The relationship between carbon dioxide fixation and chlorophyll a fluorescence during induction of photosynthesis in maize leaves at different temperatures and carbon dioxide concentrations

The rate of CO₂ fixation (F_c) and 680 nm chlorophyll fluorescence emission (F680) were measured simultaneously during induction of photosynthesis in Zea mays L. leaves under varying experimental conditions in order to assess the validity of fluorescence as an indicator of in vivo photosynthetic carbon assimilation. Z. mays leaves showed typical Kautsky fluorescence induction curves consisting of a fast rise in emission (O to P) followed by a slow quenching via a major transient (S-M) to a steady-state (T). After an initial lag, net CO₂ assimilation commenced at a point corresponding to the onset of the S-M transient on the F680 induction curve. Subsequently, F_c and F680 always arrived at a steady-state simultaneously. Decreasing the dark-adaption period increased the rate of induction of both parameters. Alteration of leaf temperature produced anti-parallel changes in induction characteristics of F_c and F680. Reducing the CO₂ level to below that required for saturation of photosynthesis also produced anti-parallel changes during induction, however, at CO₂ concentrations tenfold greater than the atmospheric level the rate of F680 quenching from P to T was appreciably reduced without a similar change in the induction of F_c. Removal of CO₂ at steady-state produced only a small increase in F680 and a correspondingly small decrease in F680 occurred when CO₂ was re-introduced. The complex relationship between chlorophyll fluorescence and carbon assimilation in vivo is discussed and the applicability of fluorescence as an indicator of carbon assimilation is considered.Abbreviations F_c rate of CO₂ fixation - F680 fluorescence emission at 680 nm     

The cyanobacterium Synechococcus modulates Photosystem II function in response to excitation stress through D1 exchange

In this minireview we discuss effects of excitation stress on the molecular organization and function of PS II as induced by high light or low temperature in the cyanobacterium Synechococcus sp. PCC 7942. Synechococcus displays PS II plasticity by transiently replacing the constitutive D1 form (D1:1) with another form (D1:2) upon exposure to excitation stress. The cells thereby counteract photoinhibition by increasing D1 turn over and modulating PS II function. A comparison between the cyanobacterium Synechococcus and plants shows that in cyanobacteria, with their large phycobilisomes, resistance to photoinhibition is mainly through the dynamic properties (D1 turnover and quenching) of the reaction centre. In contrast, plants use antenna quenching in the light-harvesting complex as an important means to protect the reaction center from excessive excitation.Abbreviations D1 reaction center protein of Photosystem II - P₆₈₀ the reaction center of Photosystem II - Q_A the primary quinone acceptor of Photosystem II - Tyr_Z tyrosine electron donor to P₆₈₀     

The role of carbon dioxide and oxygen in determining chlorophyll fluorescence quenching during leaf development

Chlorophyll fluorescence emission at 680 nm (F680) and the rate of CO₂ fixation were measured simultaneously in sections along the length of wheat and maize leaves. These leaves possess a basal meristem and show a gradation in development towards the leaf tip. The redox state of the primary electron acceptor, Q, of photosystem II was estimated using a non-invasive method. Distal mature leaf sections displayed typical F680 induction curves which were generally anti-parallel with CO₂ fixation and during which Q became gradually oxidised. In leaf-base sections net assimilation of CO₂ was not detectable, F680 quenched slowly and monotonously without displaying any of the oscillations typical of mature tissue and Q remained relatively reduced. Sections cut from mid-regions of the leaf showed intermediate characteristics. There were no major differences between the wheat and maize leaf in the parameters measured. The results support the hypothesis that generation of the transthylakoid proton gradient and associated ATP production is not a major limitation to photosynthesis during leaf development in either C₃ or C₄ plants. Removal of CO₂ from the mature leaf sections caused little change in steady-state F680 and produced about 50% reduction of Q. When O₂ was then removed, F680 rose sharply and Q became almost totally reduced. In immature tissue unable to assimilate CO₂, removal of O₂ alone caused a similar large rise in F680 and reduction of Q whilst removal of CO₂ had negligible effects on F680 and the redox state of Q. It is concluded that in leaf tissue unable to assimilate CO₂, either because CO₂ is absent or the tissue is immature, O₂ acts as an electron acceptor and maintains Q in a partially oxidised state. The important implication that O₂ may have a role in the prevention of photoinhibition of the photochemical apparatus in the developing leaf is discussed.Abbreviations F680 chlorophyll fluorescence emission at 680 nm - PSI photosystem I - PSII photosystem II - Q PSII primary electron acceptor - pH transthylakoid proton gradient     

