Interaction of respiratory and photosynthetic electron transport, and evidence for membrane-bound pyridine-nucleotide dehydrogenases in Anabaena variabilis

Membranes isolated from vegetative cells of Anabaena variabilis (ATCC 29413) oxidine NADPH and NADH in a cyanide-sensitive reaction, NADPH being the better donor. In addition, both act as reductants for photosystem I in the light and in the presence of DCMU. An NADH-regenerating system has been introduced experimentally, functioning as donor system for photosystem I of Anabaena membranes. This NADH dehydrogenation is strictly light-dependent, sensitive to DBMIB, establishes a transhydrogenase system with NADP⁺ was hydrogen acceptor and competes with oxygen. Kinetic analysis of oxygen uptake with reference to nucleotide concentration exhibited different K_m- and V_max-values for NADPH and NADH in the light and in the dark.     

Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes

Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I.     

N,N-diethylhydroxylamine: a new electron donor to photosystem II

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. Diethylhydroxylamine supports dichlorophenolindophenol photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II before the herbicide binding site. The stimulation of the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed that the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamine. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations with diethylhydroxylamine either in light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that diethylhydroxylamine is a useful electron donor to the photosystem II.     

Lipids in oxygen-evolving photosystem II complexes of cyanobacteria and higher plants

Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes.     

Transient homodimer interactions studied using the electron self-exchange reaction

Transient homodimer protein interactions have been investigated by analyzing the influence of ionic strength (NaCl) on the electron self-exchange (the bimolecular reaction whereby the two oxidation states of a redox protein interconvert) rate constant (k(ese)) of four plastocyanins. The k(ese) values for the plastocyanins from spinach, Dryopteris crassirhizoma (a fern), and the green alga Ulva pertusa, which possess acidic patches of varying size and locations, increase 190-, 29-, and 21-fold, respectively, at elevated ionic strength (I = 2.03 M). In contrast, the k(ese) for the almost neutral cyanobacterial plastocyanin from Anabaena variabilis exhibits very little dependence on ionic strength. The temperature dependence of the k(ese) for spinach plastocyanin (I = 0.28 M) provides evidence for poor packing at the homodimer interface. Representative structures of the transient homodimers involved in electron self-exchange, which are consistent with fits of the ionic strength dependence of k(ese) to van Leeuwen theory, have been obtained from protein modeling and docking simulations. The Coulombic energy of the docked homodimers follows the order spinach > D. crassirhizoma > U. pertusa > A. variabilis, which matches that of the overall influence of ionic strength on k(ese). Analysis of the homodimer structures indicates that poor packing and high planarity are features of the interface that favor transient interactions. The physiologically relevant Mg2+ ion has a much more pronounced influence on the k(ese) of spinach plastocyanin, which along with the known properties of the thylakoid lumen suggests a biological role for electron self-exchange.     

A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.     

Interaction of a phenolic inhibitor with photosystem II particles

Photosystem II particles have been prepared from spinach and Chlamydomonas reinhardii CW 15 thylakoids. Photosynthetic electron transport in these particles is inhibited by phenolic compounds like dinoseb, but not by atrazine and diuron. The labeling patterns obtained by photoaffinity labels derived from either atrazine (azido-atrazine) or the phenolic herbicide dinoseb (azido-dinoseb) were compared in photosystem II particles and thylakoids. Whereas azido-atrazine in thylakoids of spinach as well as of Chlamydomonas labels a 32-kilodalton peptide, this label does not react in photosystem II particle preparations. Azido-dinoseb, however, labels both the thylakoid membranes and the particles, predominantly polypeptides in the 40-53 kilodalton molecular weight region. Since the latter polypeptides are probably part of the reaction center of photosystem II, it is suggested that phenolic compounds have their inhibition site within the reaction center complex. This indicates that the atrazine-binding 32-kilodalton peptide is either absent or functionally inactive in photosystem II particles, whereas the phenol inhibitor-binding peptides are not.     

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.     

Substrate water exchange in photosystem II depends on the peripheral proteins.

The (18)O exchange rates for the substrate water bound in the S(3) state were determined in different photosystem II sample types using time-resolved mass spectrometry. The samples included thylakoid membranes, salt-washed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria. For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O(2)-evolving catalytic site in the S(3) state. However, the fast phase of exchange became somewhat slower (by a factor of approximately 2) in NaCl-washed membrane fragments and core complexes from spinach in which the 16- and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples. For CaCl(2)-washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of approximately 3). Interestingly, the slow phase of exchange was little affected in the samples from spinach. For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected. Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803. This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms. In all samples, however, the differences in the exchange rates are relatively small. Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site. Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered. We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.     

Immunologically cross-reactive and redox-competent cytochrome b6/f-complexes in the chlorophyll-free plasma membrane of cyanobacteria

Plasma and thylakoid membranes were separated and purified from cell-free extracts of the cyanobacteria Anacystis nidulans, Synechocystis 6714, Anabaena variabilis and Nostoc sp. strain Mac. Immunoblots of the membrane proteins using antisera raised against subunits I-IV of the chloroplast b6/f-complex gave evidence for the presence of a homologous complex in both plasma and thylakoid membranes from the four species of cyanobacteria investigated. Both plasma and thylakoid membranes catalyzed the electron transfer from (exogenous) plastoquinol-9 and NADH to horse heart ferricytochrome c. However, while with plasma membranes these reactions were severely inhibited by low concentrations of antimycin A and rotenone, respectively, the inhibitors were without major effect on thylakoid membranes. The results will be discussed in terms of a possible similarity (analogy and/or homology?) of cyanobacterial plasma membranes to the inner mitochondrial membrane.     

