Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S₂Q_B ^- (B band) and S₂Q_A ^– (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of Q_A ^- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block Q_A to Q_B electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced Q_B. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the Q_B/DCMU binding site. The preferential loss of dark stable Q_B ^- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.Abbreviations Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A first quinone electron acceptor of PS II - Q_B second quinone electron acceptor of PS II - Tyr-D accessory electron donor of PS II - S₀-S₄ charge storage states of the water-oxidizing complex     

Further characterization of a photosystem ii particle isolated from spinach chloroplasts by triton treatment delayed light emission

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.     

Identification of a 32–34-kilodalton polypeptide as a herbicide receptor protein in Photosystem II

Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32–34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a ¹⁴C-labeled photoaffinity triazine.In Photosystem II particles depleted of the 32–34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.     

Modulation of herbicide-binding by the redox state of Q400, an endogenous component of Photosystem II

Preincubation of isolated chloroplasts with ferricyanide, prior to addition of DCMU, unmasks a high-potential electron acceptor (Q₄₀₀) in Photosystem II that acts as an additional quencher and prolongs the fluorescence induction curve in the presence of DCMU (Ikegami, I. and Katoh, S. (1973) Plant Cell Physiol. 14, 829–836). This study confirms that Q₄₀₀ is endogenous to Photosystem II and is not a bound ferricyanide, and several new characteristics of this high potential acceptor are established. (a) It is accessible to ferricyanide even in the presence of DCMU. The rate of oxidation, however, is very slow, consistent with access only via Q_A. Accessibility may be enhanced by magnesium, reminiscent of the oxidation of Q⁻_A by ferricyanide. (b) Oxidation of Q₄₀₀ drastically suppresses the binding of DCMU at neutral and alkaline pH. Below pH 6, however, DCMU binding is essentially normal. The pH dependence of DCMU binding is consistent with the known pH dependence of the redox midpoint potential of Q₄₀₀. (c) Binding of many other inhibitors of Q_A-to-Q_B electron transfer is much less affected or even completely unaffected. These results have implications for current notions of herbicide binding and may also bear on the origin of slow phases of fluorescence induction in the presence of DCMU.     

In vitro reassociation of phycobiliproteins and membranes to form functional membrane-bound phycobilisomes

A membrane-bound phycobilisome complex has been isolated from the cyanobacterium Fremyella diplosiphon grown in green light, thus containing phycoerythrin in addition to phycocyanin and allophycocyanin. The complex was dissociated by lowering the salt concentration. In the mixture obtained, no energy transfer from phycoerythrin to chlorophyll (Chl) a was observed. Reassociation of the phycobiliproteins and membrane mixture was carried out by a gradual increase of the salt concentration. The complex obtained after reassociation was characterized by polypeptide composition, absorbance and fluorescence emission spectra and electron microscopy. These analyses revealed similar composition and structure for the original and reconstituted membrane-bound phycobilisomes. Fluorescence emission spectra and measurements of Photosystem II activity demonstrated energy transfer from phycoerythrin to Chl a (Photosystem II) in the reconstituted complex. Reassociation of mixtures with varying phycoerythrin / Chl ratio showed that the phycobiliprotein concentration was critical in the reassociation process. Measurements of the amount of phycobilisomes reassociated with the photosynthetic membrane did not show saturation of binding when increasing the phycobiliprotein concentration. The ratio phycoerythrin / Chl a in the native complex was 7:1 (mg / mg). When the phycobiliprotein concentration was increased during the reassociation process, a ratio of 13–15 mg phycoerythrin / mg Chl a could be obtained. Under these conditions, only part of the phycobilisomes attached to the thylakoids was able to transfer energy to Photosystem II.     

Reduction kinetics of the photo-oxidized chlorophyll a+II in the nanosecond range: Measurements of the absorption changes at 688 nm under repetitive flash excitation

The 688 nm absorption changes (ΔA₆₈₈), indicating the photochemical turnover of chlorophyll a_II (Chl a_II) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a⁺_II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a⁺_II becomes re-reduced in the microsecond range.     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

A viewpoint: Why chlorophyll <Emphasis Type="Italic">a</Emphasis>?