Acidosis,glycolysis and energy state in anaerobic heart tissue from rainbow trout

With focus on metabolism not depending on contractility in myocardial tissue from rainbow trout, Oncorhynchus mykiss, the effects of high CO₂ on lactate production, phosphocreatine, creatine, ATP, ADP, AMP and intracellular pH were examined under a blockage of cell respiration either alone or in combination with a glycolytic inhibition. Irrespective of metabolic interventions, a change in CO₂ from 1 to either 11 or 5% of the gas mixture perfusing the muscle bath with 15 mmol·l^-1 HCO _- ³ caused a drop of intracellular pH from 7.4 to either 6.5 or 7.0, respectively. An elevation of CO₂ to 11% diminished the rate of anaerobic lactate formation and slightly lowered anaerobic energy degradation. The further addition of 1 mmol·l^-1 iodoacetate to inhibit glycolysis strongly enhanced the tendency of acidosis to lower energy degradation. Moreover, iodoacetate induced a parallel decrease in ATP and total concentration of phosphorylated adenylates and an increase in resting tension. These effects were all substantially dampened by acidosis and could not immediately be related to tissue content of energy-rich phosphates. Tentatively, the depression of resting tension was the prime effect and a cause of the other effects acidosis. However, these were not affected by an inhibition of resting tension development with 2,3-butadione monoxime. The results suggest that glycolysis protects the anaerobic myocardium also by means not immediately related to tissue energy state. Acidosis exerts a similar protection, which is marginal as long as glycolysis is fully active, but substantial with an inhibited glycolysis.Abbreviations Cr _t total tissue concentration of creatine - G _PCr energy liberated per mol PCr hydrolyzed - IAA iodoacetate - PCr phosphocreatine - PE total tissue concentration of energy-rich phosphate bonds - pH _i intracellular pH - P _i inorganic phosphate - TAN total tissue concentration of phosphorylated adenylates - 2,3-BDM 2,3-butadione monoxime - SE standard error of the mean     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Key cofactors of Photosystem II cores from four organisms identified by 1.7-K absorption, CD and MCD

Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98–103]. Electrochromic shifts resulting from Q_A reduction by 1.7-K illumination were recorded, and the Q_x and Q_y absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Q_x transition is ∼3 nm (100 cm⁻¹) to higher energy in cyanobacteria than in the plants. The predominant Q_y shift appears in the range 683–686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Q_y region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.     

Bacteriochlorophyll c monomers,dimers, and higher aggregates in dichloromethane,chloroform, and carbon tetrachloride

Bacteriochlorophyll c in vivo is a mixture of at least 5 homologs, all of which form aggregates in CH₂Cl₂, CHCl₃ and CCl₄. Three homologs exist mainly in the 2-R-(1-hydroxyethyl) configuration, whereas the other two homologs, 4-isobutyl-5-ethyl and 4-isobutyl-5-methyl farnesyl bacteriochlorophyll c, exist mainly in the 2-S-(1-hydroxyethyl) configuration (Smith KM, Craig GW, Kehres LA and Pfennig N (1983) J. Chromatograph. 281: 209–223). In CCl₄ the S-homologs form an aggregate of 2–3 molecules whose absorption (747 nm maximum) and circular dichroism spectra resemble those of the chlorosome. In CH₂Cl₂, CHCl₃ and CCl₄ the 4-n-propyl homolog (R-configuration) forms dimers absorbing at ca. 680 nm and higher aggregates absorbing at 705–710 nm. In CCl₄ the dimerization constant is approx. 10 µM^–1 (1000 times that for chlorophyll a). The difference between the types of aggregates formed by the 4-n-propyl and 4-isobutyl homologs is attributed to the difference between the R- and S-configurations of the 2-(1-hydroxyethyl) groups in each chlorophyll.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - MEF 4-methyl-5-ethyl farnesyl - PEP 4-n-propyl-5-ethyl farnesyl     

