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1.
In strong illumination, 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while P-700 turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undectectable. When 10muM N-methylphenazonium methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 muM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in P-700 turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline. When 10muM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), P-700 turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurrs by a mechanism unrelated to the generation of a phosphyorylation potential. With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.  相似文献   

2.
Using steady-state relaxation spectrophotometric technique a P700 component (t 12 ~20 ms) has been detected which was sensitized by low concentration (10?7M) DCMU in isolated broken chloroplasts of pea. The relative quantum yield of electron flux through DCMU-sensitized P700 was similar to that with methyl viologen or NADP as terminal electron acceptor and water as electron donor. Kinetic analysis showed that a small fraction (10%) of the total P700 reaction centers was sensitized by low DCMU.  相似文献   

3.
S. Köster  U. Heber 《BBA》1982,680(1):88-94
Upon illumination of suspensions of intact chloroplasts, fluorescence of 9-aminoacridine was quenched, light scattering was increased, chlorophyll fluorescence was decreased after an initial increase, and chloroplast ATPADP ratios were increased. The response of 9-aminoacridine fluorescence quenching and light scattering to light intensity, anaerobiosis and inhibition of electron transport by DCMU was similar to that shown by chloroplast ATPADP ratios. It is discussed under what conditions 9-aminoacridine fluorescence quenching or light scattering can be used to monitor changes in the phosphorylation state of the chloroplast adenylate system.  相似文献   

4.
An O2-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O2 evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O2 uptake activity. The preparation showed a chlorophyll ab ratio of 1.9 and a P-700chlorophyll ratio of 12400. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O2-evolving Photosystem II preparation.  相似文献   

5.
E. Tel-Or  W.D.P. Stewart 《BBA》1976,423(2):189-195
Isolated heterocysts of the N2-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll ah) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll ah). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.  相似文献   

6.
J. Haveman  P. Mathis 《BBA》1976,440(2):346-355
A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O2 evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays (t12 about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II (t12 about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.  相似文献   

7.
Hans J. Rurainski  Hans J. Hoppe 《BBA》1976,430(1):105-112
The heterotrophically grown, P-700-free mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or P-700 turn-over could not be detected.When grown mixotrophically, the mutant showed increasing P-700 activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good P-700 turn-over and reasonable rates of NADP reduction.The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of P-700 in linear electron transport.  相似文献   

8.
Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10–30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose.The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 μmol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains β-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N2 fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome b-559 ratio of 50 : 1. The Photosystem I particle is highly enriched in P-700, with a chlorophyll to P-700 ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.  相似文献   

9.
The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol → methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N′,N′-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.  相似文献   

10.
Addition of 1mM ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the ADPO ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O2 uptake or ADPO ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O2 uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O2 uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O2 uptake by replacing water as the photosystem II donor.  相似文献   

11.
R.L. Pan  S. Izawa 《BBA》1979,547(2):311-319
NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100–250 μequiv · h?1 · mg?1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 → Photosystem II → dimethylquinone reaction supports phosphorylation with a Pe2 ratio of 0.25–0.35 and proton uptake with H+e values of 0.67 (pH 8)–0.85 (pH 6). These are close to the Pe2 value of 0.3–0.38 and the H+e values of 0.7–0.93 found in parallel experiments for the H2O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I?, ferrocyanide).  相似文献   

12.
The membrane potential generated at pH 8.5 by K+-depleted and Na+-loaded Vibrioalginolyticus is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na+-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na+ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na+-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na+ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na+ chemical gradient.  相似文献   

13.
G. Renger  Ch. Wolff 《BBA》1976,423(3):610-614
In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of ≈ 1 μs. It has been found that X 320 is reduced by a flash in ? 1 μs. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.  相似文献   

14.
The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA820) in the microsecond time range. The extent of ΔA820 induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg2+ free medium (plus 15 mM K+) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll ab-protein complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg2+ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.  相似文献   

15.
Joseph T. Warden 《BBA》1976,440(1):89-97
A 300 μs decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 μs actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375±10 mV (pH = 7.5). These data suggest the assignment of the 300-μs decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171–1174 (1971)).  相似文献   

16.
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   

17.
The addition of ATP to thylakoids isolated from Chlorella vulgaris is shown to lead to a quenching of fluorescence originating from Photosystem II and phosphorylation of chlorophyll achlorophyll b light-harvesting protein (LHCP) directly analogous to that reported for higher-plant chloroplasts. The time courses of these two processes are shown to be identical. Parallel measurements of ATP-induced changes in the fluorescence properties of isolated algal thylakoids and light-driven (State 1 / State 2 changes) in whole cells strongly support the idea that LHCP phosphorylation plays an important role in State 2 adaptation under in vivo conditions.  相似文献   

18.
We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φf) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg2+ causes an increase in φf. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φf. The PMS triggered decrease in φf is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg2+ and Ca2+ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg2+-induced increase of φf, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg2+ causes any change in φf. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg2+-induced increase of φf. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (kh), whereas the Mg2+ effect is due to a decrease in the rate of energy transfer (kt), between the two photosystems.4. From the relative ratio of φf with DCMU and DCMU plus Mg2+, we have calculated kt (the rate constant of energy transfer between Photosystems II and I to be 4.2·108 s?1, and φt (quantum yield of this transfer) to be 0.12.  相似文献   

19.
The phosphorylation of five E.gracilis thylakoid membrane polypeptides was studied, in isolated chloroplasts. Using [32P] labelling, in the light, we found that phosphorylation was inhibited by ethanol and DCMU. Inhibition curves were characteristic of photosynthetic inhibition. [γ-32P] ATP labelling was used to distinguish between two groups of phosphoproteins: the first one, includes protein I, II, V which require only ATP for phosphorylation while the second one includes protein III and IV whose phosphorylation is light-requiring. Phosphorylation of protein III and IV was inhibited by CCCP, NH4Cl and DCMU, and was reversible in the dark.  相似文献   

20.
In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (g ≈ 2.0045, ΔHpp ≈ 19 G). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination (λ < 680 nm and occurs stoichiometrically in a 1 : 1 ratio with P-700+. The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the t12 for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol?1. Inhibition of electron transport through Photosystem II by o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, P-680+.  相似文献   

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