Photochemically induced dynamic nuclear polarization in the reaction center of the green sulphur bacterium Chlorobium tepidum observed by 13C MAS NMR

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by (13)C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced (13)C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.     

Photosynthetic reaction centres: variations on a common structural theme?

From their hybrid properties, the reaction centres of green sulphur bacteria and heliobacteria seem to be the missing links between the two branches of the reaction centre family, typified by higher plant photosystem I and the purple bacterial reaction centre. This suggests that all of the diverse types of photosynthetic reaction centres have closer structural resemblances than was previously thought.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of < 5 μs and ≈ 50 μs). We believe that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll.We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below ?450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD⁺ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Characteristics of the Photosystem II reaction centre. I. Electron acceptors

The EPR characteristics of Photosystem II electron acceptors are described, in membrane and detergent-treated preparations from a mutant of Chlamydomonas reinhardii lacking Photosystem I and photosynthetic ATPase. The relationship between the quinone-iron and pheophytin acceptors is discussed and a heterogeneity of reaction centres is demonstrated such that only a minority of reaction centres were capable of secondary electron donation at temperatures below 100 K. Only these centres were therefore able to stabilise a reduced acceptor below 100 K. Parallel experiments using a barley mutant (viridis zb63) which also lacks Photosystem I, provide similar results indicating that the C. reinhardii system can provide a general model for the Photosystem II electron acceptor complex. The similarity of the system to that of the purple photosynthetic bacteria is discussed.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

Type I photosynthetic reaction centres: structure and function

We review recent advances in the study of the photosystem I reaction centre, following the determination of a spectacular 2.5 A resolution crystal structure for this complex of Synechococcus elongatus. Photosystem I is proving different to type II reaction centres in structure and organization, and the mechanism of transmembrane electron transfer, and is providing insights into the control of function in reaction centres that operate at very low redox potentials. The photosystem I complex of oxygenic organisms has a counterpart in non-oxygenic bacteria, the strictly anaerobic phototrophic green sulphur bacteria and heliobacteria. The most distinctive feature of these type I reaction centres is that they contain two copies of a large core polypeptide (i.e. a homodimer), rather than a heterodimeric arrangement of two related, but different, polypeptides as in the photosystem I complex. To compare the structural organization of the two forms of type I reaction centre, we have modelled the structure of the central region of the reaction centre from green sulphur bacteria, using sequence alignments and the structural coordinates of the S. elongatus Photosystem I complex. The outcome of these modelling studies is described, concentrating on regions of the type I reaction centre where important structure-function relationships have been demonstrated or inferred.     

Observation of the solid-state photo-CIDNP effect in entire cells of cyanobacteria Synechocystis

Cyanobacteria are widely used as model organism of oxygenic photosynthesis due to being the simplest photosynthetic organisms containing both photosystem I and II (PSI and PSII). Photochemically induced dynamic nuclear polarization (photo-CIDNP) ¹³C magic-angle spinning (MAS) NMR is a powerful tool in understanding the photosynthesis machinery down to atomic level. Combined with selective isotope enrichment this technique has now opened the door to study primary charge separation in whole living cells. Here, we present the first photo-CIDNP observed in whole cells of the cyanobacterium Synechocystis.     

Effect of crowding on the electron transfer process from plastocyanin and cytochrome c<Subscript>6</Subscript> to photosystem I: a comparative study from cyanobacteria to green algae

Plastocyanin and cytochrome c ₆, the alternate donor proteins to photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I vary accordingly for cyanobacteria, algae and plants. The effect of different crowding agents on the kinetics of the reaction between plastocyanin or cytochrome c ₆ and photosystem I from three different cyanobacteria, Synechocystis PCC 6803, Nostoc PCC 7119 and Arthrospira maxima, and a green alga, Monoraphidium braunii, has been investigated by laser flash photolysis, in order to elucidate how molecular crowding affects the interaction between the two donor proteins and photosystem I. The negative effect of viscosity on the interaction of the two donors with photosystem I for the three cyanobacterial systems is very similar, as studied by increasing sucrose concentration. Bovine serum albumin seems to alter the different systems in a specific way, probably by means of electrostatic interactions with the donor proteins. Ficoll and dextran behave in a parallel manner, favouring the interaction by an average factor of 2, although this effect is somewhat less pronounced in Nostoc. With regards to the eukaryotic system, a strong negative effect of viscosity is able to overcome the favourable effect of any crowding agent, maybe due to stronger donor/photosystem I electrostatic interactions or the structural nature of the eukaryotic photosystem I-enriched membrane particles.     

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.     

Seasonal changes in the structure of the anoxygenic photosynthetic bacterial community in Lake Shunet,Khakassia

Seasonal studies of the anoxygenic phototrophic bacterial community of the water column of the saline eutrophic meromictic Lake Shunet (Khakassia) were performed in 2002 (June) and 2003 (February–March and August). From the redox zone down, the lake water was of dark green color. Green sulfur bacteria predominated in every season. The maximum number of green sulfur bacteria was 10⁷ cells/ml in summer and 10⁶ cells/ml in winter. A multi-syringe stratification sampler was applied for the study of the fine vertical distribution of phototrophs in August 2003; the sampling was performed every 5 cm. A 5-cm-thick pink-colored water layer inhabited by purple sulfur bacteria was shown to be located above the layer of green bacteria. The species composition and ratio of purple bacterial species depended on the sampling depth and on the season. In summer, the number of purple sulfur bacteria in the layer of pink water was 1.6 × 10⁸ cells/ml. Their number in winter was 3 × 10⁵ cells/ml. In the upper oxygen-containing layer of the chemocline the cells of purple nonsulfur bacteria were detected in summer. The maximum number of nonsulfur purple bacteria, 5 × 10² cells/ml, was recorded in August 2003. According to the results of the phylogenetic analysis of pure cultures of the isolated phototrophic bacteria, which were based on 16S rDNA sequencing, green sulfur bacteria were close to Prosthecochloris vibrioformis, purple sulfur bacteria, to Thiocapsa and Halochromatium species, and purple nonsulfur bacteria, to Rhodovulum euryhalinum and Pinkicyclus mahoneyensis.     

