INHIBITION OF ELECTRON TRANSPORT IN CHLOROPLASTS BY CHAOTROPIC AGENTS AND THE USE OF MANGANESE AS AN ELECTRON DONOR TO PHOTOSYSTEM II*

Abstract— Incubating spinach chloroplasts with various chaotropic agents results in inhibition of photosynthetic electron transport between water and Photosystem II similar to the inhibition caused by washing chloroplasts with a high concentration of Tris buffer. Partial restoration of NADP photoreduction and fluorescence of variable yield is achieved by adding hydroquinone or Mn²⁺, either of which donates electrons to Photosystem II in the inhibited chloroplasts. The inhibitory treatments cause the release of Mn from its bound state in the chloroplast, thus allowing the measurement of the ESR signal of Mn²⁺. The ESR measurement is used to follow the photooxidation of Mn²⁺ as it donates electrons to photosystem II.     

NEW PHOTOSYSTEM I ELECTRON ACCEPTORS: IMPROVEMENT OF HYDROGEN PHOTOPRODUCTION BY CHLOROPLASTS *

Abstract Four novel electron carriers (two zwitterionic bipyridyls, dicarboxyl colbalticinium and sodium metatungstate), which are negatively charged in their reduced form, have been tested as photo-system I acceptors and as mediators of H₂ evolution. Measurements of O₂ uptake, anaerobic photoreduction rates and stationary concentrations of reduced species under continuous illumination indicate that Coulombic interactions control the electron transfer between the photosynthetic membrane and the mediators. Both rates of forward transfer and back reaction (electron cycling) seem to depend on the charge of the electron carrier. The low concentration of anionic species in the diffuse layer associated with the membrane could explain our results. Hydrogen evolution rates obtained with these four mediators used as electron relays between the photosynthetic membrane and colloidal platinum catalyst are higher than with methylviologen. This improvement of the conversion efficiency parallels the high steady state accumulation of reduced carriers favoured by their negative charge. It is also shown that these synthetic mediators, except metatungstate, are able to evolve hydrogen with an hydrogenase isolated from Desulfovibrio desulfuricans.     

THE LIGHT DEPENDENT QUENCHING OF CHLOROPLAST FLUORESCENCE BY COFACTORS OF CYCLIC ELECTRON FLOW IN PHOTOSYSTEM I

Abstract—When 3–(3',4'-dichlorophenyl)-1,1-dimethylurea poisoned, intact thylakoids of isolated chloroplasts are illuminated in salt free suspension media, N -methylphenazinium cations (MP⁺) are reversibly taken up. Simultaneously, the chlorophyll fluorescence is reversibly lowered. When inorganic salts in the reaction medium provide membrane permeant charge balancing ions, the extent of the MP⁺ association with the thylakoids is strongly increased, but the fluorescence lowering is hardly affected. lonophoretically active agents inhibit specifically the salt dependent increment of the MP⁺ interaction with the thylakoids, but have only insignificant effects on the fluorescence lowering provided the experimental conditions do not allow the formation of a proton gradient across the thylakoid membrane. On the basis of these results, and of data obtained from comparative studies with other cofactors of cyclic electron transport in PS I, it is suggested that the 'energy dependent' fluorescence lowering is linked to a binding of cationic cofactors to nucleophilic sites in or at the thylakoid membrane. Such sites appear to become exposed in the wake of a light dependent transport of the cofactor, or of protons, into the thylakoid.     

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.     