Chlorophyll a (Chl a) serves a dual role in oxygenic photosynthesis: in light harvesting as well as in converting energy of absorbed photons to chemical energy. No other Chl is as omnipresent in oxygenic photosynthesis as is Chl a, and this is particularly true if we include Chl a ₂, (=[8-vinyl]-Chl a), which occurs in Prochlorococcus, as a type of Chl a. One exception to this near universal pattern is Chl d, which is found in some cyanobacteria that live in filtered light that is enriched in wavelengths >700 nm. They trap the long wavelength electronic excitation, and convert it into chemical energy. In this Viewpoint, we have traced the possible reasons for the near ubiquity of Chl a for its use in the primary photochemistry of Photosystem II (PS II) that leads to water oxidation and of Photosystem I (PS I) that leads to ferredoxin reduction. Chl a appears to be unique and irreplaceable, particularly if global scale oxygenic photosynthesis is considered. Its uniqueness is determined by its physicochemical properties, but there is more. Other contributing factors include specially tailored protein environments, and functional compatibility with neighboring electron transporting cofactors. Thus, the same molecule, Chl a in vivo, is capable of generating a radical cation at +1 V or higher (in PS II), a radical anion at −1 V or lower (in PS I), or of being completely redox silent (in antenna holochromes).

Target tissue uptake selectivity of three fluorine-substituted progestins: Potential imaging agents for receptor-positive breast tumors

We have studied three new fluorine-substituted progestins (1–3) as potential imaging agents for progesterone receptor (PgR)-positive human breast tumors. Two of these are fluorine-substituted analogs of the potent progestin R5020 (promegestone), derived from (21S)-hydroxy R 5020 (RU 27987) and (21R)-hydroxy R 5020 (RU 27988), known metabolites of R 5020, which have affinities for PgR that are 116 and 4%, respectively (relative to R 5020 = 100%). These precursors were protected as their 3,3-dioxolane derivatives and converted to the 21-trifluoromethanesulfonate derivatives. Fluoride ion displacement, followed by acid-catalyzed deprotection, furnished in good yield the epimeric fluoroanalogs, (21S)- and (21R)-fluoro R 5020 (1 and 2, affinities for PgR, 11 and 45%, respectively). These compounds were also prepared in ¹⁸F labeled form by the same route, in 14–32% overall radiochemical yield (decay corrected; synthesis time 90 min; sp. act. 370–1060 Ci/mmol). In tissue distribution studies in estrogen-primed immature rats, uterus-to-muscle ratios were 4.3 at 1 h for the 21S-epimer and 1.1 for the 21R-epimer, paralleling their relative binding affinities. Considerable metabolic defluorination was observed. The third fluorine-substituted progestin, DU 41165, has a novel retroprogesterone (9β, 10α) structure, substituted with fluorine at C-6; its binding affinity is 145% relative to R 5020, and it was prepared in tritium-labeled form by acetylation of DU 41231, the 17α-hydroxy precursor, with [³H]acetic anhydride. In estrogen-primed immature rats, this compound shows uterus-to-muscle ratios of 15 at 1 h, and 18–71 between 2 and 6 h, suggesting that compounds in this retroprogesterone series may be very promising candidates for selective imaging of PgR-positive tissues and tumors.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 °;K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth ～ 8 gauss) which decays with a half-time of 5.0 ms at 35 °;K. The half-time of decay is independent of temperature in the range of 10–77 °;K. This reversible signal can be eliminated by preillumination of the sample at 35 °;K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to — 150 mV prior to freezing. In the presence of ferricyanide (20–50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 °;K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680⁺ and the primary electron acceptor.     

Excitation-energy redistribution in the cryptomonad alga Cryptomonas ovata

We have studied the redistribution of excitation energy in the cryptomonad alga Cryptomonas ovata. Low-temperature fluorescence emission spectra from cells preilluminated with light 1 and light 2 show that preferential excitation of Photosystem II (PS II) leads to decreased fluorescence emission from chlorophyll (Chl) a associated with PS II relative to the emission following the preferential excitation of Photosystem I (PS I). The fluorescence change is indicative of a light-state transition by the cells. However, comparision of measurements of the kinetics of P-700 photooxidation by cells fixed with glutaraldehyde following illumination with light 1 or light 2 shows that the relative activity of PS I is lower in cells fixed in light 2. This is in contrast to the expectation for cells in State 2. Excitation spectra for the fluorescence emission from PS II Chl a show that preferential excitation of PS II leads to a decreased probability for energy transfer from phycoerythrin and Chl c₂ to PS II when compared to cells in which PS I is preferentially excited. This result is in accordance with recent picosecond time-resolved fluorescence studies (Bruce, D., Biggins, J., Charbonneau, S. and Thewalt, M. (1987) in Progress in Photosynthesis Research (Biggins, J., ed.), Vol. II, pp. 777–780, Martinus Nijhoff, Dordrecht) and we, therefore, suggest that C. ovata does not undergo a classical light-state transition. However, preferential excitation of PS II or PS I appears to cause pigment-protein conformational changes which change the probability for energy transfer from phycoerythrin to PS II, and we suggest that this may be a mechanism for photoprotection of PS II. Studies of the kinetics of excitation-energy redistribution, and of the effects of electron-transport inhibitors and uncouplers of photophosphorylation indicate that the mechanism for excitation-energy redistribution in C. ovata and phycobilisome-containing organisms may be similar.     

