Photochemical cooperativity in photosystem II. Characterization of oxygen evolution discontinuities in the light-response curves.

In two previous papers (Fragata et al., J. Phys. Chem. B, 2005, 109, 14707-14714; Fragata et al., J. Phys. Chem. B, 2007, 111, 3315-3320), it was shown that the variation of oxygen evolution with the light intensity (I) in photosystem II (PSII) in steady state conditions can be formulated according to the Langmuir adsorption isotherm for heterogeneous catalysis. This yielded the expression OEth = OEth(max) I/(L1/2 + I), where OEth is the theoretical oxygen evolution, OEth(max) the maximum oxygen evolution, and L1/2 the irradiance giving OEth(max)/2. In this approximation, the photons interaction with the chlorophylls in the PSII reaction center is assumed to be a heterogeneous reaction in which the light is represented as a stream of particles instead of an electromagnetic wave. That is, the chlorophyll molecules are the adsorption surfaces (or heterogeneous catalysts), and the incident (or exciting) photons are the substrate, or the reagent. Recently, the examination of new experimental data obtained with 2,6-dichloro-p-benzoquinone (DCBQ) and p-benzoquinone (pBQ) as exogenous electron acceptors, disclosed the presence of oxygen evolution discontinuities (or transitions) in the light-response curves. The new data were fitted with a mathematical summation of hyperbola of order n(i) > 1, OEth = Sigma(i) [OEth(max)]iIn(i)/[(L1/2)i(n(i)) + I(n(i))], where the n(i)'s are the number of sites used by the incident photons in their interaction with the photosynthetic pigments in each population i of PSII centers open for photochemistry. The mathematical simulations yielded only three distinct n(i)'s, that is, 1.8, 4.8, 8.5 and 1.8, 4.2, 8.4 for isolated PSII particles incubated with DCBQ and pBQ, respectively. Implicitly, this means the simultaneous excitation of each PSII reaction center with more than one photon, that is, the excitation of more than one pigment molecule. It is suggested that these transitions have their origin in the cooperative interaction of the photons and the chlorophylls, and most likely also the pheophytins. This indicates that the discontinuities (or transitions) observed in the light-response curves of oxygen evolution are consistent with the hypothesis of photochemical cooperativity in photosystem II.

Investigation of the electron transfer site of p-benzoquinone in isolated photosystem II particles and thylakoid membranes using alpha- and beta-cyclodextrins.

The electron transfer sites of p-benzoquinone (pBQ) and 2,6-dichloro-p-benzoquinone (DCBQ) were investigated in thylakoid membranes and isolated photosystem II (PSII) particles from barley (Hordeum vulgare) using alpha- and beta-cyclodextrins (CD) at concentrations up to 16 mM. In CD-treated thylakoid membranes incubated with DCBQ the electron transport through PSII, estimated as oxygen evolution (OE), is largely enhanced according to a S-shaped (sigmoidal) dose-response curve displaying a sharp inflection point, or transition. The maxima percent OE enhancement at cyclodextrin concentrations above 14 mM are about 100% (alpha-CD) and 190% (beta-CD). On the contrary, in thylakoid membrane preparations incubated with pBQ as electron acceptor one observes an OE inhibition of about 30% which might result from the depletion of the thylakoid membrane of its plastoquinone content. It was also found that in isolated PSII particles incubated with either pBQ or DCBQ the cyclodextrins induce only a small OE enhancement. Moreover, the observation in CD-treated thylakoid membranes incubated with pBQ of a residual, non-inhibited oxygen-evolving activity of about 70% puts a twofold question. That is, either the plastoquinone depletion was not complete, or, pBQ binds to electron acceptor sites of different nature. From this and data published in the literature, it is concluded that in the thylakoid membrane (i) DCBQ binds to Q(B), as is generally accepted, and (ii) pBQ binds to the plastoquinol molecules in the PQ pool and most likely also to Q(B), thereby in accord with Satoh et al.'s model [K. Satoh, M. Ohhashi, Y. Kashino, H. Koike, Plant Cell Physiol. 36 (1995) 597-605]. An attractive alternative hypothesis is the direct interaction of pBQ with the non-haem Fe(2+) between Q(A) and Q(B).

