Membrane-bound NADPH dehydrogenase- and ferredoxin: NADP oxidoreductase activity involved in electron transport during acetate oxidation to CO2 in Desulfobacter postgatei

Desulfobacter postgatei grows on acetate and sulfate as energy source. The oxidation of acetate to 2 CO₂ proceeds via the citric acid cycle involving membrane-bound succinate dehydrogenase and membrane-bound malate dehydrogenase. We report here that the organism contains membrane-bound NADPH dehydrogenase and ferredoxin: NADP oxidoreductase for the reoxidation of NADPH and reduced ferredoxin generated during isocitrate- and 2-oxoglutarate oxidation, respectively. The presence of proton translocating ATPase activity is also described.NADPH dehydrogenase and succinate dehydrogenase were found to be electrically connected within the membrane and electron transfer between these two enzymes was shown to be coupled with proton translocation. The membrane fraction catalyzed the oxidation of NADPH with fumarate and the reduction of NADP with succinate. NADPH oxidation with fumarate was stimulated by protonophores and inhibited by the proton translocating ATPase inhibitor dicyclohexylcarbodiimide (DCCD) and by heptylhydroxyquinoline-N-oxide (HQNO); inhibition by DCCD was relieved by protonophores. NADP reduction with succinate was dependent on ATP and inhibited by protonophores, DCCD, and HQNO. The membrane fraction also mediated the oxidation of NADPH with the water soluble menaquinone analogue dimethylnaphthoquinone (DMN) and the reduction of fumarate with DMNH₂. Only the former reaction was stimulated by protonophores and only the latter reaction was inhibited by HQNO. This suggests that the NADPH dehydrogenase reaction is the site of energy conservation and the succinate dehydrogenase is the site of HQNO inhibition.Non-standard abbreviations APS Adenosine 5-phosphosulfate - DCCD N,N-dicyclohexylcarbodiimide - DCPIP 2,6-dichloroindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - HQNO 2(n-heptyl)-4-hydroxyquinoline-N-oxide - TCS 3,5,3,4-tetrachlorosalicylanilide - Tricine N-tris-(hydroxymethyl)methylglycine - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - SF-6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile     

Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity

Absorbance changes of ferredoxin measured at 463 nm in isolated thylakoids were shown to arise from the activity of the enzyme ferredoxin-plastoquinone reductase (FQR) in cyclic electron transport. Under anaerobic conditions in the presence of DCMU and an appropriate concentration of reduced ferredoxin, a light-induced absorbance decrease due to further reduction of Fd was assigned to the oxidation of the other components in the cyclic pathway, primarily plastoquinone. When the light was turned off, Fd was reoxidised and this gave a direct quantitative measurement of the rate of cyclic electron transport due to the activity of FQR. This activity was sensitive to the classical inhibitor of cyclic electron transport, antimycin, and also to J820 and DBMIB. Antimycin had no effect on Fd reduction although this was inhibited by stigmatellin. This provides further evidence that there is a quinone reduction site outside the cytochrome bf complex. The effect of inhibitors of ferredoxin-NADP⁺ reductase and experiments involving the modification of ferredoxin suggest that there may be some role for the reductase as a component of FQR. Contrary to expectations, NADPH₂ inhibited FQR activity; ATP and ADP had no effect.Abbreviations AQS 9,10-anthraquinone-2-sulphonate - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - dimaleimide N,N-p-phenylenedimaleimide - EDC N-(dimethylaminopropyl)-N-ethylcarbodiimide - Fd ferredoxin - FNR Fd-NADP⁺ oxidoreductase - FQR Fd-PQ reductase - GME glycine methyl ester - J820 tetrabromo-4-hydroxypyridine - PC plastocyanin - PMS N-methylphenazinium methyl sulphate - PS Photosystems I and II - PQ plastoquinone - Q quinone - Q_r and Q_o sites of quinone reduction and oxidation, respectively - sulpho-DSPD disulphodisalicylidenepropane-1,2-diamine     