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 effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.     

The C550 photoresponse at room temperature observed in membrane fragments of the blue-green alga Anabaena variabilis

The C550 photoresponse at room temperature was studied withmembrane fragments of the blue-green alga Anabaena variabilis.The kinetics, light minus dark difference spectrum and DCMUeffect were the same as those reported for spinach chloroplasts.The photoresponse size suggested that the number of the photosystemII center is half that for the photosystem I center in thisorganism grown under our autotrophic culture conditions. (Received September 16, 1975; )     

Heptameric association of light-harvesting complex II trimers in partially solubilized photosystem II membranes.

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of seven trimeric light-harvesting complex II proteins. The complex was readily observed in partially-solubilized Tris-washed photosystem II membranes from spinach but was also found to occur, with a low frequency, in oxygen-evolving photosystem II membranes. The structure reveals six peripheral trimers with the same rotational orientation and a central trimer with the opposite orientation. We conclude that the heptamer represents a naturally occurring aggregation state of part of the light-harvesting complex II trimers in the thylakoid membranes.     

The supramolecular architecture, function, and regulation of thylakoid membranes in red algae: an overview

Red algae are a group of eukaryotic photosynthetic organisms. Phycobilisomes (PBSs), which are composed of various types of phycobiliproteins and linker polypeptides, are the main light-harvesting antennae in red algae, as in cyanobacteria. Two morphological types of PBSs, hemispherical- and hemidiscoidal-shaped, are found in different red algae species. PBSs harvest solar energy and efficiently transfer it to photosystem II (PS II) and finally to photosystem I (PS I). The PS I of red algae uses light-harvesting complex of PS I (LHC I) as a light-harvesting antennae, which is phylogenetically related to the LHC I found in higher plants. PBSs, PS II, and PS I are all distributed throughout the entire thylakoid membrane, a pattern that is different from the one found in higher plants. Photosynthesis processes, especially those of the light reactions, are carried out by the supramolecular complexes located in/on the thylakoid membranes. Here, the supramolecular architecture, function and regulation of thylakoid membranes in red algal are reviewed.     

Inactive photosystem II centers: A resolution of discrepencies in photosystem II quantitation

The abundance of photosystem II in chloroplast thylakoid membranes has been a contentious issue because different techniques give quite different estimates of photosystem II titer. This discrepancy led in turn to disagreements regarding the stoichiometry of photosystem II to photosystem I in these membranes. We believe that the discrepancy in photosystem II quantitation is resolved by evidence which shows that a large population of photosystem II centers with negligible turnover rates are present in isolated thylakoid membranes as well as in normally developed leaves of healthy plants.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

Extrinsic polypeptides of spinach photosystem I

By combining Triton X-114 partitioning with alkaline-salt and chaotropic washings of thylakoid membrane vesicles and photosystem I particles, we have studied the protein subunit composition and organization of spinach photosystem I. Upon fractionation of photosystem I particles with Triton X-114, 6 polypeptides of 5.0, 8.2 (psaE), 10.5, 16.6 (psaG), 19.3 and 22.1 kDa (psaD) were considered to be extrinsic membrane proteins. By combining this partitioning with salt washes of thylakoid membranes, the polypeptides of 8.2, 11.6 (psaH), 19.3 and 22.1 kDa were directly shown to be stromally oriented and extrinsic while no extrinsic subunits were identified at the inner thylakoid surface. The 5.0, 8.2, 10.5, 17.2, 19.3 and 22.1 kDa polypeptides appear to have regulatory rather than catalytic functions as their release from photosystem I particles upon high salt-alkali treatment does not affect photosystem I-mediated electron transport.Abbreviations DCIP 2.6-dichlorophenol indophenol - DCMU dichlorophenyl dimethyl urea - LHC light harvesting complex - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine (N-tris[Hydroxymethyl]-methylglycine; N-[2-Hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine) - Tris (tris[Hydroxymethyl]aminomethane)     

Studies on Cation-induced Thylakoid Membrane Stacking, Fluorescence Yield, and Photochemical Efficiency

Trypsin digestion of photosynthetic membranes isolated from spinach (Spinacia oleracea L.) leaves eliminates the cation stimulation of chlorophyll fluorescence. High concentrations of cations protect the fluorescence yield against trypsin digestion, and the cation specificity for this protection closely resembles that required for the stimulation of fluorescence by cations. Trypsin digestion reverses cation-induced thylakoid stacking, and the time course of this effect seems to parallel that of the reversal of cation fluorescence. High concentrations of cations protect thylakoid stacking and cation-stimulated fluorescence alike. The cation stimulation of photosytem II photochemistry remains intact after trypsinization has reversed both cation-induced thylakoid stacking and fluorescence yield. It is concluded that cation-stimulated fluorescence yield, and not the cation stimulation of photosystem II photochemistry, is associated with thylakoid membrane stacking.     

Study on the effects of chloride depletion on photosystem II using different chloride depletion methods

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.