Effect of alpha-, beta- and gamma-cyclodextrins on oxygen evolution by the thylakoid membrane. influence of pH and temperature.

The present work investigates the effect of alpha-, beta- and gamma-cyclodextrins (CD), i.e., alpha-CD, beta-CD and gamma-CD, on the oxygen evolution activity, the protein content and the uv-vis spectroscopic characteristics of thylakoid membranes. To study the pH-dependence, the thylakoids were incubated with the cyclodextrins at 273 K for a period of 10 min in the pH range from 5.5 to 9.0. To study the temperature-dependence the membranes were incubated at 273 and 293 K at pH 6.5, that is, the pH which induces a maximal oxygen evolution in the thylakoid preparations. The major observations are: (i) a stimulation of oxygen evolution in thylakoids incubated with alpha- and beta-CD either in acidic or alkaline conditions, (ii) a low inhibitory effect induced by gamma-CD on oxygen evolution, (iii) a significant decrease of the stimulatory effect of alpha- and beta-CD on oxygen evolution as the incubation temperature is raised from 273 to 293 K, (iv) the apparent inability of the cyclodextrins to change the protein contents of the thylakoids, and (v) a significant CD-induced red-shift from 681 to 683 nm observed in the absorption and second derivative spectra of the thylakoid membranes treated with beta-CD. First, it was found that the temperature effect described here is in accord with the general trend of the chemical effect of various cyclodextrins, i.e., the increase of the CD efficiency with decreasing temperature. Secondly, the CD effect is related to the size of the inner cavity diameter of the cyclodextrin molecules. An important conclusion in this work is that the molecular targets of the cyclodextrins are not limited to the thylakoid lipids as was described previously [Rawyler A. and Siegenthaler PA. (1996) Biochim. Biophys. Acta 1278, 89-97], but are located as well in other molecular species exposed at the stromal side of the thylakoid membrane. In particular, the CD-induced red-shift from 681 to 683 nm in the absorption and second derivative spectra of the thylakoid membranes indicates that the cyclodextrins targets might be either the exposed heme macrocycle in cytochrome b559, or the chlorophylls and pheophytins in the pigment-proteins of the photosystems I and II.

Oxygen evolution loss and structural transitions in photosystem II induced by low intensity UV-B radiation of 280 nm wavelength.

UV-B radiation of 280 nm wavelength (UV280) and low intensity (2.0 W/m2) gives rise to an important oxygen evolution (OE) loss in photosystem II (PSII) particles isolated from the thylakoid membrane of plant chloroplasts on the one hand, and to structural changes, or transitions, in the proteins of the PSII complex on the other hand. The latter UV280 effect was studied in this work by Fourier transform infrared (FT-IR) spectroscopy. First, irradiation of the PSII particles with UV280 for about 40 min causes an almost complete loss of OE activity. The remaining OE after 15, 20, 30 and 40 min is respectively 52, 44, 27 and 12% of the OE activity in control PSH particles kept in darkness. Secondly, difference FT-IR spectra of PSII particles irradiated for 30 min, i.e., [PSII irradiated with UV280]-minus-[PSII non-irradiated], show that the UV280 light is at the origin of significant IR absorbance changes in several spectral regions: (i) amide I (1696-1620 cm(-1)) and amide II (1580-1520 cm(-1)), (ii) tyrosine side chain (1620-1580 cm(-1) and 1520-1500 cm(-1), i.e., the v8a, v8b and v19a vibrational modes, respectively), and (iii) chlorophylls (1750-1696 cm(-1)). Thirdly, comparison of the UV-B effect reported here with structural changes induced by heat-stress in PSII proteins [M. Joshi, M. Fragata, Z. Naturforsch. 54c (1999) 35-43] clearly indicates that the stability of the functional centers in the PSII complex is dependent on a dynamic equilibrium between a-helix conformers and extended chain (beta-strand) structures. In this framework, transient 'alpha-helix-to-beta-strand transitions' are susceptible of giving rise in vivo to recurrent changes in the activity of the PSII complex, and as such act as a control mechanism of the photosynthetic function in the thylakoid membrane.

Heat-induced changes in the photochemical centres and the protein secondary structures of photosystem II studied by variable fluorescence and difference FT-IR spectroscopy.

Variable fluorescence (Fv), i.e., Fv = Fm-Fo where Fo is the minimal fluorescence and Fm the maximum fluorescence, and difference Fourier transform infrared (FT-IR) spectroscopy were used to study the effect of heat stress in the 25-55 degrees C range on photosystem II (PSII) structure and function. First, the Fv intensity reflects accurately the changes in the number of open photochemical centers in PSII. Secondly, the use of Fv in combination with FT-IR spectroscopy can disclose structure-function correlations in the heat inactivation of the PSII complex. Analysis of the midpoint temperatures of thermal denaturation, i.e., 50% inactivation, reported so far in investigations of the thylakoid membrane components has revealed that most of the thermal transitions attributed to PSII are in the 39-46 degrees C range. In this work, it is shown specifically that the midpoint temperature of PSII inactivation is at about 40 degrees C. Moreover, it was clearly demonstrated that the heat-induced changes above 40 degrees C are the result of a marked decrease in the number of open photochemical centers in PSII. It was also seen that above this same temperature the loss of photochemical centers has its structural counterpart in overall modifications of the secondary structures of the PSII proteins resulting from the decrease in the alpha-helix content concomitant with the increase in extended chain (beta-strand) conformations. In brief, a novel finding reported here is that the number of open photochemical centers in PSII is dependent on a dynamic equilibrium between the contents of the PSII proteins in alpha-helix and extended chains (beta-strands), but not in beta-sheets and beta-turn structures except for the antiparallel-beta-sheet conformations. This therefore associates the thermal inactivation of the photochemical centers in photosystem II with distinct conformational changes in the proteins of the PSII supramolecular complex. In the particular context of the present study, these findings constitute a significant contribution to the investigation of structure-function correlations in the photosynthetic membrane. In a broader context, this information might be essential for the comprehension of the molecular arrangements or local structure order that are involved directly or indirectly in biological catalysis.

Fourier transform infrared spectroscopic study of ion binding and intramolecular interactions in the polar head of digalactosyldiacylglycerol.

Lipid bilayers composed of digalactosyldiacylglycerol (DGDG), that is, Galp alpha 1-6Galp beta 1-3DAG, a non-ionic lipid of the thylakoid membrane of chloroplasts, aggregate in aqueous media containing mono- and divalent cations in amounts above a threshold concentration (Ct) of about 1.0, 4.7 and 10.0 mM for Ca2+, Mg2+ and Na+, respectively. In this work, we found that above Ct the DGDG membranes do not undergo fusion and that the aggregation can be reversed or disrupted. This means that the perturbation induced by the salts results from adsorption, or complexation of the ions in the polar head of DGDG. To investigate this question, we used Fourier transform infrared (FTIR) spectroscopy to identify the molecular sites in DGDG which are modified by interaction, or adduct formation with CaCl2, MgCl2 and NaCl. We also determined whether the ions affect the intramolecular hydrogen bonding between the sn2 ester C = O and the carbon-6 of the alpha-anomer of galactose (Gal). The major conclusions are: (i) the salts do not affect, at least directly, the ester carbonyl region of DGDG, (ii) the most probable sites of binding, or adsorption, for the ions are the ring oxygen, and (iii) the ring hydroxyls are the sites of either ion complexation or intra- and intermolecular H-bonding in interacting DGDG membranes. Within this framework, the complexation of the ions with Gal might induce total or partial dehydration of the galactolipid headgroup and thus provides the means to overcome the repulsive hydration forces that hinder aggregation of the DGDG membranes.

Fluorescence characteristics of pyrene and phosphatidylethanolamine-bound pyrene incorporated into lipid vesicles solubilized in media of differing NaCl concentrations.

We used the excimer/monomer ratio of pyrene (PY) and N-(1-pyrenesulfonyl)dipalmitoyl-L-alpha-phosphatidylethanolamine (DPPE-PY) fluorescence intensities (IE/IM), and the polarity ratio I/III to investigate the state of the polar head group region of small, unilamellar phosphatidylcholine vesicles (SUV-PC) solubilized in media of differing NaCl concentrations. PY or DPPE-PY excimer formation resulting from vesicles' collisions is not affected by the presence of monovalent ions. In addition, the ionic strength does not alter the dielectric environment in the neighborhood of PY incorporated into SUV-PC. Since IE/IM of both PY and DPPE-PY is insensitive to variations in the ionic strength, we conclude that the probes' diffusion in SUV-PC, and consequently the membrane fluidity, are independent of NaCl concentration at least up to 0.5 M. The vesicles' concentration in the aqueous solution was the only factor which induced a rise of IE/IM. To explain the results in the context of the transient-fusion model developed previously (G.P. L'Heureux and M. Fragata, Biophys. Chem. 30 (1988) 293) and the hypothesis of repulsive hydration forces, we postulate a heterogeneous distribution of dehydrated domains, or contact areas, on the outer surfaces of colliding vesicles.

Micropolarities of lipid bilayers and micelles. 5. Localization of pyrene in small unilamellar phosphatidylcholine vesicles.

The excimer/monomer ratio of emission intensities (IE/IM) and the enhancement of the 0-0 vibronic transition in the fluorescence spectra of pyrene (PY) and 16-(1-pyrenyl)hexadecanoic acid (C16PY) were used to investigate the localization of PY in the bilayers of small unilamellar vesicles constituted of phosphatidylcholine (SUV-PC). First, from comparison of the fluorescence characteristics of PY in water with those of PY incorporated into the SUV-PC membranes, we concluded that the probe is incorporated preferentially in the lipid phase of the vesicles and not in the bulk aqueous phase. In addition, we found that, contrary to what happens with the pyrenyl moiety of C16PY, the location of PY varies with its relative concentration in the membrane space. The critical concentration was observed to be around 1.0 mol% of incorporated PY. At concentrations below this value, PY is located in the hydrocarbon core of the lipid bilayers. Above 1.0 mol%, the PY molecules reside preferentially in the neighbourhood of the glyceryl moiety region of the PC vesicles.

Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy.

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K3 Fe (CN)6, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at ∼ 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-QA interactions.

Mixing of single-chain amphiphiles in two-chain lipid bilayers. 2. Characteristics of chlorophyll a and alpha-tocopherol incorporation in unilamellar phosphatidylcholine vesicles.

As a step toward the elucidation of the biological significance of the isoprenic chains found ubiquitously in single-chain lipids involved in electron and energy transfer of chloroplasts and mitochondria, we undertook a comparative study of the incorporation of chlorophyll a (Chl a) and alpha-tocopherol (alpha T) in unilamellar phosphatidylcholine (PC) vesicles. We observed that while Chl alpha is added to the PC bilayers in a simple, almost linear way, the inclusion of alpha T is a sigmoid-like function characterized first by a slow variation of alpha T incorporation followed by a rather steep increase till saturation occurs at initial mol% alpha T of approx. 5%. Owing to the likeness of the isoprenic chains of Chl a and alpha T the simplest interpretation of the data is that the different mixing behaviours of the two lipids in PC vesicles are due to the special arrangement of the tetrapyrrole macrocycle and the chromanol ring in the lipid bilayers. However, no satisfactory explanation of the mechanisms involved can be given as yet. One possibility is that the inclusion of Chl a and alpha T in the membranes results from adsorption differences brought about by the extent of penetration of the molecule into the monolayers of the unilamellar vesicles.