Inhibition of photosynthetic oxygen evolution by protonophoric uncouplers

The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K₃[Fe(CN)₆] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I₅₀ values were approx. 1–2, 4–6 and 0.04–0.10 M, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Q_p) which is also the electron donor for K₃ [Fe(CN)₆] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y - ANT2p- 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene - CCCP- carbonyl cyanide m-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl 6-isopropyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT- 2-iodo-6-isopropyl-3-methyl 2,4,4-trinitrodiphenyl ether - DPC- 1,5-diphenylcarbazide - DPIP- 2,6-dichlorophenolindophenol - FCCP- carbonyl cyanide p-trifuoromethoxyphenylhydrazone - FeCy- potassium ferricyanide - HQNO- 2-n-heptyl-4-hydroxyquinoline N-oxide - (MN)₄- the tetranuclear Mn cluster of water oxidizing complex - P680- photoactive Chl of the reaction center of Photosystem II - PCP- 2,3,4,5,6-pentachlorophenol - PS- photosystem - Q_A and Q_B- primary and secondary plastoquinones of PS II - Q_C and Q_Z- plastoquinone binding sites in the cytochrome blf complex - Q_p- membrane pool of plastoquinone - SiMo- sodium silicomolybdate - TMPD- N,N,N-tetramethyl-p-phenylenediamine - TTFB- 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - WOC- water oxidixing complex - Y_Z- tyrosine-161 of the Photosystem II D1 polypeptide     

NADH as electron donor for the photosynthetic membrane of Chlamydomonas reinhardii

A chloroplast fraction from Chlamydomonas reinhardii cells can oxidize NADH in the light, unlike chloroplasts of higher plants. The Chlamydomonas preparation catalyzes electron flow from NADH to methylviologen or ferredoxin to evolve hydrogen (in the presence of a hydrogenase) or take up oxygen. The NADH photooxidation is sensitive to rotenone, dibromothymoquinone and dicyclohexylcarbodiimide. This suggests that a rotenone sensitive NADH dehydrogenase is coupled on the plastoquinone reduction site of the potosynthetic electron flow system. On sonication of the particles NADH photooxidation is lost but may be restored by a protein fraction from an acetone extract plus plastocyanin.Abbreviations DAD diaminodurene - DCCD dicyclohexylcarbodiimide - DCMU (3,3-dichlorphenyl)-N·N dimethyl urea - DBMIB dibromothymoquinone - DNP-INT dinitro-phenylether of 2-iodo-4-nitrothymol - MV methylviologen - chl chlorophyll Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Glycolytic Flux and Hexokinase Activities in Anoxic Maize Root Tips Acclimated by Hypoxic Pretreatment

In vitro cyclic electron transport around PSI was studied in thylakoids isolated from barley (Hordeum vulgare L.). Redox poising was obtained by using anaerobic conditions, preillumination, and the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Postillumination rates of P700+ re-reduction of 1 to 5 electrons s-1 were observed, depending on the conditions. The thylakoids supported two parallel paths of cyclic electron transport that were distinguishable by differences in antimycin sensitivity, saturation characteristics, and substrate specificity. The pathway most sensitive to antimycin was not saturated at ferredoxin concentrations up to 50 [mu]M, whereas the more insensitive pathway was saturated at 5 [mu]M ferredoxin. At the lower concentration of reduced ferredoxin, the antimycin-sensitive rate of P700+ re-reduction was lower than the antimycin-insensitive rate. The lower range of reduced ferredoxin concentrations are closer to in vivo conditions. Flavodoxin is shown to mediate cyclic electron transport. Flavodoxin was less efficient in mediating the antimycin-sensitive pathway but mediated the antimycin-insensitive pathway as efficiently as ferredoxin. Antibodies raised against ferredoxin:NADP+ oxidoreductase had no effect on either pathway for re-reduction of P700+. However, the ferredoxin: NADP+ oxidoreductase inhibitor 2[prime]-monophosphoadenosine-5[prime]-diphosphoribose was able to inhibit the antimycin-sensitive as well as the antimycin-insensitive pathway.     

