Anisotropy measurements of intrinsic fluorescence of prenyllipids reveal much higher mobility of plastoquinol than alpha-tocopherol in model membranes

As an alternative to a fluorescent probe approach, the intrinsic fluorescence of reduced forms of prenylquinones has been exploited, which offers a convenient means of determining directly motional properties of these molecules. The steady-state fluorescence anisotropy measurements of plastoquinols (PQH(2)) and alpha-tocopherol (alpha-Toc) incorporated into phospholipid liposomes have been performed. The effect of prenyllipid concentration, PQH(2) side chain length and the composition of the membranes has been studied. For the data interpretation, the fundamental anisotropy of alpha-Toc, PQH(2), ubiquinol-10 and alpha-tocopherolquinol, as well as the angles between the absorption and emission transition moments have been also determined. It was concluded that alpha-Toc shows very low mobility in the lipid bilayer, whereas PQH(2)-9 displays significant motional freedom in dipalmitoylphosphatidylcholine vesicles and even higher in egg yolk lecithin membranes.     

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     

Tocochromanols, plastoquinol, and other biological prenyllipids as singlet oxygen quenchers-determination of singlet oxygen quenching rate constants and oxidation products

Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and alpha-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, alpha-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the gamma-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-gamma-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.     

Structural variability of plant photosystem II megacomplexes in thylakoid membranes

Plant photosystem II (PSII) is organized into large supercomplexes with variable levels of membrane‐bound light‐harvesting proteins (LHCIIs). The largest stable form of the PSII supercomplex involves four LHCII trimers, which are specifically connected to the PSII core dimer via monomeric antenna proteins. The PSII supercomplexes can further interact in the thylakoid membrane, forming PSII megacomplexes. So far, only megacomplexes consisting of two PSII supercomplexes associated in parallel have been observed. Here we show that the forms of PSII megacomplexes can be much more variable. We performed single particle electron microscopy (EM) analysis of PSII megacomplexes isolated from Arabidopsis thaliana using clear‐native polyacrylamide gel electrophoresis. Extensive image analysis of a large data set revealed that besides the known PSII megacomplexes, there are distinct groups of megacomplexes with non‐parallel association of supercomplexes. In some of them, we have found additional LHCII trimers, which appear to stabilize the non‐parallel assemblies. We also performed EM analysis of the PSII supercomplexes on the level of whole grana membranes and successfully identified several types of megacomplexes, including those with non‐parallel supercomplexes, which strongly supports their natural origin. Our data demonstrate a remarkable ability of plant PSII to form various larger assemblies, which may control photochemical usage of absorbed light energy in plants in a changing environment.     

The interaction of thylakoid lipids with everted photosystem II membranes

Spinach ( Spinacia oleracea L. cv. Viking II) thylakoid membranes enriched in photosystem II were disrupted by incubation at 35°C for 5 min followed by sonication. This treatment caused the loss of manganese and the release of extrinsic photosystem II proteins of 16, 23 and 33 kdaltons, resulting in retention of only 12–18% of the oxygen-evolving activity. Prior addition of exogenous digalactosyldiacylglycerol to the membranes afforded protection against the release of manganese and extrinsic proteins and enabled the retention of up to 47% of the activity. Monogalactosyldiacylglycerol was less effective in both respects and enabled retention of only 30% of the activity. Addition of charged thylakoid lipids had a severely damaging effect on photosystem II.     

Differential thermal effects on the energy distribution between photosystem II and photosystem I in thylakoid membranes of a psychrophilic and a mesophilic alga

Sensitivity of the photosynthetic thylakoid membranes to thermal stress was investigated in the psychrophilic Antarctic alga Chlamydomonas subcaudata. C. subcaudata thylakoids exhibited an elevated heat sensitivity as indicated by a temperature-induced rise in F_o fluorescence in comparison with the mesophilic species, Chlamydomonas reinhardtii. This was accompanied by a loss of structural stability of the photosystem (PS) II core complex and functional changes at the level of PSI in C. reinhardtii, but not in C. subcaudata. Lastly, C. subcaudata exhibited an increase in unsaturated fatty acid content of membrane lipids in combination with unique fatty acid species. The relationship between lipid unsaturation and the functioning of the photosynthetic apparatus under elevated temperatures is discussed.     

Differential thermal effects on the energy distribution between photosystem II and photosystem I in thylakoid membranes of a psychrophilic and a mesophilic alga

Sensitivity of the photosynthetic thylakoid membranes to thermal stress was investigated in the psychrophilic Antarctic alga Chlamydomonas subcaudata. C. subcaudata thylakoids exhibited an elevated heat sensitivity as indicated by a temperature-induced rise in F(o) fluorescence in comparison with the mesophilic species, Chlamydomonas reinhardtii. This was accompanied by a loss of structural stability of the photosystem (PS) II core complex and functional changes at the level of PSI in C. reinhardtii, but not in C. subcaudata. Lastly, C. subcaudata exhibited an increase in unsaturated fatty acid content of membrane lipids in combination with unique fatty acid species. The relationship between lipid unsaturation and the functioning of the photosynthetic apparatus under elevated temperatures is discussed.     

Spectroelectrochemistry of P700 in native photosystem I particles and diethyl ether-treated thylakoid membranes from spinach and Thermosynechococcus elongatus

The redox potentials (E(composite function')) of P700 in intact and diethyl ether-treated thylakoid membranes as well as native photosystem (PS) I particles from spinach and Thermosynechococcus elongatus have been measured by a spectroelectrochemistry with an error range of +/-2-3 mV. Stepwise removal of antenna pigments by ether treatment caused distinct shifts of the E( composite function') value with increasing degree of water saturation in ether; negatively from +471 to +428 mV for spinach, but positively from +423 to +436 mV for T. elongatus. Such a contrasting behavior is discussed by invoking the mode of action of ether on the microenvironments around P700.     

Site-specific interaction of ATPase-pumped protons with photosystem II in chloroplast thylakoid membranes

The chloroplast thylakoid ATPase proton pump-driven H+ accumulation in the dark was compared to the light-dependent proton pump driven by either photosystem II or I, in regard to the effects of the resultant acidity on chemical modification reactions. The assays used to detect the acidity effects were: (a)the incorporation of [3H]-acetic anhydride into membrane protein -NH2 groups, and (b) the effect of a certain level of that chemical modification on inhibition of photosystem II water oxidation activity. Based on labeling data with [3H]-acetic anhydride, 20-30 nmol.(mg chl)-1 of -NH3+ groups appear to be metastable in the dark in untreated membranes. The term metastable is used because proton leak-inducing treatments in the dark lead to about 20-30 nmol . (mg chl)-1 increase in acetic anhydride labeling probably due to reaction with the -NH2 form of amine groups. Addition of low levels of uncoupler or a brief thermal treatment caused a loss of protons from the membrane equivalent to the increase in acetic anhydride derivatization. The increase in acetic anhydride derivatization caused inhibition of water oxidation activity. Using thermally sensitized membranes, photosystem II but not photosystem I electron transport (each giving a steady-state proton accumulation of about 50 nmol H+ . (mg chl)-1 restored the lower level of acetic anhydride reactivity as in previous results (Baker et al., 1981). In dark-maintained, thermally treated membranes, ATPase activity, i.e., the proton pump associated with it, also restored the lower level of acetic anhydride labeling, and again acetic anhydride no longer inhibited water oxidation. Because photosystem I activity did not elicit this type of response to acetic anhydride, there appears to be a pathway for ATPase pumped protons which allows them to reach a restricted domain, perhaps intramembrane, common with the photosystem II water oxidation mechanism and unavailable to protons pumped by photosystem I. The membrane structure(s) which determines this site specificity is not yet understood.     

Heat-induced changes in photosystem I activity as measured with different electron donors in isolated spinach thylakoid membranes.

Heat-induced changes in photosystem I (PSI) have been studied in terms of rates of oxygen consumption using various donors (DCPIPH2, TMPDred and DADred), formation of photo-oxidized P700 and changes in Chl a fluorescence emission at 77 K. Linear heating of thylakoid membranes from 35 degrees C to 70 degrees C caused an enhancement in PSI-mediated electron transfer rates (DCPIPH2-->MV) up to 55 degrees C. However, no change was observed in PSI rates when other electron donors were used (TMPDred and DADred). Similarly, Chl a fluorescence emission spectra at 77 K of heat-treated thylakoid membranes did not show any increase in peak at 735 nm, however, a significant decrease was observed as a function of temperature in the peaks at 685 and 694 nm. In DCMU-treated control thylakoid membranes maximum photo-oxidized P700 was generated at g = 2.0025. In heat-treated thylakoid membranes maximum intensity of photo-oxidized P700 signal was observed at approximately 50-55 degrees C without DCMU treatment. The steady-state signal of the photo-oxidized P700 was studied in the presence of DCPIPH2 and TMPDred as electron donors in DCMU-treated control and in 50 degrees C treated thylakoid membranes. We present here the first of such comparative study of PSI activity in terms of the rates of oxygen consumption and re-reduction kinetics of photo-oxidized P700 in the presence of different electron donors. It appears that the formation of the P700+ signal in heat-treated thylakoid membranes is due to an inhibited electron supply from PSII and not due to spillover or antenna migration.     

Increased thermal stability of photosystem II and the macro-organization of thylakoid membranes,induced by co-solutes,associated with changes in the lipid-phase behaviour of thylakoid membranes

Photosynthetica - The principal function of the thylakoid membrane depends on the integrity of the lipid bilayer, yet almost half of the thylakoid lipids are of non-bilayer-forming type, whose...     

Double reduction of plastoquinone to plastoquinol in photosystem 1

In Photosystem 1 (PS1), phylloquinone (PhQ) acts as a secondary electron acceptor from chlorophyll ec(3) and also as an electron donor to the iron-sulfur cluster F(X). PS1 possesses two virtually equivalent branches of electron transfer (ET) cofactors from P(700) to F(X), and the lifetime of the semiquinone intermediate displays biphasic kinetics, reflecting ET along the two different branches. PhQ in PS1 serves only as an intermediate in ET and is not normally fully reduced to the quinol form. This is in contrast to PS2, in which plastoquinone (PQ) is doubly reduced to plastoquinol (PQH(2)) as the terminal electron acceptor. We purified PS1 particles from the menD1 mutant of Chlamydomonas reinhardtii that cannot synthesize PhQ, resulting in replacement of PhQ by PQ in the quinone-binding pocket. The magnitude of the stable flash-induced P(700)(+) signal of menD1 PS1, but not wild-type PS1, decreased during a train of laser flashes, as it was replaced by a ~30 ns back-reaction from the preceding radical pair (P(700)(+)A(0)(-)). We show that this process of photoinactivation is due to double reduction of PQ in the menD1 PS1 and have characterized the process. It is accelerated at lower pH, consistent with a rate-limiting protonation step. Moreover, a point mutation (PsaA-L722T) in the PhQ(A) site that accelerates ET to F(X) ~2-fold, likely by weakening the sole H-bond to PhQ(A), also accelerates the photoinactivation process. The addition of exogenous PhQ can restore activity to photoinactivated PS1 and confer resistance to further photoinactivation. This process also occurs with PS1 purified from the menB PhQ biosynthesis mutant of Synechocystis PCC 6803, demonstrating that it is a general phenomenon in both prokaryotic and eukaryotic PS1.     

Reevaluation of the stoichiometry of cytochrome b559 in photosystem II and thylakoid membranes.

The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donation in PSII, and the stoichiometry of the other redox-active, reaction center components. We have reinvestigated the stoichiometry of cytochrome b559 in PSII-enriched and thylakoid membranes, using differential absorbance and electron paramagnetic resonance spectroscopies. The data from both quantitation procedures strongly indicate only one copy of cytochrome b559 per reaction center in PSII-enriched membranes and also suggest one copy of cytochrome b559 per reaction center in thylakoid membranes.     

Effects of high temperature and low pH on photosystem 2 photochemistry in spinach thylakoid membranes

The effects of temperature (25–45 °C) and pH (7.5–5.5) on photosystem (PS) 2 was studied in spinach (Spinacia oleracea L.) thylakoid membranes using chlorophyll a fluorescence induction kinetics. In high temperature and low pH treated thylakoid membranes a decline in the variable to maximum fluorescence ratio (F_v/F_m) and PS 2 electron transport rate were observed. More stacking in thylakoid membranes, studied by digitonin fractionation method, was observed at low pH, while the degree of unstacking increased under high temperature conditions. We conclude that the change in pH does not significantly affect the donor/acceptor side of PS 2 while high temperature does. Fluorescence emission spectra at 77 K indicated that low pH is associated with energy redistribution between the two photosystems while high temperature induced changes do not involve energy re-distribution. We suggest that both, high temperature and low pH, show an inhibitory effect on PS 2 but their mechanisms of action are different.     

Supramolecular photosystem II organization in grana thylakoid membranes: evidence for a structured arrangement

The distribution of photosystem (PS) II complexes in stacked grana thylakoids derived from electron microscopic images of freeze-fractured chloroplasts are examined for the first time using mathematical methods. These characterize the particle distribution in terms of a nearest neighbor distribution function and a pair correlation function. The data were compared with purely random distributions calculated by a Monte Carlo simulation. The analysis reveals that the PSII distribution in grana thylakoids does not correspond to a random protein mixture but that ordering forces lead to a structured arrangement on a supramolecular level. Neighboring photosystems are significantly more separated than would be the case in a purely random distribution. These results are explained by structural models, in which boundary lipids and light-harvesting complex (LHC) II trimers are arranged between neighboring PSII. Furthermore, the diffusion of PSII was analyzed by a Monte Carlo simulation with a protein density of 80% area occupation (determined for grana membranes). The mobility of the photosystems is severely reduced by the high protein density. From an estimate of the mean migration time of PSII from grana thylakoids to stroma lamellae, it becomes evident that this diffusion contributes significantly to the velocity of the repair cycle of photoinhibited PSII.     

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

Scavenging of superoxide anion radical by chaparral

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accomodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.     

Scavenging of superoxide radical by ascorbic acid

Using acetaldehyde and xanthine oxidase as the source of suPeroxide radical, the second order rate constant for the reaction between ascorbic acid and superoxide radical was estimated to be 8.2 X 10⁷ M^-1 s^-1. In rats, the average tissue concentration of ascorbic acid was of the order of 10^-3 M and that of superoxide dismutase was of the order of 10^-6 M. So, taking together both the rate constants and the tissue concentrations, the efficacy of ascorbic acid for scavenging superoxide radical in animal tissues appears to be better than that of suPeroxide dismutase. The significance of ascorbic acid as a scavenger of superoxide radical has been discussed from the point of view of the evolution of ascorbic acid synthesizing capacity of terrestrial vertebrates.