Site of salicylaldoxime interaction with photosystem II

The reversible inhibition of Photosystem II by salicylaldoxime was studied in spinach D-10 particles by fluorescence, optical absorption, and electron spin resonance spectroscopy. In the presence of 15 mM salicylaldoxime, the initial fluorescence yield was raised to the level of the maximum fluorescence, indicating efficient charge recombination between reduced pheophytin (Ph) and P680⁺. In agreement with the rapid (ns) backreaction expected between Ph^– and P680⁺, the optical absorption transient at 820 mm was not observed. When the particles were washed free of salicylaldoxime, the optical absorption transient resulting from the rereduction of P680⁺ was restored to the µs timescale. These results, along with the previously observed inhibition of electron transport reactions and diminution of the 515-nm absorption change in chloroplasts [Golbeck, J.H. (1980) Arch Biochem Biophys 202, 458–466], are consistent with a site of inhibition between Ph and QA in Photosystem II. ESR Signal IIf and Signal Its were abolished in the presence of 25 mM salicylaldoxime, but both signals could be recovered by washing the D-10 particles free of the inhibitor. The loss of Signal Ilf is most likely a consequence of the inhibition between Ph and Q_A; the rapid charge recombination between Ph^– and P680⁺ would preclude electron transfer from an electron donor on the oxidizing side of Photosystem II. The loss of Signal Its may be due to a change in the environment of the donor complex such that the semiquinone radical giving rise to Signal Its interacts with a nearby reductant.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited chloroplasts - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F₀ prompt chlorophyll a fluorescence yield - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_var variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino) ethanesulfonic acid - P680 reaction center chlorophyll a of photosystem II - Ph pheophytin intermediate electron acceptor - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - Tris tris(hydroxymethyl)aminomethane - Z electron donor to P680⁺     

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y _z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680⁺ was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680⁺ reduction by Y _z under conditions where the catalytic site of water oxidation stays in the redox state S₁. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca²⁺. The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the Q_B-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680⁺ reduction by Y _z , and (c) Ca²⁺ could play a functional role for the electronic and vibrational coupling between the redox groups Y _z and P680. The electron transport from Y _z to P680⁺ is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - ANT 2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5 dinitrothiophene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2[N-Morpholino]ethanesulfonic acid - PS II photosystem II - Q_A, Q_B primary and secondary plastoquinone acceptor of photosystem II - S _i redox states of the catalytic site of water oxidation - Y _z redox active Tyr-161 of polypeptide D-1     

Aggregation of bacteriochlorophyll c homologs to dimers,tetramers, and polymers in water-saturated carbon tetrachloride

Three homologs of BChl c, 2-(R)-(1-hydroxyethyl)-4-n-propyl-5-ethyl-farnesyl BChl c (PEF-BChl c), 2-(R)-(1-hydroxyethyl)-4-ethyl-5-ethyl-farnesyl BChl c (EEF-BChl c), and 2-(S)-(1-hydroxyethyl)-4-isobutyl-5-methyl/ethyl-farnesyl BChl c (iBM/EF-BChl c), formed aggregates in water-saturated carbon tetrachloride (H₂O-satd CCl₄). The water content was about 100 times higher than that of the dried CCl₄ previously used. Absorption spectra were recorded for 8 concentrations for the three homologs of BChl c and were deconvoluted in terms of standard spectra of monomer, dimer, tetramer and polymer (747-nm aggregate, Olson and Pedersen (1990) Photosynthe Res 25: 25). PEF- and EEF-BChl c formed dimers (680 nm maximum) and tetramers (705–710 nm maximum), but iBM/EF-BChl c formed polymers. Inhibition of dimer formation by water faciliated the study of the initial stages of the polymerization of BChl c. When the logarithm of polymer concentration was plotted versus the logarithm of the monomer concentration for iBM/EF-BChl c, the initial slope was 30±10 and indicated the cooperation of 20–40 BChl c molecules to form a polymer from a seed. Circular dichroism spectra of the polymers with positive and negative bands at 743 and 760 nm, respectively, were similar to those for chlorosomes (Brune et al. (1990) Photosynth Res 24: 253).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - H₂O-satd CCl₄ water saturated carbon tetrachloride - PEF 4-n-propyl-5-ethyl farnesyl     

