Monitoring electron transfer by photoacoustic spectroscopy in native and immobilized thylakoid membranes

Photoacoustic spectroscopy was used to monitor photo synthetic electron transfer in native and immobilized thylakoid membranes. The photoacoustic parameter phi(r)' (the percentage of absorbed energy that is stored in photo chemical intermediates) and i(50) (the half-saturation modulated light intensity) were directly correlated to electron transfer rates. As previously shown, thylakoids immobilized in an albumin-glutaraldehyde matrix were more resistant to aging. The inhibitory effects of the immobilization procedure and of aging at 4 degrees C were detected as a decrease in i(50) values. In analogy with enzyme kinetic analysis, the effect could be characterized as a competitive type of inhibition. Photoacoustic measurements are performed in conditions similar to a working bioreactor cell with regards to the sample preparation.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Sites of inhibition by disulfiram in thylakoid membranes

Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between Q_A, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, Q_B. This site of inhibition is shown by a severalfold increase in the half-time of Q⁻_A oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II; disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from H₂O to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.     

Photopolymers as an immobilized matrix in biosensing

Biosensors are perspective devices for analysis of substances in chemistry, biological chemistry, medicine, biotechnology and also for environment status monitoring. Their advantages are compact size, short time of analysis, display high response and simplicity in usage. The working characteristics of biosensors often depend on efficacy of a biological stuff immobilization on the surface of transducer. In this context there is a need for development of immobilization methods capable to provide for execution of the following demands: 1) compatibility of this process with technology of building transducer; 2) simplicity in fulfillment, cheapness and expressness at manufacture of biomembranes; 3) ability to provide the maximal safety of a biological stuff activity and its high adhesion to a surface of transducer; 4) reproducibility at serial application and capacity of standardizing. Usage of photocrosslinked and photopolymerized compound at the immobilization of a biological stuff allows to provide execution of the listed above demands. The present review is devoted to features of application of the given class of compound at building of biosensors.     

Architectural switches in plant thylakoid membranes

Recent progress in elucidating the structure of higher plants photosynthetic membranes provides a wealth of information. It allows generation of architectural models that reveal well-organized and complex arrangements not only on whole membrane level, but also on the supramolecular level. These arrangements are not static but highly responsive to the environment. Knowledge about the interdependency between dynamic structural features of the photosynthetic machinery and the functionality of energy conversion is central to understanding the plasticity of photosynthesis in an ever-changing environment. This review summarizes the architectural switches that are realized in thylakoid membranes of green plants.     

Sigmoidal stimulation by solutes of light-induced proton translocation in thylakoid membranes

A sigmoidal curve was obtained for the relationship betweenthe stimulation of light-induced proton uptake and the concentrationof salts in a suspending medium for thylakoid membranes. Substitutionof sucrose for the salts also resulted in a sigmoidal curve.It changed into a hyperbolic curve with salts when the mediumalready contained sucrose. The results are discussed in relationto the structural arrangement of the thylakoid membranes bythe osmotic effect of the solutes. (Received February 10, 1975; )     

Removal of heavy metals by an Aspergillus terreus strain immobilized in a polyurethane matrix

AIMS: The aim was to investigate the biosorption of chromium, nickel and iron from metallurgical effluents, produced by a steel foundry, using a strain of Aspergillus terreus immobilized in polyurethane foam. METHODS AND RESULTS: A. terreus UFMG-F01 was immobilized in polyurethane foam and subjected to biosorption tests with metallurgical effluents. Maximal metal uptake values of 164.5 mg g(-1) iron, 96.5 mg g(-1) chromium and 19.6 mg g(-1) nickel were attained in a culture medium containing 100% of effluent stream supplemented with 1% of glucose, after 6 d of incubation. CONCLUSIONS: Microbial populations in metal-polluted environments include fungi that have adapted to otherwise toxic concentrations of heavy metals and have become metal resistant. In this work, a strain of A. terreus was successfully used as a metal biosorbent for the treatment of metallurgical effluents. SIGNIFICANCE AND IMPACT OF THE STUDY: A. terreus UFMG-F01 was shown to have good biosorption properties with respect to heavy metals. The low cost and simplicity of this technique make its use ideal for the treatment of effluents from steel foundries.     

