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PubMed1978

Determination and modification of the redox state of the secondary acceptor of photosystem II in the dark.

The redox state of the secondary electron acceptor B of Photosystem II was studied using flourescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given B/B-ratio in the dark-adapted samples. We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts. Benzoquinone treatment modifies this distribution in Clorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark. The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B- is generated and stabilized in the dark.

Chlorella↗
PubMed1990

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments.

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.

Acetates↗
PubMed1989

Retinal isomer ratio in dark-adapted purple membrane and bacteriorhodopsin monomers.

On the basis of data obtained by spectroscopic analysis and chromatography of retinal extracts, a consensus has been adopted that dark-adapted purple membrane (pm) contains 13-cis- and all-trans-retinal in equal amounts, whereas the light-adapted membrane contains all-trans-retinal only. We have developed an improved extraction technique which extracts up to 70% of the retinal in pm within 4 min. In the extracts from dark-adapted pm at room temperature, we consistently find 66-67% 13-cis-retinal and 33-34% all-trans-retinal, and more than 98.5% all-trans isomer in light-adapted samples. The spectrum obtained by reconstitution of bacterioopsin with 13-cis-retinal at 2 degrees C (to minimize isomerization) shows an absorbance maximum at 554 nm and agrees well with the spectrum for the 13-cis component calculated from the dark-adapted and light-adapted bR spectra with our extraction data. The ratio of 13-cis:all-trans isomer in dark-adapted pm is 2:1 and nearly constant between 0 and 38 degrees C but begins to decrease distinctly above 40 degrees C, and more rapidly near 70 degrees C, reaching 0.75 at 90 degrees C. The van't Hoff plot of the isomer ratio shows a nonlinear temperature dependence above 40 degrees C, indicating a more complex system than a simple thermal 13-cis/all-trans isomer equilibrium. We attribute the broadening, absorbance decrease, and blut shift of the visible absorption band with increasing temperature to the appearance of at least one and possibly two or three new chromophores which contain, mainly or exclusively, the all-trans isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1987

Selected ion monitoring analysis of monoamine metabolites in cerebrospinal fluid. Application to the study of in vivo effects of alpha 2-antagonists.

The technique of isotope dilution mass spectrometry has been used for the measurement of biogenic amine metabolites in cerebrospinal fluid (CSF). CSF samples were collected from rabbits treated with alpha 2-antagonists. The aim of our study was to determine the specificity of these drugs on the central nervous noradrenergic, dopaminergic and serotonergic activity as measured by the release of corresponding monoamine metabolites. 3-Methoxy-4-hydroxyphenylethylene glycol (MHPG) and vanilmandelic acid (VMA) were used as parameters for the noradrenergic activity, whereas homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) were employed to follow the dopaminergic and serotonergic activity, respectively. For the measurement of the biogenic amine metabolites a published GCMS method has been adapted. Samples of 200 microliters CSF were processed. Following addition of deuterated internal standards and acidification, extraction was carried out with ethyl acetate. Preliminary experiments with the analysis of MHPG using diethyl ether for extraction gave rise to emulsion formation and resulted in poor recoveries for MHPG and in irreproducibility problems due to a preferential extraction of non-labelled MHPG, effects which were not observed with ethyl acetate extraction. Derivatization was done with a mixture of pentafluoropropionic anhydride/pentafluoropropanol (or hexafluoroisopropanol) in order to derivatize both hydroxyl and carboxylic acid groups. The derivatization procedure was optimized for the analysis of 5-HIAA by carrying out a second reaction step with pentafluoropropionic anhydride alone in order to complete the derivatization for the indolic NH moiety. The molecular ions of the derivatized products were selected for detection.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed1985

Adenine nucleotide and P-creatine levels in layers of frog retina as a function of dark and light adaptation.

Eight layers of frog retina were analyzed for ATP, P-creatine, ATP + ADP, and AMP under conditions of dark, 2 sec, 2 min, and in the case of AMP, 2 hr of light adaptation. Samples of each layer, usually ranging between 5 and 50 ng, were dissected from lyophilized frozen sections. After brief light exposure, ATP dropped while ADP rose sharply in the pigment epithelium, outer segments, and inner segments; ADP was too low to be measured accurately in the inner retina. The profile of ATP, P-creatine, and ATP + ADP concentrations showed peaks in the inner nuclear and ganglion layers. AMP, by contrast, was highest in the two plexiform layers. Levels in the inner retina dropped after only 2 sec of light but rose after 2 hr to levels that were higher than dark values in all retinal layers. AMP was often characterized by a non-uniform distribution: adjacent areas of a layer agreed very closely in value to each other but could vary several-fold from a different section of the same frog or from another frog exposed to the same conditions. This distribution produced clusters of values, particularly prominent in dark-adapted animals, something not observed with the other metabolites measured. The peaks of AMP in the plexiform layers suggest that AMP may be a by-product of dopamine-stimulated adenylate cyclase which also has peaks in these same layers.