The involvement of lipids in light-induced charge separation in the reaction center of photosystem II

Extraction of PS II particles with 50 mM cholate and 1 M NaCl releases several proteins (33-, 23-, 17- and 13 kDa) and lipids from the thylakoid membrane which are essential for O₂ evolution, dichlorophenolindophenol (DCIP) reduction and for stable charge separation between P680⁺ and Q_A ^-. This work correlates the results on the loss of steady-state rates for O₂ evolution and PS II mediated DCIP photo-reduction with flash absorption changes directly monitoring the reaction center charge separation at 830 nm due to P680⁺, the chlorophyll a donor. Reconstitution of the extracted lipids to the depleted membrane restores the ability to photo-oxidize P680 reversibly and to reduce DCIP, while stimulating O₂ evolution minimally. Addition of the extracted proteins of masses 33-, 23- and 17- kDa produces no further stimulation of DCIP reduction in the presence of an exogenous donor like DPC, but does enhance this rate in the absence of exogenous donors while also stimulating O₂ evolution. The proteins alone in the absence of lipids have little influence on charge separation in the reaction center. Thus lipids are essential for stable charge separation within the reaction center, involving formation of P680⁺ and Q_A ^-.Abbreviations A₈₃₀ Absorption change at 830 nm - Chl Chlorophyll - D₁ primary electron donor to P680 - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - MOPS 3-(N-morpholino)propanesulfonic acid - P680 reaction center chlorophyll a molecule of photosystem II - PPBQ Phenyl-p-benzoquinone - PS II Photosystem II - Q_A, Q_B first and second quinone acceptors in PS II - V-DCIP rate of DCIP reduction - V-O₂ rate of oxygen evolution - Y water-oxidizing enzyme system - CHAPS 3-Cyclohexylamino-propanesulfonic acid     

Light saturation response of inactive photosystem II reaction centers in spinach

Oxygen evolving photosystem II particles were exposed to 100 and 250 W m^–2 white light at 20°C under aerobic, anaerobic and strongly reducing (presence of dithionite) conditions. Three types of photoinactivation processes with different kinetics could be distinguished: (1) The fast process which occurs under strongly reducing (t _1/21–3 min) and anaerobic conditions (t _1/24–12 min). (2) The slow process (t _1/215–40 min) and (3) the very slow process (t _1/2>100 min), both of which occur under all three sets of conditions.The fast process results in a parallel decline of variable fluorescence (F _v) and of Hill reaction rate, accompanied by an antiparallel increase of constant fluorescence (F _o). We assume that trapping of Q_A in a negatively charged stable state, (Q_A ^–)_stab, is responsible for the effects observed.The slow process is characterized by a decline of maximal fluorescence (F _m). In presence of oxygen this decline is due to the well known disappearance of F _v which proceeds in parallel with the inhibition of the Hill reaction; F _o remains essentially constant. Under anaerobic and reducing conditions the decline of F _m represents the disappearance of the increment in F _o generated by the fast process. We assume that the slow process consists in neutralization of the negative charge in the domain of Q_A in a manner that renders Q_A non-functional. The charge separation in the RC is still possible, but energy of excitation becomes thermally dissipated.The very slow photoinactivation process is linked to loss of charge separation ability of the PS II RC and will be analyzed in a forthcoming paper.Abbreviations F chlorophyll a fluorescence - F _o, F _v, F _m constant, variable, maximum fluorescence - F _o, F _v, F _m the same, measured in presence of dithionite (F _v suppression method) - PS II photosystem II - RC reaction centre (P680. Pheo) - P680 primary electron donor - Pheo pheophytin, intermediary electron acceptor - Q_A, Q_B the primary and secondary electron acceptor - Z, D electron donors to P680 - (Q_A)_stab, (Q_A H)_stab hypothetical modifications of Q_A resulting from photoinactivation - O-, A- and R-conditions aerobic, anaerobic and strongly reducing (presence of dithionite) conditions - MES 2-(N-morpholine) ethanesulphonic acid - DCPIP 2,6-dichlorphenolindophenol - GGOC mixture of glucose, glucose oxidase and catalase - DT-20 oxygen-evolving PS II particles     

Tetranitromethane modification of photosystem 2

Inhibition of photosystem 2 by the peptide-modification reagent, tetranitromethane, has been investigated with spinach digitonin particles. In the presence of tetranitromethane, (1) the initial fluoresence yield is suppressed with a concomitant elimination of the variable component of fluorescence; (2) the optical absorption transient at 820 nm, attributed to P680⁺, is greatly attenuated; (3) diphenylcarbazide-supported photoreduction of dichlorophenol indophenol is abolished; and (4) electron spin resonance Signal 2f and Signal 2s are eliminated. These results are consistent with multiple sites of modification in photosystem 2 by tetranitromethane, and suggest further that this reagent can inhibit charge stabilization in the reaction center.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited photosystem 2 preparations - DPIP 2,6-dichlorophenol indophenol - esr electron spin resonance - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_v variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino)ethanesulfonic acid - P680 primary electron donor chlorophyll of photosystem 2 - Ph pheophytin - PS 2-photosystem 2 - Q_a primary quinone electron acceptor - Q_b secondary quinone acceptor - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine - TNM tetranitromethane     

Thermoluminescence study of the in vivo effects of bicarbonate depletion and acetate/formate presence in the two algae Chlamydobotrys stellata and Chlamydomonas reinhardtii

We investigated the influence of CO₂/HCO₃ ^–-depletion and of the presence of acetate and formate on the in vivo photosynthetic electron transport in the two green algae Chlamydobotrys stellata and Chlamydomonas reinhardtii by means of thermoluminescence technique and mathematical glow curve analysis. The main effects of the removal of CO₂ from the algal cultures was: (1) A shift of the glow curve peak position to lower temperatures resulting from a decrease of the B band and an increase of the Q band. (2) Treatment of CO₂-deficient Chl. stellata with DCMU yielded two thermoluminescence bands in the Q band region peaking at around +12°C and +5°C; in case of Chl. reinhardtii DCMU treatment induced only one band with an emission maximum at +5°C. The presence of acetate or formate in CO₂-depleted algal cultures lowered the intensities of all of the individual TL bands but that of a HT band (TL+37). The effects of CO₂-depletion and of the presence of anions were fully reversible.Abbreviations DCMU 3-(3,4)-dichlorophenyl-1,1-dimethylurea - HT band high temperature TL band - P₆₈₀ reaction center chlorophyll of PS II - Q_A and Q_B primary and secondary quinone acceptors of PS II, respectively - PS II Photosystem II - S_2/3 redox states of the oxygen evolving complex of PS II - TL thermoluminescence     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

The donor side of Photosystem II as the copper-inhibitory binding site

We have measured, under Cu (II) toxicity conditions, the oxygen-evolving capacity of spinach PS II particles in the Hill reactions H₂OSiMo (in the presence and absence of DCMU) and H₂OPPBQ, as well as the fluorescence induction curve of Tris-washed spinach PS II particles. Cu (II) inhibits both Hill reactions and, in the first case, the DCMU-insensitive H₂O SiMo activity. In addition, the variable fluorescence is lowered by Cu (II). We have interpreted our results in terms of a donor side inhibition close to the reaction center. The same polarographic and fluorescence measurements carried out at different pHs indicate that Cu (II) could bind to amino acid residues that can be protonated and deprotonated. In order to reverse the Cu (II) inhibition by a posterior EDTA treatment, in experiments of preincubation of PS II particles with Cu (II) in light we have demonstrated that light is essential for the damage due to Cu (II) and that this furthermore is irreversible.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenilcarbazide - F_o initial non-variable fluorescence - F_I intermediate fluorescence yield - F_m maximum fluorescence yield - F_v variable fluorescence yield - Mes 2,-(N-morpholino)ethanosulfonic acid - OEC oxygen-evolving complex - P680 Primary electron donor chlorophyll - Pheo pheophytin - PPBQ phenyl-p-benzo-quinone - PS II Photosystem II - SiMo Silicomolybdate - Q_B secondary quinone acceptor - Q_A primary quinone aceptor - Tris N-tris(hydroxymethyl)amino ethane - Tyr_z electron carrier functioning between P680 and the Mn cluster This article is dedicated to Prof. Dr. Harmut Lichtenthaler on the occasion of his 60th birthday.     