Calcium depletion alters energy transfer and prevents state changes in intact Anacystis

A time-dependent loss of Photosystem II (PS II) activity seen in Anacystis nidulans grown without Ca²⁺ was paralleled by a loss in chlorophyll (Chl) a fluorescence of variable yield which reflects inhibition of Q reduction and of state changes. Both inhibitions were fully reversed by the addition of Ca²⁺ to the growth medium. The lack of state changes in Ca²⁺-depleted cells was confirmed in 77 K fluorescence difference spectra of light versus dark-adapted cells.Absorption spectra of control and of Ca²⁺-depleted cells were identical whether measured at room temperature or at 77 K. Fluorescence emission spectra measured at 39°C (cell growth temperature) demonstrated higher yields in Ca²⁺-depleted cells compared to controls. Fluorescence emission spectra at 77 K also produced higher yields in Ca²⁺-depleted cells but the increased fluorescence at this temperature occurred principally at 683 nm. The increased relative fluorescence yield in Ca²⁺-depleted samples results from light absorbed by phycocyanin (PC), but not from light absorbed almost exclusively by Chl. The 683 run fluorescence peak probably represents increased allophycocyanin (APC) emission as intact phycobilisomes become energetically disassociated from the photosynthetic apparatus. This inferred disassociation occurred only after PSII activity was mostly inhibited in Ca²⁺-depleted cells, and was not fully reversible.Abbreviations APC Allophycocyanin - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA ethylenediaminotetraacetic acid - PC phycocyanin - PS photosystem - Q primary quinone electron acceptor of Photosystem II also a quencher of Chl a fluorescence DPB-CIW Publ. No. 817     

Stereoselective epoxidation of divinylmethanol: A synthetic approach to the pentitols

Peroxy-acid epoxidation of divinylmethanol (9), followed by acetylation afforded the acetylated monoepoxides 1 and 2 having the erythro (53%) and threo (47%) configurations. Peroxy-acid epoxidation of 1 and 2 yielded the acetylated diepoxides 3 (erythro-erythro, 36%) and 4 (erythro-threo, 64%) (from 1), and 4 (erythro-threo, 47%) and 5 (threo-threo, 53%) (from 2). Relative configurational assignments were made to 1–5 on the basis of (a) ¹H-n.m.r. chemical-shift and coupling-constant data, (b) the observation that 1 gave only 3 and 4, and that 2 gave only 4 and 5 on epoxidation, and (c) the fact that 3–5 separately undergo epoxide-ring opening preferentially at their primary carbon atoms with acetate ion in acetic anhydride, to afford the penta-acetates of ribitol, dl-arabinitol, and xylitol, respectively, as major products. Epoxide-ring formation favours the erythro configuration when either peroxy acids or tert-butyl hydroperoxide with catalytically active Ti⁴⁺, V⁵⁺, or Mo⁶⁺ complexes are employed as epoxidation reagents. However, the diastereoselectivities characterising the epoxidations and the regioselectivities governing the epoxide-ring openings are not sufficiently high to constitute an attractive synthesis of either ribitol, dl-arabinitol, or xylitol from divinylmethanol.

