-free preparation from the blue-green alga Nostoc muscorum. The steady-state oxidation levels of both cytochromes were affected by noncyclic electron acceptors and by inhibitors of noncyclic electron transport. These results are consistent with the hypothesis that the mechanism of NADP reduction by water involves a Photosystem II and a Photosystem I light reaction operating in series and linked by a chain of electron carriers that includes cytochrome f and cytochrome b558.2. Phosphorylation cofactors shifted the steady-state of cytochrome f to a more reduced level under conditions of noncyclic electron transport but had no effect on cytochrome b558. These observations suggest that the noncyclic phosphorylation site lies before cytochrome f (on the Photosystem II side) and that cytochrome f is closer to this site than is cytochrome b558.3. A Photosystem II photoreduction of C550 at 77 °K was observed, suggesting that in blue-green algae, as in other plants, C550 is closely associated with the primary electron acceptor for Photosystem II. A Photosystem I photooxidation of P700 at 77 °K was observed, consistent with P700 serving as the primary electron donor of Photosystem I.  相似文献   

10.
Yocum CF 《Plant physiology》1977,60(4):597-601
A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH2OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.  相似文献   

11.
The light-dependent quenching of 9-aminoacridine fluorescence was used to monitor the state of the transthylakoid proton gradient in illuminated intact chloroplasts in the presence or absence of external electron acceptors. The absence of appreciable light-dependent fluorescence quenching under anaerobic conditions indicated inhibition of coupled electron transport in the absence of external electron acceptors. Oxygen relieved this inhibition. However, when DCMU inhibited excessive reduction of the plastoquinone pool in the absence of oxygen, coupled cyclic electron transport supported the formation of a transthylakoid proton gradient even under anaerobiosis. This proton gradient collapsed in the presence of oxygen. Under aerobic conditions, and when KCN inhibited ribulose bisphosphate carboxylase and ascorbate peroxidase, fluorescence quenching indicated the formation of a transthylakoid proton gradient which was larger with oxygen in the Mehler reaction as electron acceptor than with methylviologen at similar rates of linear electron transport. Apparently, cyclic electron transport occured simultaneously with linear electron transport, when oxygen was available as electron acceptor, but not when methylviologen accepted electrons from Photosystem I. The ratio of cyclic to linear electron transport could be increased by low concentrations of DCMU. This shows that even under aerobic conditions cyclic electron transport is limited in isolated intact chloroplasts by excessive reduction of electron carriers. In fact, P700 in the reaction center of Photosystem I remained reduced in illuminated isolated chloroplasts under conditions which resulted in extensive oxidation of P700 in leaves. This shows that regulation of Photosystem II activity is less effective in isolated chloroplasts than in leaves. Assuming that a Q-cycle supports a H+/e ratio of 3 during slow linear electron transport, vectorial proton transport coupled to Photosystem I-dependent cyclic electron flow could be calculated. The highest calculated rate of Photosystem I-dependent proton transport, which was not yet light-saturated, was 330 mol protons (mg chlorophyll h)–1 in intact chloroplasts. If H+/e is not three but two proton transfer is not 330 but 220 mol (mg Chl H)–1. Differences in the regulation of cyclic electron transport in isolated chloroplasts and in leaves are discussed.  相似文献   

12.
Rita Khanna  T. Wydrzynski 《BBA》1977,462(1):208-214
Using artificial electron donors and acceptors, it is shown here that the major HCO3? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.  相似文献   

13.
With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K+. NH4Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH4Cl plus valinomycin but not by either alone. K+ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH4Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg2+-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H+/e ratio. These contrasting activities in chloroplasts are not easily explained.Contribution number 389 of the Charles F. Kettering Research Laboratory.  相似文献   

14.
15.
R.C. Jennings  G. Forti 《BBA》1975,396(1):63-71
Under conditions in which the Photosystem II quencher is rapidly reduced upon illumination, either after a preillumination or following treatment with dithionite, the fluorescence-induction curve of intact spinach chloroplasts (class I type) displays a pronounced dip. This dip is probably identical with that observed after prolonged anaerobic incubation of whole algal cells (“I-D dip”). It is inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea and occurs in the presence of dithionite, sufficient to reduce the plastoquinone pool. It is influenced by far red light, methylviologen, anaerobiosis and uncouplers in a manner consistent with the interpretation that it represents a photochemical quenching of fluorescence by an electron transport component situated between the Photosystem II quencher and plastoquinone. Glutaraldehyde inhibition may indicate that protein structural changes are involved.  相似文献   

16.
R. Tiemann  G. Renger  P. Gräber  H.T. Witt 《BBA》1979,546(3):498-519
The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H+ + e?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H+ uptake and the internal H+ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H+ uptake, the electron translocation through the plastoquinone pool and the internal H+ release (corrected for H+ release due to H2O oxidation) is valid (pHout = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H+ + e?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.  相似文献   

17.
The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP+. Magnesium ions were required not only for light-induced electron transport from water to NADP+ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg2+ lost the capacity to photoreduce NADP+ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP+ by DCIPH2 was independent of Mg2+ (or Cl?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl2 produced a strong inhibition of NADP+ photoreduction with DCIPH2 without significantly affecting the rate of NADP+ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP+ with different electron donors are discussed.  相似文献   

18.
When chloroplasts are aged for 5 min at pH 9.6, or are exposed to uncouplers at pH 8.5–9.0, electron flow from water to Hill acceptors is inhibited. Both treatments induce rapid millisecond dark decay of delayed light emission. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-sensitive electron transport through Photosystem II can be regenerated in both types of inhibited chloroplasts by the artificial electron donor, 1,5-diphenylcarbohydrazide. Neither treatment inhibits electron flow through Photosystem I. Uncouplers at alkaline pH, when added in the light, are less effective in producing the inhibition than when added in the dark. These results are interpreted as indicating inhibition of the oxygen-evolving apparatus by alkaline intrathylakoid pH.  相似文献   

19.
The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1.Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.  相似文献   

20.
Delayed light emission from the Triton-fractionated Photosystem II subchloroplast fragments (TSF-IIa) was measured between 0.5 and 10 ms after the termination of illumination. The delayed light emission was diminished by Photosystem II inhibitors, DCMU and o-phenanthroline, which act between the reduced primary acceptor and the plastoquinone pool.Secondary electron donors to Photosystem II, diphenylcarbazide, phenylenediamine, Mn2+, and ascorbate inhibited delayed light emission. Secondary electron acceptors such as ferricyanide, dichlorophenol indophenol, and dimethyl benzoquinone enhanced delayed light emission. The addition of secondary electron acceptors to TSF-IIa particles containing Mn2+ restored delayed light emission to almost the control level. The plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl p-benzoquinone, increased delayed light emission at low concentrations but decreased it at higher concentrations. Silicomolybdate enhanced the delayed light emission of TSF-IIa particles markedly, and reversed the inhibition by DCMU. Silicomolybdate showed a similar stimulatory effect on the delayed-light intensity in broken spinach chloroplasts at shorter times after the termination of illumination. Carbonyl cyanide m-chloro (or p-trifluoromethoxy) phenylhydrazones inhibited the delayed light emission, but NH4Cl had no effect.  相似文献   

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