Involvement of the donor tyrosine-D1 (Yd) in Photosystem II electron transport in the green alga, Chlamydobotrys stellata

The light-induced oxidation of the accessory donor tyrosine-D (Y_D) has been studied by measurements of the EPR Signal II_slow at room temperature in the autotrophically and photoheterotrophically cultivated alga Chlamydobotrys stellata. After illumination and dark adaptation, Y_D Signal II_slow was observed only in autotrophic algae, i.e. under conditions of a linear photosynthetic electron transfer from water to NADP⁺. The addition of artificial electron acceptors phenyl-p-benzoquinone (PPQ) or dichloro-p-benzoquinone (DCQ) to the autotrophic cells caused an almost negligible increase of this signal. When photosynthetic electron flow and oxygen evolution were diminished by removal of the carbon source CO₂ and addition of acetate (photoheterotrophy), a pronounced Y_D Signal II_slow was seen only in presence of DCQ or PPQ. Several possibilities are discussed to explain the absence of Y_D Signal II_slow in photoheterotrophic Chl. stellata such as the existence of a cyclic PS II electron flow very effectively reducing P₆₈₀ and thereby preventing the possibility of Y_D oxidation. Artificial electron acceptors withdraw electrons from this cycle thus keeping the primary quinone acceptor, Q_A, oxidized and thereby diminishing the reduction of P₆₈₀ ⁺ by cyclic PSII. This leads to the appearance of the Y_D Signal II_slow also in the photoheterotrophically grown algae.Abbreviations A-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - DCQ- 2,5-dichlorobenzoquinone - D₂- structure protein of Photosystem II - EPR- electron paramagnetic resonance - OEC- oxygen evolving complex - PPQ- phenyl-p-benzoquinone - PS II- Photosystem II - P₆₈₀- reaction center of Photosystem II - Q-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - S_i- oxidation levels of the OEC - Y_D- tyrosine-D accessory donor to P₆₈₀ - Y_Z- tyrosine-Z electron donor to P₆₈₀ Dedicated to Prof. Dr E. Schnepf/Heidelberg.     

Functional size of Photosystem II determined by radiation inactivation

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy -rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the Q_B site: H₂O (Mn)₄ Y_z P680 Pheophytin Q phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the and subunits of cytochrome b ₅₅₉and the 4.5 kDa psbI gene product.Abbreviations Cyt cytochrome - PS Photosystem - P680 primary electron donor of Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II - Y_z tyrosine donor to P680     

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 131-deoxo-131-hydroxy-pheophytin a and comparison of their 6 K absorption spectra

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13¹-deoxo-13¹-hydroxy-pheophytin a (13¹-OH-Pheo). The absorption bands of 13¹-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC_1×) or twice (RC_2×). HPLC analysis and Pheo Q_X absorption at 543 nm show that in RC_1× about 50% of Pheo is replaced and in RC_2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo⁻ is unmodified in RC_1× and decreased only in RC_2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the Q_Y region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC_1× or RC_2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength. This revised version was published online in June 2006 with corrections to the Cover Date.     

pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome (Cyt b ₅₅₉). At pH 6.5 it was found that Cyt b ₅₅₉ was reversibly photoreduced by red actinic light when Mn²⁺ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b ₅₅₉ was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942–10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b ₅₅₉ is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b ₅₅₉ increases, with increasing pH, the rate of irreversible photo-oxidation of -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680