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H₂O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H₂O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-Q_B-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1,-dimethylurea - DCPIP dichlorophenol-indophenol (2,6-dichloro-4((4-hydroxyphenyl)imino)-2,5-cyclohexadien-1-one) - DPC diphenyl carbazide (2,2-diphenylcarbonic dihydrazide) - FeCN ferricyanide (hexacyanoferrate(III)) - _app apparent quantum yield of photosynthetic oxygen evolution - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - PPBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PQ pool plastoquinone - Q_B secondary quinone acceptor of PS II - RT room temperature - WC whole chain electron transfer     

Electron transport chain of Zymomonas mobilis

The interaction of the membrane-bound glucose dehydrogenase from the anaerobic but aerotolerant bacterium Zymomonas mobilis with components of the electron transport chain has been studied. Cytoplasmic membranes showed reduction of oxygen to water with the substrates glucose or NADH. The effects of the respiratory chain inhibitors piericidin, capsaicin, rotenone, antimycin, myxothiazol, HQNO, and stigmatellin on the oxygen comsumption rates in the presence of NADH or glucose as substrates indicated that a complete and in the most parts identical respiratory chain is participating in the glucose as well as in the NADH oxidation. Furthermore, the presence of coenzyme Q₁₀ (ubiquinone 10) in Z. mobilis was demonstrated. Extraction from and reincorporation of the quinone into the membranes revealed that ubiquinone is essential for the respiratory activity with glucose and NADH. In addition, a membrane-associated tetramethyl-p-phenylene-diamine-oxidase activity could be detected in Z. mobilis.Abbreviations ABTS 2,2-Azino-di-[3-ethyl-benzthiazolinesulfonate (6)] - GDH glucose dehydrogenase - HQNO 2-heptyl-4-hydroxy-quinoline-N-oxide - PQQ pyrroloquinoline quinone - TMPD N,N,N,N-tetramethyl-p-phenylene-diamine     

Spectrophotometric measurement of plastoquinone photoreduction in the blue-green alga,Phormidium uncinatum

The redox state of plastoquinone was measured in vivo in the blue-green alga, Phormidium uncinatum by means of a double beam UV-spectrophotometer. The difference in absorbance of the oxidized and the reduced forms of plastoquinone was amplified, and stored and averaged in a computer. The redox state was changed by two alternating actinic light beams. When one actinic wavelength was kept constant at 700 nm (PSI) variation of the other yielded an action spectrum representing photosystem II. The inhibitors of the photosynthetic electron transport chain, DCMU and DBMIB, reduced the difference in absorbance between the oxidized and reduced forms of plastoquinone.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

Divergent pathways of photosynthetic electron transfer: The autonomous oxygenic and anoxygenic photosystems

The aim of this article is to assemble and integrate, from a personal perspective of a research participant, seldom examined evidence that is incompatible with some basic tenets of photosynthetic electron transport, the cornerstone of which is the Z scheme. The nonconforming evidence pertaining to the mode of ferredoxin reduction and the role of the copper redox protein, plastocyanin, indicates that contrary to the Z scheme ferredoxin is reduced in two experimentally distinguishable ways: oxygenically by PS II (renamed the oxygenic photosystem), without the participation of PS I, and anoxygenically by PS I (renamed the anoxygenic photosystem). It also indicates that plastocyanin is not only, as the Z scheme asserts, the electron donor to the reaction center chlorophyll of PS I (P700) but also to the reaction center chlorophyll of PS II (P680). Other unconventional findings include evidence that the fully functional oxygenic photosystem, when operating separately from the anoxygenic photosystem, reduces plastoquinone to plastoquinol and subsequently oxidizes plastoquinol by two pathways acting in concert: one being the universally recognized DBMIB-sensitive pathway via the Rieske iron-sulfur center of the cytochrome bf complex and the other, a hitherto unrecognized, DBMIB-insensitive electron transport pathway around P680 that centers on cytochrome b-559. These nonconforming findings form the basis of an alternate hypothesis of photosynthetic electron transport that modifies and complements the Z scheme.Abbreviations PS photosystem - PQ oxidized plastoquinone - PQH₂ reduced plastoquinone (plastoquinol) - Q_A and Q_B specialized membrane-bound forms of PQ - PC plastocyanin - Fd ferredoxin - BISC F_AF_B, membrane-bound iron-sulfur centers of PS I - DBM1B 2,5-dibromo-3-methyl-6-isopropyl-n-benzoquinone (dibromothymoquinone) - DNP-INT dinitrophenol ether of iodonitrothymol - NADP⁺ NADPH, oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate - FCCP carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone - CCCP carbonyl cyanide-3-chlorophenylhydrazone - SF 6847 2,6,-di-(t-butyl)-4-(2,2-dicyanovinyl) phenol - diuron (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - DCIP 2,6-dichloro-phenolindophenol - UHDBT 5-(n-undecyl)-6-hydroxy-4-7-dioxobenzothiazole; cytochrome b-559_HP-cytochrome b-559_LP, high- and low potential states of cytochrome b-559 - oxygenic reductions reductions in which water is the electron donor - BBY PS II preparation made according to Berthold et al. (1981) Dedicated to Professor Achim Trebst on his 65th birthday.Based in part on lecture in Advanced Course on Trends in Photosynthesis Research, Palma de Mallorca, Spain, September 18, 1990.     