Adenine Nucleotides↗
PubMed1991

Unusual low reactivity of the water oxidase in redox state S3 toward exogenous reductants. Analysis of the NH2OH- and NH2NH2-induced modifications of flash-induced oxygen evolution in isolated spinach thylakoids.

The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].

Hydrazines↗
PubMed1990

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin.

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.

Bacteriorhodopsins↗
PubMed1980

The orientation of the magnetic axes of the membrane-bound iron-sulfur clusters of spinach chloroplasts.

Spinach chloroplast membranes were oriented onto mylar sheets by partial dehydration, and the orientation of the magnetic axes of membrane-bound paramagnetic clusters determined by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the reduced Rieske iron-sulfur cluster signal is of orthorhombic symmetry oriented with th gy = 1.90 axis orthogonal to the membrane plane and with the gz = 2.03 axis in the membrane plane; the gx-axis is undetectable, presumably due to its broadness. If the Rieske center is a two-iron iron-sulfur cluster, we conclude that the iron-iron axis lies in the plane of the membrane. Illumination reduces the two bound chloroplast iron-sulfur proteins known as Clusters A and B. Center A is oriented such that gx = 1.86 and gy = 1.94 lie at an angle of about 40, and gz = 2.05 is at approximately 25, to the membrane plane. There are two possible orientations of Cluster B depending on the set of g-values assigned to this cluster. For one set of g-values, gz = 2.04 and gx = 1.89 are oriented in the plane of the membrane while gy = 1.92 is orthogonal to the plane. Alternatively, gz = 2.07 and gy = 1.94 are oriented approximately 50 and 40 to the membrane plane respectively, and gx = 1.80 is in the plane of the membrane. An additional light-induced signal at g = 2.15 oriented orthogonal to the plane is currently unexplained, as are other membrane perpendicular signals seen at g = 2.3 and g = 1.73 in dark-adapted samples.

Cell Membrane↗
PubMed1984

Interaction of linolenic acid with bound quinone molecules in Photosystem II. Time-resolved optical and electron spin resonance studies.

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.

Chloroplasts↗
PubMed1992

Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942.

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40 degrees C in wild-type thylakoids to about 30 degrees C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak termperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of QB. (e) Mutations at the QB site not only influence the binding of QB and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S2 and S3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the QB site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and QB, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect QB or DCMU binding in Synechococcus PCC7942.

Atrazine↗
PubMed1995

Photocycle of halorhodopsin from Halobacterium salinarium.

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.

Bacteriorhodopsins↗
PubMed1998

On the origin of the '35-mus kinetics' of P680(+.) reduction in photosystem II with an intact water oxidising complex.

The origin of the '35-micros kinetics' of P680(+.) reduction in photosystem II (PS II) with an intact water oxidising complex has been analysed by comparative measurements of laser flash induced changes of the 830-nm absorption and the relative quantum yield of chlorophyll (Chl) fluorescence. The latter parameter was monitored at a time resolution of 500 ns by using newly developed home built equipment [Reifarth, F., Christen, G. and Renger, G. (1997) Photosynth. Res. 51, 231-2421. It was found that: (i) the amplitudes of the unresolved ns-kinetics of both 830-nm absorption changes and the rise of fluorescence yield exhibit virtually the same period four oscillation pattern when dark adapted samples are excited with a train of saturating laser flashes; (ii) the corresponding oscillation patterns of the normalised extent of the 35-micros kinetics under identical excitation conditions are strikingly different with maxima after the 3rd and 5th flash for the 830-nm absorption changes vs. pronounced maxima after the 4th and 8th flash for the rise of the fluorescence yield. The period four oscillations unambiguously show that the '35-micros kinetics' of P680(+.) reduction are characteristic for reactions in PS II entities with an intact water oxidising complex. However, the disparity of the oscillation patterns of (ii) indicates that in contrast to the ns components of P680(+.) reduction the 35-micros kinetics do not reflect exclusively an electron transfer from Y(Z) to P680(+.). It is inferred that a more complex reaction takes place which comprises at least two processes: (a) P680(+.) reduction by Y(Z) and (b) coupled and/or competing reaction(s) which give rise to additional changes of the chlorophyll fluorescence yield.

Chlorophyll↗
PubMed1997

Stochastic optimization algorithms of a Bayesian design criterion for Bayesian parameter estimation of nonlinear regression models: application in pharmacokinetics.

This article proposes three stochastic algorithms to optimize a Bayesian design criterion for Bayesian estimation of the parameters of nonlinear regression models; this criterion is the information expected from an experiment. The first algorithm is based on a stochastic version of the simplex with an adaptive sampling procedure. The others are stochastic approximation algorithms: the Kiefer-Wolfowitz and the pseudogradient algorithms. We first present the information criterion and the optimization algorithms. The efficiency of each algorithm for optimizing this Bayesian design criterion is then assessed by a simulation study for a nonlinear model assuming a discrete prior distribution. An application for designing an experiment to estimate the kinetics of radioiodine thyroid uptake is then proposed.

Algorithms↗
PubMed1998

High-throughput solid-phase extraction for the determination of cimetidine in human plasma.

For the implementation and validation of an automated 'high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C18 sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.

Chromatography, High Pressure Liquid↗
PubMed1997

Radioimmunoassay for the measurement of S9788 in serum and microdialysis samples.

S9788, 6-[4-(2,2-di-(fluorophenyl)-ethylaminol-1-piperdinyl]-N,N'-d i-2-propenyl-1, 3,5-triazine- 2, 4-diamine, is a novel compound designed to reverse tumour multidrug resistance associated with cancer chemotherapy. A specific and sensitive radioimmunoassay has been developed for the analysis of S9788 in serum samples and adapted for samples obtained by microdialysis. The limit of quantitation is 0.2 ng ml-1 in perfusion medium and there is no cross reactivity of the antibody with known metabolites of the parent compound or with certain cytotoxic compounds likely to be coadministered with S9788. Maximum probe recovery during microdialysis was 66% at a flow of 1 microliter min-1, using Ringer/BSA (70 mg ml-1) as the perfusion medium. The assay has sufficient sensitivity, precision, accuracy and specificity for the analysis of rat and human serum and microdialysis perfusate samples. The assay has been successfully applied to the determination of S9788 in rat plasma (total concentration) and the microdialysate of the same samples.

Animals↗
PubMed1995

Reconstitution of the endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles, and PS II core complexes from spinach.

The possibility of reconstituting a functionally competent endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles (ISO-vesicles), and PS II core complexes was analyzed by measuring (i) the characteristic period four oscillation of the oxygen yield due to excitation of dark-adapted samples with a train of short flashes and (ii) laser flash-induced transients of the relative quantum yield of chlorophyll fluorescence. The data obtained revealed that (a) an endogenous pool capacity comparable to that of intact thylakoids can be restored in PS II membrane fragments and ISO-vesicles by a sonication treatment using native plastoquinone-9, (b) a more pronounced oxygen oscillation pattern arises in PS II core complexes after application of the same reconstitution procedure, (c) the extent of the endogenous pool restoration at a ratio of 15 quinone molecules per PS II in the reconstitution assay strongly depends on the nature of the quinone molecule [maximum effects can be only achieved with PQ-9, while at the same concentration ubiquinone-45 (UQ-9) is almost inefficient], and (d) a sonication step is required for stable insertion of PQ-9 into PS II preparations. Measurements of the reconstruction degree as a function of the structure of different quinones with selected properties lead to the conclusion that specific binding domains exist in PS II in addition to the QB site. These domains exhibit a surprisingly high specificity for the type of quinone that can be bound. On the basis of a comparison of the results obtained, the structure of the quinone head group seems to be more important than the large hydrophobic side chain and/or the general lipophilicity of the compound.

Fluorescence↗
PubMed1993

Structure-function relations in photosystem II. Effects of temperature and chaotropic agents on the period four oscillation of flash-induced oxygen evolution.

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured in dark-adapted samples as a function of temperature and upon addition of chaotropic agents. The following results were obtained: (a) Within the range of 0 < theta < 35 degrees C, the ratio of the oxygen yield induced by the 4th and 3rd flashes of the train, Y4/Y3, and the oxygen yield induced by the 2nd flash, Y2, exhibit similar dependencies on the temperature in isolated thylakoids, PS II membrane fragments, and inside-out vesicles. (b) Below a characteristic temperature theta c of 20-25 degrees C, the values of Y4/Y3 and Y2, which reflect (at constant S0 dark population) the probabilities of misses and double hits, respectively, remain virtually independent of temperature, whereas above theta c these parameters increase. (c) The dark decays of S2 and S3 via fast and slow kinetics due to reduction of the water oxidase by YD and other endogenous electron donor(s), respectively, exhibit comparatively strong temperature dependencies in thylakoids with the following activation energies: EA(S2fast) = 55 kJ/mol, EA(S3fast) = 50 kJ/mol, EA(S2slow) = 85 kJ/mol, and EA(S3slow) = 75 kJ/mol. The activation energy of S0 oxidation to S1 by YDox was found to be markedly smaller with a value of EA(S0) = 30 kJ/mol. (d) Incubation with chaotropic agents at concentrations which do not significantly impair the oxygen evolution capacity leads to modifications of the oscillation pattern with remarkable differences for various types of agents: Tris and urea are practically without effect; guanidine hydrochloride affects Y4/Y3 in a similar way as elevated temperature but without significant changes of Y2 and the decay kinetics of S2 and S3; and anions of the Hofmeister series (SCN-, ClO4-, I-) cause a drastic destabilization of YDox. Possible structure-function relations of the PS II complex are discussed on the basis of these findings.

Guanidine↗
PubMed1992

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome b Group↗