Senescence induced structural reorganization of thylakoid membranes in Cucumis sativus cotyledons; LHC II involvement in reorganization of thylakoid membranes

We report the formation and appearance of loosely stacked extended grana like structures along with plastoglobuli in the chloroplasts isolated from 27-day old senescing cucumber cotyledons. The origin and the nature of these extended grana structures have not been elucidated earlier. We isolated Photosystem I complexes from 6-day-old control and 27-day-old senescing cotyledons. The chlorophyll a/b ratio of the isolated Photosystem I complex obtained from 6-day cotyledons was 5–5.5 as against a ratio of 2.9 was found in Photosystem I complexes obtained from 27-day-old senescing cotyledons. We also found that the presence of LHC II in the Photosystem I complexes isolated from 27-day cotyledonary chloroplasts. The presence of LHC II in Photosystem I complexes in senescing and not in control samples, clearly suggest the detachment and diffusion of LHC II complexes from stacked grana region to Photosystem I enriched stroma lamellar region thereby, forming loose disorganized extended grana structures seen in the transmission electron microscope. Furthermore, we show that under in vitro condition the senescing cotyledon chloroplasts exhibited lower extent of light induced phosphorylation of LHC II than the control samples suggesting a possible irreversible phosphorylation and diffusion of LHC II in vivo during the progress of senescence in Cucumis cotyledons. From these findings, we suggest that the senescence induced phosphorylation of LHC II and its migration towards Photosystem I may be a programmed one some how causing the destruction of the thylakoid membrane. The released membrane components may be stored in the plastoglobuli prior to their mobilization to the younger plant parts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low pH induced structural reorganization in thylakoid membranes

By using low temperature fluorescence spectroscopy, it has been shown that exposing chloroplast thylakoid membranes to acidic pH reversibly decreases the fluorescence of photosystem II while the fluorescence of photosystem I increases [P. Singh-Rawal et al. (2010) Evidence that pH can drive state transitions in isolated thylakoid membranes from spinach, Photochem Photobiol Sci, 9 830-837]. In order to shed light on the origin of these changes, we performed circular dichroism (CD) spectroscopy on freshly isolated pea thylakoid membranes. We show that the magnitude of the psi-type CD, which is associated with the presence of chirally ordered macroarrays of the chromophores in intact thylakoid membranes, decreases gradually and reversibly upon gradually lowering the pH of the medium from 7.5 to 4.5 (psi, polymer or salt induced). The same treatment, as shown on thylakoid membranes washed in hypotonic low salt medium possessing no psi-type bands, induces no discernible change in the excitonic CD. These data show that while no change in the pigment-pigment interactions and thus in the molecular organization of the bulk protein complexes can be held responsible for the observed changes in the fluorescence, acidification of the medium significantly alters the macro-organization of the complexes, hence providing an explanation for the pH-induced redistribution of the excitation energy between the two photosystems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

The formation of an inverted hexagonal phase from thylakoid membranes upon heating

Barley thylakoid membranes were studied with FTIR and EPR spectroscopy. Thylakoids were exposed to elevated temperatures in order to induce structural changes. As temperatures increased through physiological to even higher levels, no features changed, but upon heating to above 45 degrees C, the fraction of lipid acyl chain segments with gauche-type vibration increased, accompanied by a sharp drop in the membranous spin probe component. These apparently conflicting observations in fact concur with the formation of an inverted hexagonal (H(II)) phase, supporting its putative role in protecting the photosynthetic machinery in thylakoid membranes against thermally-induced disassembly.     

Freezing of isolated thylakoid membranes in complex media

Chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw treatment in a buffered medium containing 70 mM KCl, 30 mM NaNO₃ and 20 mM K₂SO₄ in different combinations. In the presence of the three predominant inorganic electrolytes, inactivation of photophosphorylation was mainly caused by a decrease in the capacity of the photosynthetic electron transport; release of proteins from the membranes was not manifest and light-induced H⁺ gradient and proton permeability were largely unaffected. Omission of nitrate from the medium had little effect. When either sulfate or chloride or both were omitted prior to freezing, inactivation of photophosphorylation was correlated with stimulation of the phosphorylating electron flow, marked increase in H⁺ permeability and loss of the ability of the thylakoids to accumulate protons in the light. In the absence of sulfate, uncoupling was mainly a consequence of the dissociation of chloroplast coupling factor (CF₁). Partial restoration of proton impermeability and pH gradient occurred upon the addition of N,N-dicyclohexylcarbodiimide (DCCD). When sulfate was present but chloride omitted, CF₁ remained attached to the membranes and the addition of DCCD had no effect, indicating that the increase in proton efflux was caused by a different mechanism. It is concluded that sulfate stabilizes the CF₁ and prevents its release from the membranes, but KCl is also necessary for maintaining the low permeability of the membranes to protons. The importance of complex media for investigations on isolated biomembrane systems is stressed.Abbreviations CF₁ chloroplast coupling factor - DCCD N,N-dicyclohexylcarbodiimide - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid I=Santarius 1986 b     

Protein determination of cells immobilized in cross-linked synthetic gels

Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.     

Lateral heterogeneity of photosystems in thylakoid membranes studied by Brownian dynamics simulations

The aggregation and segregation of photosystems in higher plant thylakoid membranes as stromal cation-induced phenomena are studied by the Brownian dynamics method. A theoretical model of photosystems lateral movement within the membrane plane is developed, assuming their pairwise effective potential interaction in aqueous and lipid media and their diffusion. Along with the screened electrostatic repulsive interaction the model accounts for the van der Waals-type, elastic, and lipid-induced attractive forces between photosystems of different sizes and charges. Simulations with a priori estimated parameters demonstrate that all three studied repulsion-attraction alternatives might favor the local segregation of photosystems under physiologically reasonable conditions. However, only the lipid-induced potential combined with the size-corrected screened Coulomb interaction provides the segregated configurations with photosystems II localized in the central part of the grana-size simulation cell and photosystems I occupying its margins, as observed experimentally. Mapping of thermodynamic states reveals that the coexistence curves between isotropic and aggregated phases are the sigmoidlike functions regardless of the effective potential type. It correlates with measurements of the chlorophyll content of thylakoid fragments. Also the universality of the phase curves characterizes the aggregation and segregation of photosystems as order-disorder phase transitions with the Debye radius as a governing parameter.     

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     

Diffusion of molecules and macromolecules in thylakoid membranes

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Liquid photo-polymerized compositions as an immobilized matrix of biosensors

Series of liquid photopolimerized compositions based on oligourethanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemethacrylate (OCM-2), butilmethacrylate, methacrylic that acid, monomethacrylic ether of ethylene glycol and vinylpirrolidone (VP) were tested. It was shown the optimal variant of enzyme sensor development was a composition containing VP (a basic hydrophylic matrix), OCM-2 (crosslinked components) and OUM-2000T (crosslinked and increasing adsorption of polymer component). The blend contains 3% of enzyme. The obtained biosensors as based on immobilized beta-glucose oxidase and ureases have the following charachteristics: the linear response in the range of the concentration 0.1-10 mM, 0.05-20 mM, angle of slope of curve 30 mV/pC, 38 mV/pC, and response time 10-15, 5-10 mines, respectively. The maximal response of urease sensor was in the diapazon of pH 6.0-6.5. The increase of NaCl concentration in the solution to 300 mM caused reduction of sensor response. Under this concentration the was latter equal to half of initial response. Further increase of NaCl concentration (to 500 mM) doesn't lead to further response reduction. K(m) was calculated and it was shown, that amount of immobilized urease and beta-glucose oxidase in photopolymer material was equal 0.85 and 3.1 mM respectively.     

The photosynthetic partial reactions involved in photoelectrochemical current generation by thylakoid membranes

Summary A thylakoids containing photoelectrochemical cell was used to monitor the photocurrent under photentiostatic mode using specific electron donnors and acceptors, and inhibitors of electron transfer. It is shown that both photosystem I and II can generate a photocurrent under the appropriate conditions. The photocurrent was also monitored in the absence of oxygen evolution thus suggesting a possible application for hydrogenase catalysed hydrogen production.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol-indophenol - DCBQ 2,3-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DPC p-diphenylcarbazide - FeCN potassium ferricyanide     

Protein diffusion and macromolecular crowding in thylakoid membranes

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 +/- 0.4 x 10(-11) cm(2) s(-1)), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.