Evidence for a branched respiratory system with two cytochrome oxidases in autotrophically grown Alcaligenes latus

The respiratory system of chemolithoautotrophically-grown Alcaligenes latus contains a, b, and c type cytochromes. Two cytochrome oxidases were identified by their carbon monoxide difference spectra and their differing sensitivities to cyanide and carbon monoxide. The oxidases were cytochrome o and an a-type cytochrome. Ubiquinone was present in A. latus membranes and could be reduced by H₂. The quinone analogue, 2-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), was a strong inhibitor of the H₂ oxidase reaction, but did not prevent the reduction of either ubiquinone or the cytochromes.Abbreviations HQNO 2-heptyl-4-hydroxy-quinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Reconstitution of cytoplasmic streaming inCharaceae

Summary The active sites of actin of oneCharaceae species were found to interact with the endoplasmic factor from a different species. Protoplasm was suqueezed out of cells ofChara australis with vacuoles that had been perfused beforehand with a medium containing EGTA and Mg · ATP. Centrifugation of this protoplasmic mixture divided it into the supernatant composed of endoplasmic granules and the precipitate composed of chloroplasts and nuclei. When the endoplasmic granular aggregates were introduced into a tonoplast-freeNitella axilliformis cell treated with NEM to inactivate the endoplasmic factor, they became attached to theNitella gel and streamed longitudinally with the polarity. Treatment of the endoplasmic granules with the strong Mg²⁺chelator CyDTA (1,2-cyclohexane diamineN, N-tetraacetic acid) irreversibly inhibited reconstitution of the cytoplasmic streaming.Abbreviations APW artificial pond water - ATP adenosine-5-triphosphoric acid - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - HMM heavy meromyosin - NEM N-ethylmaleimide - PEP phosphoenolypyruvate - PIPES piperazine-N,N-bis-(2-ethane-sulfonic acid) - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride     

Localization of electron transport inhibition in plastoquinone reactions

Reduction kinetics of P700 following a short flash are measured in spinach chloroplasts after oxidation of the electron carriers between the two photoreactions by far-red light. Three features of the kinetics allow us to localize simultaneously inhibition at different sites between photoreaction II and the reducing site of plastoquinol. These are the initial lag, the halftime, and the area under the transient of the P700 absorbance change, which indicate the electron transfer time from photoreaction II to the reducing site of plastoquinol, the rate of plastoquinol oxidation, and the number of electrons transferred to the special plastoquinone B functioning as secondary electron acceptor of photosystem II, respectively. As an additional diagnostic parameter for inhibition before and after the plastoquinone pool, the area under the transient of the P700 absorbance change is used after long flashes. This area is proportional to the amount of reduced plastoquinone as shown by the absorbance change at 265 nm. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) are compared with those of 2-bromo-4-nitrothymol, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, and Illoxan as representatives for new classes of inhibitors. While 2-halogeno-4-nitrothymols inhibit the reduction of plastoquinone similarly to DCMU, their diphenyl ether derivatives inhibit selectively the oxidation of plastoquinol.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