Toward a multistep mechanism of cytochrome c reactivity. Answer to a comment.

Koppenol's rejection (Biophys. Chem. 18 (1983) 203) of a model of polarity-dependent ferrocytochrome c oxidation (M. Fragata and F. Bellemare, Biophys. Chem. 15 (1982) 111) places emphasis on the role of the protein surface charges in reactivity but is at the same time too restrictive as it neglects largely the polarity (dielectric constant) of the aqueous and hydrophobic interfaces of the exposed heme edge and the inner cleft (heme crevice) of cytochrome c which appear to be the oxidation-reduction sites. It is suggested that a more general model should take into account (i) a recognition (or diffusion) step where the distance travelled by cytochrome c at the membrane surface and/or the Brownian displacements in the bulk solution are greatly influenced by ionic strength, and (ii) a redox step where low polarity effects prevail with concomitant weakening of ionic activity.

Dielectric constant dependence of biological oxidation-reduction. 1. A model of polarity-dependent ferrocytochrome c oxidation.

A theoretical model for the effect of the dielectric constant (c) of the solvent medium on ferrocytochrome c oxidation by ferricyanide is developed to account for the observed variations of the rate constant (k) of reactions in aqueous binary mixtures with alcohols (less than 5-10 mol% ethanol and propranolol). A correlation between k and c is found if ln k is expressed as a function of the Kirkwood parameter (c-1)(2c+1). The results of calculations indicate that the use of the 'overall dipole moment' of cytochrome c in oxidoreduction studies is likely to be unreliable. Instead, the decrease in k in alcohol/water mixtures is best explained--in conformity with Onsager's theory of the reaction field--by a polarity effect on the dipole moment of the cytochrome c heme upon diffusion of the polar solvent molecules into the low dielectric constant heme crevice.

Transmembrane distribution of alpha-tocopherol in single-lamellar mixed lipid vesicles.

A study of the molar ratio dependence of the incorporation of alpha-tocopherol into single-lamellar vesicles showed that the number of molecules which the bilayers can accommodate increased linearly with increasing alpha-tocopherol/phosphatidylcholine initial molar ratios till about 0.05, then approached a saturation limit. At 5 mol%, one alpha-tocopherol molecule per 60 phospholipids can be incorporated into the membranes. Up to this limit the distribution of alpha-tocopherol in the bilayers is uniform, while at initial molar ratios higher than 0.05 a disproportionation toward the inner monolayer of the vesicles is observed. The average outer/total ratio is found to be 0.27 +/- 0.03 at alpha-tocopherol/phosphatidylcholine molar ratios above 0.07 and is similar to asymmetrical distributions that have been reported in vesicles containing other one-chain amphiphiles (e.g., cholesterol). This large disproportionation is in contrast with the packing distribution of certain two-chain amphiphiles, and indicates that one of the driving forces for asymmetry formation in lipid bilayers might be dependent on the number of hydrocarbon chains per amphiphile molecule. A possible reason for the disproportionation effect observed in our experiments is the displacement of unsaturated phospholipids to the outer monolayer of the single-lamellar vesicles, by the more rigid isoprene units of alpha-tocopherol.

On the location of the tetrapyrrole macrocycle of chlorophyll a in phospholipid vesicles and in hexadecane.

The state of chlorophyll a in phosphatidylcholine vesicles was examined. The results indicate that the chlorophylls are present in monomeric form. A kinetic study of chlorophyll reactions with K2S2O8 and piperidine showed that these substances react with the porphyrin rings of pigments located on both vesicle faces, most probably within the polar headgroup region.