ALTERNATIVE MEASURES OF PHOTOSYSTEM II ELECTRON TRANSFER INHIBITION IN ANTHRAQUINONE-TREATED CHLOROPLASTS

We have previously used chlorophyll fluorescence measurements at Fmax conditions (i.e. with Photosystem II electron acceptor QA reduced) to monitor the action of 9,10-anthraquinones on photosynthetic electron transport in plant chloroplasts. The present investigation employs two additional techniques to characterize the extent of electron transport inhibition induced by the addition of substituted anthraquinones to the suspending medium of spinach chloroplasts. Results are presented for spectrophotometric assays of the rate of electron transfer to an exogenous electron acceptor, 2,6-dichloroindophenol (DCIP) and for electrochemical determinations of the rate of oxygen evolution in anthraquinone-treated chloroplasts. In general, amino-substituted anthraquinones are ineffective inhibitors, maintaining electron transfer rates to DCIP at levels ranging from 50 to 90% of normal rates and yielding rates of O2 evolution averaging at 70% of the rate in untreated chloroplasts. In contrast, hydroxy-substituted anthraquinones efficiently block Photosystem II electron transport, resulting in low rates of DCIP photoreduction ranging from 0 to 20% of normal values and reducing O2 evolution rates to an average of 30% of the rate observed for untreated chloroplasts. Relative rates of DCIP photoreduction for anthraquinone-treated chloroplasts show a strong linear correlation with the reported relative Fmax chlorophyll fluorescence intensities. Relative O2 evolution rates are observed to correlate with the Stern-Volmer fluorescence quenching parameter Ksv. We suggest that slight differences in the extent of inhibitory activity of an anthraquinone as measured by the three techniques are consistent with certain known Photosystem II heterogeneities. The similarities in relative rankings of inhibitory effects for the 9, 10-anthraquinones, however, demonstrate that the three techniques employed (measurements of Fmax chlorophyll fluorescence, DCIP photoreduction rates, and O2 evolution rates) are alternative assays of anthraquinone-induced Photosystem II electron transport inhibition.     

A LASER FLASH SPECTROSCOPY STUDY OF THE KINETICS OF ELECTRON TRANSFER FROM SPINACH PHOTOSYSTEM I TO SPINACH AND ALGAL FERREDOXINS

Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from P700 in Photosystem I (PSI)-enriched particles from spinach to the ferredoxins from spinach and the green alga Monoraphidium braunii. Very similar behavior for the interaction of both ferredoxins with the PSI complex was observed, although the algal ferredoxin appears to be slightly more effective as an electron acceptor. For both proteins, a non-linear protein concentration dependence of the rate constant for reduction was obtained, indicating complex formation preceding electron transfer. Estimates of 3 times 10⁷M^?1 s^?1 and 140–180 s^?l were obtained from these data for the second order rate constants for complex formation, and the limiting first order rate constants for electron transfer, respectively. At neutral pH, a biphasic dependence of the rate constant for ferredoxin reduction on the concentration of NaCl or MgCl₂ was observed. This was interpreted in terms of the electrostatic interactions which occur between ferredoxin and the PSI membrane. In addition, magnesium cations appear to play a specific role in the interaction between PSI and ferredoxin. Thus, the addition of these ions under optimal conditions induces a 6-f-old increase in the electron transfer reaction rate constant, compared with a 2-f-old increase in the presence of an optimal amount of NaCI. This cannot be explained as arising from ionic strength effects. To our knowledge, this is the first time that a direct measurement of the rate constant for the reduction of ferredoxin by the PSI complex has been reported.     

乳状液膜分离肌酐的研究

以液体石蜡为膜相、以氧化环糊精为内相捕捉剂制备一种新的乳状液膜，尝试运用其清除分离肌酐。实验发现当外相肌酐溶液浓度为1．3g/L，载体月桂醛含量为5％，内相氧化环糊精浓度为10％时，乳状液膜吸附容量为22．5mg／g。内相氧化环糊精的浓度对单位吸附量起决定作用。此种吸附剂对氨基酸吸附量仅为肌酐的1／10，这为其在含大量氨基酸的人体环境中有效的清除肌酐提供了优良的选择性。     

酰基二茂铁在液相中的电子传递性质

酰基二茂铁在液相中的电子传递性质蒋朝阳邰子厚＊季斌（南京大学配位化学国家重点实验室，南京２１００９３）关键词：酰基二茂铁三相氧化还原体系电子传递二茂铁及其衍生物在生物模拟和光电材料中有着极高的应用价值［１－４］。本文合成了一系列的酰基二茂铁ＦｃＸ（Ｘ...     

EFFECTS OF PHOTOSYSTEM II INHIBITORS ON ELECTRON PARAMAGNETIC RESONANCE SIGNAL II OF SPINACH CHLOROPLASTS

Abstract— In isolated spinach chloroplasts the light-induced electron paramagnetic resonance signal (signal II) associated with the oxygen evolving photosystem (photosystem II) decays slowly and incompletely in the dark. Tris-washing, hydroxylamine, or carbonylcyanide m -chlorophenylhydrazone (CCCP) enhance the decay of signal II, which can still be induced by red (645 nm) but not by far-red (735 nm) radiation. Although 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) alone has no effect on signal II, it blocks the induction of signal II in the presence of hydroxylamine or CCCP. These data suggest that signal II is an indicator of an oxidized intermediate on the water-splitting side of photosystem II.     

PICOSECOND TRANSIENT BEHAVIOR OF REACTION-CENTER PARTICLES OF PHOTOSYSTEM I ISOLATED FROM SPINACH CHLOROPLASTS. ENERGY AND ELECTRON TRANSFER UPON SINGLE AND MULTIPLE PHOTON EXCITATION

Abstract— Both [15-¹³C] and [14-¹³C] all-trans-retinals were synthesized. Bacteriorhodopsin containing [14-¹³C]retinal as a chromophore, when solubilized with octyl-β-D-glucoside, showed characteristic resonances at 125 and 118 ppm from tetramethyl silane. The former was assigned to the signal from free retinal and the latter from protonated Sehiff base. When the bacteriorhodopsin was denatured in sodium dodecyl sulfate, the signal at 118 ppm disappeared, while the signal at 125 ppm rather increased.
In the case of bacteriorhodopsin containing [15-¹³C]retinal, when solubilized with Triton X-100, a characteristic resonance at 169 ppm was distinguishable as a shoulder peak superimposed on the broad signal of carbonyl carbons and it was assigned to the signal from the protonated Sehiff base. The other signal observed at 191 ppm was from free retinal.
These results suggested that the Sehiff base of bacteriorhodopsin is protonated in the dark.     

PROTEIN COMPOSITION OF SPINACH CHLOROPLASTS AND THEIR PHOTOSYSTEM I AND PHOTOSYSTEM II SUBFRAGMENTS*

Abstract— The proteins of spinach chloroplasts and their subfragments containing photosystem I and photosystem II, obtained by Triton X-100 treatment or French-pressure rupture, were separated by sodium dodecyl sulfate (SDS)-acrylamide electrophoresis at pH 7·0 in phosphate buffer. The individual protein bands were identified where possible by comparing them with known, isolated and characterized proteins from chloroplasts, and their molecular weights were determined. The protein composition of the chloroplast fragments were correlated to the functional properties of these fragments. Distinct patterns were obtained for photosystem I and photosystem II particles. The major protein of photosystem II is expressed in the 23 kilodalton range and photosystem I proteins seem to be clustered mainly in the 50–70 kilodalton range.     

STRUCTURE OF PHOTOSYSTEM I AND PHOTOSYSTEM II OF PLANT CHLOROPLASTS*,†,§

Abstract— The action of Triton X-100 upon photosynthetic membranes which are devoid of carotenoids produces a small Photosystem I particle (HP700 particle) which is active in N ADP photoreduction and has a [Chl]/[P700] ratio of 30. The properties of the HP700 particle indicate that it is a reaction center complex which is served by an accessory complex containing the additional light-harvesting chlorophyll of Photosystem I as well as the cytochromes and plastoquinone. When Photosystem II particles obtained by the action of Triton X-100 are further washed with a solution 0.5 M in sucrose and 0.05 M in Tris buffer (pH 8.0), chlorophyll-containing material is released. After centrifugation, the supernatant contains about 1 per cent of the chlorophyll and contains three types of particles which can be separated by sucrose density gradient centrifugation. One of these particles, designated TSF-2b, has the same pigment composition as the original Photosystem II fragment, contains cytochrome 559, and shows Photosystem II activity (DCMU-sensitive diphenylcarbazide-supported photoreduction of 2,6-dichlorophenolindophenol). The other two particles (TSF-2a and TSF-2a′) have a [Chl a]/[Chl b] ratio of 8, have a low concentration of xanthophylls, and show a [Chl]/[Cyt 5591 ratio of about 20. Only the TSF-2a particle is active in the Photosystem II reaction described above. On the basis of these data, it is proposed that the Photosystem II unit consists of a reaction center complex which contains Chl a, Cyt 559, and an acceptor for the photochemical reaction. The reaction center complex would be served by an accessory complex which contains the light-harvesting pigments, Chl a. Chi b, and xanthophyils.     