Reconstitution of electron transport by cytochrome c-553 in a cell-free system of Nostoc muscorum

Treatment of spheroplasts of Nostoc museorum with hypotonic buffer results in membranes depleted of cytochrome c-553, but still active in photosynthetic and respiratory electron transport. These membranes retain full photosystem II activity (H₂ODAD_ox). Complete linear electron transport (H₂ONADP⁺), however, is decreased as compared with untreated spheroplasts. Addition of basic Nostoc cytochrome c-553 to depleted membranes reconstitutes NADP⁺ reduction and redox reactions of the photosystem I region as well.Using NADPH as electron donor, respiration of depleted membranes is also stimulated by adding cytochrome c-553, indicative of its function in respiratory electron transport.Cytochrome c-553 from Bumilleriopsis filiformis, Spirulina platensis (acidic types), Phormidium foveolarum (basic type), and mitochondrial horse-heart cytochrome c-550 are not effective in reconstituting both photosynthetic and respiratory electron transport, which points to a specific role of Nostoc cytochrome c-553.Abbreviations BSA bovine serum albumin - DAD 3,6-diaminodurene - DAD_ox 3,6-diaminodurene oxidized by potassium ferricyanide - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Fd ferredoxin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(-N-morpholino)-ethanesulfonic acid - MV methylviologen (1,1-dimethyl-4,4-bipyridylium dichloride) - PS I photosystem I - PS II photosystem II - Tris tris-(hydroxymethyl)-aminomethane     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

The role of glycinebetaine in the protection of spinach thylakoids against freezing stress

The quaternary ammonium compound glycinebetaine has been tested for cryoprotective properties, using isolated spinach thylakoids as a model membrane system. The effect of a 3-h,-20°C freezing regime on different photosynthetic parameters was measured. These parameters were the light-stimulated pH formation and dark pH decay, light-stimulated proton uptake, electron flow through photosystem II, photosystem I and total linear electron flow, and pyocyanine-mediated cyclic photophosphorylation. It was shown that below 100 mM glycinebetaine was superior as a cryoprotectant to sucrose on a molar, a molal and an activity basis. At higher concentrations, glycinebetaine was less efficient in preventing inactivation of thylakoids during freezing than sucrose. These observations are discussed in relation to the permeability of biomembranes to glycinebetaine and the colligative theory of cryoprotection. It is concluded that colligative protection is modified by direct interaction between cryoprotectant and membranes.Abbreviations Asc ascorbate - cyt f cytochrome f - DAD 2,3,5,6-tetramethyl--phenylenediamine - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DCPIP 2,6-dichlorophenolidophenol - DBMIB 2,5-dibromo-3-methyl-6-isopropyl--benzoquinone - DNP-INT 1,3-dinitrophenylether of iodonitrothymol - FeCy ferricyanide - MV methylviologen (1,1-dimethyl-4-4-bipyridinium-dichloride) - PQ plastoquinone - PS I photosystem I - PS II photosystem II     

Enhancement of chloroplast energization in Chlorella by treatments inactivating the nitrate-reducing system

Inactivation of the nitrate-reducing system in whole cells of Chlorella vulgaris Bejerinck by darkening, nitrogen starvation, ammonium, or cycloheximide brings cells into a state with a high yield of the millisecond-delayed fluorescence of chlorophyll. Activation of this system by illumination, by adding glucose to dark-adapted cells or nitrate to nitrogen-starved cells brings the cells into a low-yield state. The transitions between the lowand high-yield state induced by alternating light and dark periods are suppressed by tungstate and restored by subsequent molybdate addition. The drop in the delayed-fluorescence yield upon activation of the nitrate-reducing system is associated with the decrease of the amplitude of the electrochemical proton gradient across the thylakoid membrane of the chloroplast, as evidenced by the kinetics of the light-induced adsorption changes at 520 nm. The decrease of the proton gradient may be caused by the electron flow diverting from the cyclic path in photosystem I as a result of the activation of the electron transfer from ferredoxin to nitrite.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol