Functional Structure of the Oxygen-Evolving Unit of Photosystem II as Determined by Radiation Inactivation

The size of the complex that is essential for the electron-transferactivity from the oxygen-evolving center to the secondary electronacceptor, Q_B, is about 250 kDa, as determined by target-sizeanalysis after the radiation inactivation of functions of photosystemII (PS II). Inter-Chl tranfer of excitation energy was insensitiveto the radiation inactivation indicating that the masses ofCP47, CP43, and light-harvesting Chi a/b proteins are not includedin the functional size of the oxygen-evolving PS II complex.The transfer of electrons from the secondary electron donor,Z, to Q_B was catalyzed by a unit of only 65 kDa. The sizes ofthe complexes involved in these light-induced functions of PSII were dependent on the intensity of actinic light. Under saturatingintensities of light, the functional size of the complex fortransfer of electrons from Z to Q_B was 38 kDa, with a correspondingdecrease in the size of the oxygen-evolving PS II from 250 kDato 125 kDa [Takahashi, Mano and Asada (1985) Plant Cell Physiol.26: 383]. The protein of about 30 kDa functions in the photoreductionof the pheophytin molecule, as well as in the electron transferfrom Z to Q_A. Under low-intensity light, complexes having thesame sizes as those of the basal functional complexes undersaturating-intensity light are further required, probably tostabilize separated charges in the PS II reaction center andthe oxygen-evolving center. (Received June 20, 1990; Accepted September 18, 1990)     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Two Plastoquinone A Molecules Are Required for Photosystem II Activity: Analysis in Hexane-Extracted Photosystem II Particles

Prenylquinones were extracted with hexane from lyophilized oxygen-evolvingphotosystem II particles prepared from spinach chloroplasts.Determination by high performance liquid chromatography showedthat two molecules of plastoquinone A remained per reactioncenter after the extraction, in contrast to the presence ofthree to four plastoquinone A molecules before the extraction.Electron transfer from water to phenyl-p-quinone was not inhibitedby the extraction. Measurement of EPR signal II and microsecondchlorophyll fluorescence kinetics showed that hexane did notextract quinones which were acting as the secondary electrondonor (Z) and the primary electron acceptor (Q_A) in photosystemII. These results, as well as the effect of quinone extractionon oxygen evolution, indicate that two molecules of plastoquinoneA acting as Z and Q_A are essential for the activity of photosystemII. An artificial donor phenyl-p-quinone probably accepts electronfrom Q_A at the same site as the intrinsic secondary electronacceptor (Q_B). Q_A and Z seem to be surrounded by special microenvironmentswhich differ from that of bulk quinones, and are resistant tohexane treatment. (Received November 27, 1984; Accepted April 30, 1985)     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

Simultaneous detection of spin-coupled and decoupled QA– EPR-signals in Photosystem II complexes isolated with isoelectric focusing

The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl--D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_A ^- Fe2+-signal (g = 1.84), arising from Q_A ^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_A ^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.     

Polypeptides of photosystem II and their role in oxygen evolution

The linear, four-step oxidation of water to molecular oxygen by photosystem II requires cooperation between redox reactions driven by light and a set of redox reactions involving the S-states within the oxygen-evolving complex. The oxygenevolving complex is a highly ordered structure in which a number of polypeptides interact with one another to provide the appropriate environment for productive binding of cofactors such as manganese, chloride and calcium, as well as for productive electron transfer within the photoact. A number of recent advances in the knowledge of the polypeptide structure of photosystem II has revealed a correlation between primary photochemical events and a core complex of five hydrophobic polypeptides which provide binding sites for chlorophyll a, pheophytin a, the reaction center chlorophyll (P680), and its immediate donor, denoted Z. Although the core complex of photosystem II is photochemically active, it does not possess the capacity to evolve oxygen. A second set of polypeptides, which are water-soluble, have been discovered to be associated with photosystem II; these polypeptides are now proposed to be the structural elements of a special domain which promotes the activities of the loosely-bound cofactors (manganese, chloride, calcium) that participate in oxygen evolution activity. Two of these proteins (whose molecular weights are 23 and 17 kDa) can be released from photosystem II without concurrent loss of functional manganese; studies on these proteins and on the membranes from which they have been removed indicate that the 23 and 17 kDa species from part of the structure which promotes retention of chloride and calcium within the oxygen-evolving complex. A third water-soluble polypeptide of molecular weight 33 kDa is held to the photosystem II core complex by a series of forces which in some circumstances may include ligation to manganese. The 33 kDa protein has been studied in some detail and appears to promote the formation of the environment which is required for optimal participation by manganese in the oxygen evolving reaction. This minireview describes the polypeptides of photosystem II, places an emphasis on the current state of knowledge concerning these species, and discusses current areas of uncertainty concerning these important polypeptides.Abbreviations A 23187 ionophore that exchanges divalent cations with H⁺ - Chl chlorophyll - cyt cytochrome - DCPIP dichlorophenolindophenol - DPC diphenylcarbazide - EGTA ethyleneglycoltetraacetic acid - P680 the chlorophyll a reaction center of photosystem II - pheo pheophytin - PQ plastoquinone - PS photosystem - Q_A and Q_B primary and secondary plastoquinone electron acceptors of photosystem II - Sn (n=0, 1, 2, 3, 4) charge accumulating state of the oxygen evolving system - Signals II_vf, II_f and II_s epr-detectable free radicals associated with the oxidizing side of photosystem II - Z primary electron donor to the photosystem II reaction center The survey of literature for this review ended in September, 1984.     

Photoproduction of the Azidyl Radical from the Azide Anion on the Oxidizing Side of Photosystem II and Suppression of Photooxidation of Tyrosine Z by the Azidyl Radical

Azide ions inhibited O₂ evolution in PSII membranes from spinachin a time-dependent manner in the light until all activity disappeared.Illumination in the presence of azide (azide-phototreatment)irreversibly inhibited the following processes: (1) both theoxidation of water and the electron transport between the redox-activetyrosine 161 of the D1 protein (Y_Z) and the secondary quinoneelectron acceptor (Q_B) site, to the same extent; (2) the donationof electrons to the primary quinone electron acceptor (Q_A),as measured by monitoring the maximum variable fluorescenceof Chl; and (3) the photoproduction of the Y_Z radical (Y). Thus,the primary site of inhibition appeared to lie between Y_Z andQ_A. On illumination of Tris-treated PSII membranes in the presenceof azide, production of the azidyl radical was observed by spin-trappingESR. Yield of Y in Tris-treated membranes on illumination wassuppressed by azide. Electron transport from Y_Z to Q_B in Tris-treatedmembranes was inhibited only when the azidyl radical was photoproduced,and it was inhibited more rapidly than it was in the oxygenicPSII membranes. These results indicate that the azidyl radicalwas produced via a univalent oxidation of azide by Y and thatit irreversibly inhibited the electron transport from Y_Z toQ_A in Tris-treated membranes. Although the azidyl radical wasundetectable in the oxygenic PSII membranes, probably due tosteric interference by the peripheral proteins of water-oxidizingcomplex with the access of the spin-trapping reagent to theproduction site of the radical, the participation of the azidylradical in the inhibition of the oxygenic PSII membranes issuggested since simultaneous occurrence of both electron transportand azide was required for the inhibition. Possible inhibitorymechanisms and the target sites of azidyl radical are discussed. (Received April 21, 1995; Accepted July 3, 1995)     

Antenna Sizes of Photosystem I and Photosystem II in Chlorophyll b-Deficient Mutants of Rice. Evidence for an Antenna Function of Photosystem II Centers That are Inactive in Electron Transport

Light-harvesting capacities of photosystem I (PSI) and photosystemII (PSII) in a wild-type and three chlorophyll b-deficient mutantstrains of rice were determined by measuring the initial slopeof light-response curve of PSI and PSII electron transport andkinetics of light-induced redox changes of P-700 and Q_A, respectively.The light-harvesting capacity of PSI determined by the two methodswas only moderately reduced by chlorophyll b-deficiency. Analysisof the fluorescence induction that monitors time course of Q_Aphotoreduction showed that both relative abundance and antennasize of PSII_a decrease with increasing deficiency of chlorophyllb and there is only PSII_rß in chlorina 2 which totallylacks chlorophyll b. The numbers of antenna chlorophyll moleculesassociated with the mutant PSII centers were, therefore, threeto five times smaller than that of PSII_a in the wild type rice.Rates of PSII electron transport determined on the basis ofPSII centers in the three mutants were 60–70% of thatin the normal plant at all photon flux densities examined, indicatingthat substantial portions of the mutant PSII centers are inactivein electron transport. The initial slopes of light-responsecurves of PSII electron transport revealed that the functionalantenna sizes of the active populations of PSII centers in themutants correspond to about half that of PSII in the wild typerice. Thus, the numbers of chlorophyll molecules that serveas antenna of the oxygen-evolving PSII centers in the mutantsare significantly larger than those that are actually associatedwith each PSII center. It is proposed that the inactive PSIIserves as an antenna of the active PSII in the three chlorophyllb-deficient mutants of rice. In spite of the reduced antennasize of PSII, therefore, the total light-harvesting capacityof PSII approximately matches that of PSI in the mutants. (Received July 29, 1994; Accepted February 7, 1996)     

Reaction center light harvesting B875 complexes from Rhodocyclus gelatinosus: characterization and identification of quinones

Reaction center-B875 pigment-protein complexes were purified from Rhodocyclus gelatinosus. The proteic components consist of 7–8 polypeptides among which some were identified by their apparent molecular weights: the light harvesting B875 polypeptides and of 8 and 6 kDa, reaction center L (23 kDa), M (28 kDa) and H (34 kDa), cytochrome c (43 kDa). Four c-type hemes were found per reaction center. Flash-induced absorbance changes showed the presence of both Q_A and Q_B in the complex. Charge recombination times were determined to be: 1.16±0.2 (n=30) for P⁺Q_AQ_B ^- and 7–10 ms for P⁺Q_A ^- in presence of herbicides. From quinone analysis on one hand and kinetics of charge recombination on the other hand, we proposed that in the reaction center of Rhodocyclus gelatinosus Q_A is menaquinone 8 and Q_B is ubiquinone 8.     

Comparison of Atrazine-Resistant and -Susceptible Biotypes of Senecio vulgaris L.: Effects of High and Low Temperatures on the in vivo Photosynthetic Electron Transfer in Intact Leaves

The effects of temperature on the yield of in vivo modulatedchlorophyll fluorescence were measured in intact leaves of atrazineresistantand -susceptible biotypes of the weed Senecio vulgaris L. At25 ?C, the photochemical quenching (q_Q of steady-state chlorophyllfluorescence was reduced by around 30% in the atrazine-resistantmutant as compared to the susceptible wild type, indicatinga higher reduction state of the primary electron acceptor Q_Aof photosystem II in the former biotype. Moderately elevatedtemperatures (above 30 ?C) further increased the steady-stateconcentration of reduced in the mutant. Analysis of the temperature dependence of both the photochemicalquenching q_Q and the initial fluorescence level F_o clearly indicateda drastically enhanced heat-sensitivity of the photochemicalapparatus in the atrazine-resistant Senecio leaves. The heat-inducedchanges in F₀ and q_Q were closely correlated, suggesting thatthe phenomenon responsible for the rise in F₀ was also involvedin the inhibition of the photosynthetic electron flux. Low temperaturesalso affected reoxidation but, in contrast to heating, no apparent differences were observed in the behaviourof the two biotypes exposed to chilling stress. Key words: Atrazine-resistance, chlorophyll fluorescence, heat stress, Senecio vulgaris     

Interaction of Benzoquinones with QA- and QB- in Oxygen-Evolving Photosystem II Particles from the Thermophilic Cyanobacterium Synechococcus elongatus

The interactions of benzoquinones with the reduced forms ofthe bound plastoquinone acceptors, Q_A and Q_B, were studied withoxygen-evolving photosystem II (PS II) particles from the thermophiliccyanobacterium Synechococcus elongatus, which largely lack poolplastoquinone molecules [Takahashi and Katoh (1986) Biochim.Biophys. Acta 845: 183]. Oxygen evolution in the presence ofvarious electron acceptors was determined and flash-inducedchanges in absorbance in the blue region were analyzed in termsof difference spectra, dependence on the concentration of benzoquinoneand on temperature, and sensitivity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The more hydrophobic the quinone molecule, the higherwas the rate of oxygen evolution, and the maximum rate of 3,000µmoles O₂.(mg chlorophyll)^–1.h^–1 was recordedin the presence of phenyl- and dichloro-p-benzoquinones. DCMUinhibited oxygen evolution by more than 95%. However, spectrophotometricstudies revealed that, even though electrons were transferredto benzoquinones predominantly via the direct oxidation of by added benzoquinones occurred in such a way as to indicate thatabout 40% of PS II reaction centers were not associated withfunctional Q_B sites. was very stable in the presence of ferricyanide. However, benzoquinonesinduced the slow oxidation of . The characteristics of the benzoquinone reductioin in thePS II preparation is discussed. ¹Present address: Department of Life Sciences, Faculty of Science,Himeji Institute of Technology, Shosha 2167, Himejishi, Hyogo-ken,671-22 Japan (Received May 8, 1990; Accepted August 14, 1990)     

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     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Influence of thylakoid membrane lipids on the structure and function of the plant photosystem II core complex

Main conclusion

MGDG leads to a dimerization of isolated, monomeric PSII core complexes. SQDG and PG induce a detachment of CP43 from the PSII core, thereby disturbing the intrinsic PSII electron transport. The influence of the four thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure and function of isolated monomeric photosystem (PS) II core complexes was investigated. Incubation with the negatively charged lipids SQDG and PG led to a loss of the long-wavelength 77 K fluorescence emission at 693 nm that is associated with the inner antenna proteins. The neutral galactolipids DGDG and MGDG had no or only minor effects on the fluorescence emission spectra of the PSII core complexes, respectively. Pigment analysis, absorption and 77 K fluorescence excitation spectroscopy showed that incubation with SQDG and PG led to an exposure of chlorophyll molecules to the surrounding medium followed by conversion to pheophytin under acidic conditions. Size-exclusion chromatography and polypeptide analysis corroborated the findings of the spectroscopic measurements and pigment analysis. They showed that the negatively charged lipid SQDG led to a dissociation of the inner antenna protein CP43 and the 27- and 25-kDa apoproteins of the light-harvesting complex II, that were also associated with a part of the PSII core complexes used in the present study. Incubation of PSII core complexes with MGDG, on the other hand, induced an almost complete dimerization of the monomeric PSII. Measurements of the fast PSII fluorescence induction demonstrated that MGDG and DGDG only had a minor influence on the reduction kinetics of plastoquinone Q_A and the artificial PSII electron acceptor 2,5-dimethyl-p-benzoquinone (DMBQ). SQDG and, to a lesser extent, PG perturbed the intrinsic PSII electron transport significantly.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Evolution of PS IIα and PS IIβ centers during the greening of Euglena gracilis Z: Correlations with changes in lipid content

The building up of the two types of reaction centers, PS II and PS II, was investigated during the greening of Euglena gracilis Z cells in resting medium. The maximal values in the proportion of PS II centers (55%) and in the oxygen evolved per chlorophyll were reached at the outbreak of greening, when accumulation of galactolipids (MGDG and DGDG) rich in unsaturated fatty acids occurred, and when anionic lipids (SQDG and PG) emerged. As the greening progressed, the chlorophyll accumulation corresponded to a secondary enrichment in PS II centers, which built up more rapidly than PS II centers; correlatively, a general saturation of the fatty acids constitutive of all lipid classes took place.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - FAME Tatty acid methyl esters - HEPES acide (N-[2-hydroxyethyl]piperazine-N-[2-ethane sulfonic] - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PQ plastoquinone - PS I Photosystem I - PS II Photosystem II - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - SQDG sulfoquinovosyldiacylglycerol     

Thermodynamics of the charge recombination in photosystem II

The temperature dependence of the electric field-induced chlorophyll luminescence in photosystem II was studied in Tris-washed, osmotically swollen spinach chloroplasts (blebs). The system II reaction centers were brought in the state Z⁺P⁺-Q_A ^-Q_B ^- by preillumination and the charge recombination to the state Z⁺PQ_AQ_B ^- was measured at various temperatures and electrical field strengths. It was found that the activation enthalpy of this back reaction was 0.16 eV in the absence of an electrical field and diminished with increasing field strength. It is argued that this energy is the enthalpy difference between the states IQ_A ^- and I^-Q_A and accounts for about half of the free energy difference between these states. The redox state of Q_B does not influence this free energy difference within 150 s after the photoreduction of Q_A. The consequences for the interpretation of thermodynamic properties of Q_A are discussed.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - I intermediary electron acceptor - Mops 3-(N-morpholino)propanesulphonic acid - P (P₆₈₀) primary electron donor - PS II photosystem II - Q_A and Q_B first and second quinone electron acceptors - Tricine N-tris(hydroxymethyl)methylglycine - Tris tris-(hydroxymethyl)aminomethane - Z secondary electron donor Dedicated to Professor L.N.M. Duysens on the occasion of his retirement     

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu²⁺ ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO₄ and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (A of 430–440 nm) and by the flash induced formation of P680⁺ (A at 820 nm). The relatively slow relaxation kinetics of the latter signal (t_1/2 1 ms) may suggest that in a substantial fraction of the RCs Q_A remains bound to the complex.Abbreviations Car -carotene - Chl a chlorophyll a - CP43, CP47 chlorophyll-proteins, with R_m 43 and 47 kDa - DBMIB dibromothymoquinone,2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone - DM -dodecyl-d-maltoside - HPLC high-performance liquid chromatography - OG n-octyl--d-glucopyranoside - IMAC immobilied metal affinity chromatography - Pheo a pheophytin a - PQ-9 plastoquinone-9 - P680 primary electron donor in PS II - PS II RC Photosystem II reaction centre - Q_A primary electron acceptor in PS II - SB-12 N-dodecyl-N,N-dimethyl-3-amino-1-propanesulphonate, (sulphobetain 12)     

Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors

We demonstrated recently that norepinephrine activates Ca²⁺-permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing _1A-adrenergic receptors (CHO-_1A). Moreover, extracellular Ca²⁺ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G₁₂ and G₁₃ (G₁₂G228A and G₁₃G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₃G225A were smaller than those in CHO-_1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₂G228A were similar to those in CHO-_1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca²⁺ influx. G₁₃G225A, but not G₁₂G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the G_q/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G₁₃ couples with CHO-_1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G₁₃ is involved in norepinephrine-induced arachidonic acid release in CHO-_1A. norepinephrine; _1A-adrenergic receptor; nonselective cation channel; G₁₃ protein; arachidonic acid release     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.