THE MULTIPLE PIGMENT-PROTEINS OF THE PHOTOSYSTEM I ANTENNA*

A photosystem I (PS I) holocomplex was obtained from barley by ultracentrifugation of PS I-enriched stroma lamellae on sucrose gradients. Further solubilization with glycosidic surfactants followed by Deriphat-poly-acrylamide gel electrophoresis (PAGE) fractionated the holocomplex into its core complex (CC I) and individual light-harvesting I (LHC I) pigment-protein subcomplexes. The LHC I contains chlorophyll a, all of the chlorophyll A of PS I and xanthophylls but no carotenes. Sodium dodecylsulfate PAGE analysis of the subcomplexes shows that barley LHC I is composed of at least five apoproteins having sizes between 11 and 24 kDa. Isolation of a 17 kDa LHC Ic component by Deriphat-PAGE shows it to be a photosynthetic pigment-protein. Room-temperature absorption spectra indicate that LHC Ic is enriched in chlorophyll a in comparison to the LHC Ia and Ib components. The LHC Ic apoprotein is shown to be distinct from the subunit III and IV polypeptides of CC I. Analysis of PS I fractions obtained from sucrose gradients as well as from Deriphat-PAGE indicates that in higher plants an oligomeric structure of the PS I entity exists in vitro.     

LIGHT DRIVEN TRANSPORT OF BILIRUBIN THROUGH A BULK LIQUID MEMBRANE

Abstract— Hexadecyltrimethylammonium bromide was used as a carrier in a bulk liquid membrane to construct a model system for the rapid excretion of bilirubin during phototherapy of jaundice. When bilirubin in the donor phase was irradiated its transport through the bulk membrane increased by about two orders of magnitude compared to the dark. As production of the photodiastereomers of bilirubin and their thermal reversion are fast processes compared to the carrier mediated transport, active transport against a bilirubin concentration gradient driven by the photoisomerisation reaction is observed. The kinetics of this novel transport system is discussed and approximated by a recursion algorithm.     

TEMPERATURE DEPENDENCE OF PHOTOSENSITIZED ELECTRON TRANSPORT ACROSS LIPID VESICLE WALLS

Abstract— An overall Arrhenius activation energy of 117 plusmn; 19 kJ/mol (28.0 plusmn; 4.5 kcal/mol) has been measured for photosensitized electron transport across phospholipid vesicle walls in the temperature range 18–38°C. A dynamic model for the overall process is proposed which accounts for the parabolic growth curves of the kinetically probed species, heptyl viologen radical. The temperature dependence of the initial quantum yield derived from these curves, and of fluorescence quenching behavior of the ruthenium tris-bipyridyl photosensitizer, is used to estimate an activation energy of 67 plusmn; 21 kJ/mol(16 plusmn; 5 kcal/mol) for the electron transport step itself. The activation energy for the co-transport of charge compensating ions is estimated to contribute no more than 4.6 kJ/mol to this energy.     

THE STRUCTURE OF CHLOROPHYLL RC I, A CHROMOPHORE OF THE REACTION CENTER OF PHOTOSYSTEM I

Abstract— Chlorophyll RC I is a particular chlorophyll of photosystem I common to all organisms with oxygenic photosynthesis. Its structure could be revealed by'H-NMR, FTIR, neutron activation analysis, complemented by plasma desorption mass spectrometry data. It has been identified as 13'-hydroxy-20-chloro-Chl a . Two stereoisomers of Chl RC I have also been isolated and identified. Evidence is presented that chlorination of the pigment does not occur during extraction and that artefacts due to impurities are ruled out.     

PHOTOCHEMICAL GENERATION OF REDUCED QUINONE CATALYSTS OF PHOTOSYSTEM I CYCLIC PHOTOPHOSPHORYLATION ACTIVITY

When reaction mixtures containing 9,10-anthraquinone-2-sulfonate and chloroplasts poisoned with 3–(3,4-dichlorophenyl)-l, l′-dimethylurea were illuminated with white light, photosystem I-catalyzed cyclic photophosphorylation was observed. Illumination of identical reaction mixtures with red light produced no ATP synthesis. This phenomenon is due to photoreduction of the anthraquinone which is supported by the electron donor activity of Tricine buffer. The photoreduction reaction was used to generate reduced catalysts (anthraquinone sulfonate, menadione bisulfite) of photosystem I cyclic photophosphorylation activity. The rates of ATP synthesis obtained by this method (250–300 μmol/h-mg chlorophyll) indicate that sulfonated quinones are efficient mediators of cyclic electron transport around photosystem I. Although the activity catalyzed by these compounds is highly sensitive to dibromothymoquinone, very little decrease in activity is observed with antimycin A.     

环烷酸类载体液膜体系对金属离子传输机理的研究

本文先在大块液膜体系中以环已烷甲酸为载体,通过正交设计,系统地研究了各种因素对希土离子输送作用的影响规律。比较了相同条件下RE~(3+)(希土)同Na~+,NH_4~+、Ca~(2+)和Fe~(3+)等离子的输送作用。发现在适当条件下,无皂化的羧酸载体对RE~(3+)离子具有良好的输送效果,同时证实,羧酸输送RE~(3+)离子是通过三个H~+离子与一个RE~(3+)离子的交换,而当载体皂化时,皂化的载体直接与接收相H~+离子发生交换,从而降低了羧酸对RE~(3+)离子的输送和分离效果。 在大块液膜研究的基础上,建立了一个以无皂化的环烷酸为载体的乳状液膜体系,从模拟离子矿的硫酸铵浸出液中萃取希土,通过正交试验确定了最优的液膜萃取条件,希土萃取率达96％以上,富集度30～40倍。     

CHARGE TRANSFER FROM PHOTOSYSTEM I TO THE CYTOCHROME b/f COMPLEX: DIFFUSION AND MEMBRANE LATERAL HETEROGENEITY

Abstract— We discuss here the minimum requirements for diffusion of a charge carrier between appressed and stroma-exposed membrane regions of chloroplasts based on recent models of the thylakoid membrane and flash-induced kinetic data. We have investigated the kinetics of the transfer of a positive charge from photosystem I to the cytochrome b/f complex in spinach chloroplasts by measuring the light-induced oxidation of cytochrome f. The rate and extent of cytochrome f oxidation were measured spectrophotometrically using either long actinic flashes that induced several turnovers of photosystem I or short actinic flashes that induced a single turnover of photosystem I. In the long actinic flashes, in the electron transfer reaction from water to methyl viologen, we observed the rapid oxidation of all of the cytochrome f present in the membrane. The half-time of the oxidation was 1.0 ± 0.1 ms. The total amount of the cytochrome was determined by chemical difference spectra to be one molecule of cytochrome f per 650 – 30 chlorophyll molecules. Using short actinic flashes we studied the photosystem I-driven electron transfer reaction from duroquinol to methyl viologen in the presence of the inhibitor 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Under these conditions a single turnover flash induced the oxidation of 62 ± 5% of cytochrome f with a half-time of 240 ± 30 μs. An Arrhenius plot of the temperature dependence of the cytochrome f oxidation rate revealed an activation energy between 16 and 21 kJ/mol, a value consistent with a diffusion-controlled reaction. These kinetic data are considered in the context of two models of the thylakoid membrane.