     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Effect of hydrolytic enzymes and protein-modifying reagents on gonadotropin receptors in bovine corpus luteum cell membranes

Preincubation of membranes with various concentrations of pronase, trypsin, lipase, phospholipase A from Vipera russelli and from Crotalus durissus terrificus, phospholipase C from Bacillus cereus and from Clostridium welchii, acetic anhydride, 2,4-dinitrofluorobenzene and tetranitromethane resulted in a dose-dependent inhibition of ¹²⁵I-labeled human choriogonadotropin binding. At the submaximal concentrations of enzymes and at both submaximal and maximal concentrations of protein-modifying reagents, the losses were always greater with ¹²⁵I-labeled human choriogonadotropin than with ¹²⁵I-labeled human lutropin. The inhibition of binding was a consequence of changes in the membranes rather than changes in the hormone caused by the agents being carried over to the final incubation. Inhibition of binding was non-competitive and irreversible.In untreated membranes, the ¹²⁵I-labeled human choriogonadotropin binding was homogenous (K_d = 1.7 · 10^?10 M; N = 60 fmol/mg protein). Treatment of membranes with various enzymes and protein-modifying reagents except tetranitromethane resulted in heterogeneous binding. The number of available high affinity receptors was greatly reduced in every case. However, the affinity of these sites were either unchangedd (trypsin, lipase, phospholipase A from V. russelli, dinitrofluorobenzene and the tetranitromethane) or decreased (pronase and acetic anhydride). the newly appeared second receptor site had a K_d which varied from 3.2 · 10^?10 to 7.1 · 10^?9d M depending on the agent used, and the receptor numbers were low in all cases except acetic anhydride.Receptor occupancy conferred the receptors with marked protection against various hydrolytic enzymes, dinitrofluorobenzene and tetranitromethane. These data suggest the inhibition of binding by the above agents was primarily a consequence of changes in the receptor molecules themselves.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

Reactions of the 3-oxime 2-phenylhydrazone and mixed bishydrazones of dehydro-L-ascorbic acid: Conversion into substituted triazoles and pyrazolinediones

L-threo-2,3-Hexodiulosono-1,4-lactone 2-phenylhydrazone(1) reacted with hydroxylamine to give the 3-oxime 2-phenylhydrazone(2). On boiling with acetic anhydride,2 was dehydrated to 4-[L-threo-2,3-diacetoxy-(1-hydroxypropyl)]-2-phenyl-1,2,3-triazole-5-car?ylic acid lactone(3), which was converted into 2-phenyl-4-(L-threo-1,2,3-trihydroxypropyl)-1,2,3-triazole-5-car?amide(4) with liquid ammonia. The structure of compound4 was confirmed by acetylation to 2-phenyl-4-(L-threo-1,2,3-triacetoxypropyl)-1,2,3-triazole-5-car?amide(5), and by periodate oxidation followed by reduction, to give 4-(hydroxymethyl)-2-phenyl-1,2,3-triazole-5-car?amide(6). Treatment of compound1 with aryl- or aroyl-hydrazines afforded mixed bishydrazones(7–14), which were acetylated to15–21, and treated with hydrazine to give pyrazolinediones22 and23     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Studies on dehydro-d-arabino-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

d-erythro-2,3-Hexodiulosono-1,4-lactone 2-arylhydrazones (2) were prepared by condensation of dehydro-d-arabino-ascorbic acid with the desired arylhydrazine. Reaction of 2 with hydroxylamine gave the 2-arylhydrazone 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-d-erythro-glycerol-1-yl)-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone (5), whereas the unacetylated triazole derivatives were obtained upon reaction of 3 with bromine in water. On treatment of 5 with hydrazine hydrate, 2-aryl-4-(d-erythro-glycerol-1-yl)-1,2,3-triazole-5-carboxylic acid 5-hydrazides (6) were obtained. Acetylation of 6 gave the hexaacetyl derivatives. Similarly, treatment of 5 with liquid ammonia gave the triazolecarboxamides (12). Vigorous acetylation of 12 with boiling acetic anhydride gave tetraacetates, whereas acetylation with acetic anhydride-pyridine gave triacetates. Periodate oxidation of 6 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxylic acid 5-hydrazides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxylic acid 5-hydrazides, characterized as acetates. Similarly, periodate oxidation of 12 gave the triazolealdehyde (15), and reduction of 15 gave the hydroxymethyl derivatives (16). Acetylation of 16 gave the mono- and di-acetates, and, on reaction with o-phenylenediamine, 15 afforded the triazoleimidazole. Controlled reaction of 3 with sodium hydroxide, followed by neutralization, gave 3-(d-erythro-glycerol-1-yl)-4,5-isoxazolinedione 4-arylhydrazones. Reaction of 3 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-d-erythro-2,3-hexodiulosono-1,4-lactone 2-arylhydrazone 3-oximes (21). Compounds 21 were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-d-erythro-glycerol-1-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,1¹-lactone on treatment with acetic anhydride.