Defining the Far-red Limit of Photosystem I: THE PRIMARY CHARGE SEPARATION IS FUNCTIONAL TO 840 nm*

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P₇₀₀, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P₇₀₀⁺ formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391–2401). The same far-red limit for the P₇₀₀⁺ formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers F_A and F_B by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P₇₀₀ similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.     

The primary charge separation,cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations.Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex.The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840⁺ was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.     

Long-Term Population Dynamics of Phototrophic Sulfur Bacteria in the Chemocline of Lake Cadagno, Switzerland

Population analyses in water samples obtained from the chemocline of crenogenic, meromictic Lake Cadagno, Switzerland, in October for the years 1994 to 2003 were studied using in situ hybridization with specific probes. During this 10-year period, large shifts in abundance between purple and green sulfur bacteria and among different populations were obtained. Purple sulfur bacteria were the numerically most prominent phototrophic sulfur bacteria in samples obtained from 1994 to 2001, when they represented between 70 and 95% of the phototrophic sulfur bacteria. All populations of purple sulfur bacteria showed large fluctuations in time with populations belonging to the genus Lamprocystis being numerically much more important than those of the genera Chromatium and Thiocystis. Green sulfur bacteria were initially represented by Chlorobium phaeobacteroides but were replaced by Chlorobium clathratiforme by the end of the study. C. clathratiforme was the only green sulfur bacterium detected during the last 2 years of the analysis, when a shift in dominance from purple sulfur bacteria to green sulfur bacteria was observed in the chemocline. At this time, numbers of purple sulfur bacteria had decreased and those of green sulfur bacteria increased by about 1 order of magnitude and C. clathratiforme represented about 95% of the phototrophic sulfur bacteria. This major change in community structure in the chemocline was accompanied by changes in profiles of turbidity and photosynthetically available radiation, as well as for sulfide concentrations and light intensity. Overall, these findings suggest that a disruption of the chemocline in 2000 may have altered environmental niches and populations in subsequent years.     

On the preparation of indoxyl red from indican and some new characteristics

An indole compound with a strong purple–red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, ¹³C NMR, ¹H NMR, UV–visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel⁹ 70 years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Analysis of the Heat Stress Induced Quenching of Variable Chlorophyll a Fluorescence in Beet Spinach Leaves: Possible Accumulation of Oxidized Quencher P680+ under High Illumination

Effect of preheating of beet spinach leaves on chlorophyll a fluorescence yield was analyzed with the help of additional high intensity illumination pulses using a pulse modulated fluorometer. Preheating at mildly elevated temperature (35–45°C) causes a shift in the redox state of secondary donor of photosystem II, possibly due to uncoupling of phosphorylation because of thermal induced membrane disorganization and associated alkalinization of intra thylakoid space. Also, at these preheating temperatures, a rise in photosystem I catalyzed electron transfer has been shown to occur. These two effects induce rapid quenching of Chi a fluorescence, which drops even in the presence of actinic light, below the level of initial fluorescence (F_o′ monitored by the weak modulated probing light. Preheating of leaf segments induces an increase in fluorescence in the presence of dluron, which blocks electron flow between two photosystems, and thus this increases in fluorescence yield (F_o′ as monitored by weak modulated light, is not solely due to disorganization of light harvesting Chi-protein complex but also due to a shift in the redox equilibrium of the donor at the oxidizing side of photosystem II resulting in rapid reduction of Q_A the stable primary acceptor of photosystem II. In 50°C preheated DCMU treated samples, the fluorescence yield increases in weak modulated light and it approaches that of maximal steady state (F_max) level. At preheating temperature of 48°–50°C, the inactivation of enzymes in the reducing side of photosystem I, causes an impairment of the reoxidation of QA and under this condition, a strong illumination causes quenching of Chi a fluorescence. This quenching seems to arise because of accumulation of the P680⁺, the oxidized physiological donor of photosystem which is a quencher of Chi a fluorescence. This quenching depended on the pulse intensity and duration which saturates P680⁺ accumulation and is greatly manifested when water oxidation complex is damaged.     

Characterization and In Situ Carbon Metabolism of Phototrophic Consortia

A dense population of the phototrophic consortium “Pelochromatium roseum” was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescence in situ hybridization revealed that the brown epibionts of “P. roseum” constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low. The 465-bp 16S rRNA gene sequence of the epibiont of “P. roseum” was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. Maximum light-dependent H¹⁴CO₃⁻ fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (δ¹³C) of their specific biomarkers. Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline. Unexpectedly, isorenieratene and β-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their δ¹³C values were determined. Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in ¹³C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria. The difference between the δ¹³C values of farnesol, the major esterifying alcohol of BChl e, and CO₂ was −7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of “P. roseum” in situ is photoautotrophic.     

Structures of proteins and cofactors: X-ray crystallography

Protein crystallography is the predominately used technique for the determination of the three-dimensional structures of proteins and other macromolecules. In this article, the methodology utilized for the measurement and analysis of the diffraction data from crystals is briefly reviewed. As examples of both the usefulness and difficulties of this technique, the determination of the structures of several photosynthetic pigment–protein complexes is described, namely, the reaction center from purple bacteria, photosystem I and photosystem II from cyanobacteria, the light-harvesting complex II from purple bacteria, and the FMO protein from green bacteria.     

State 1-State 2 Transitions in a Unicellular Green Algae : Analysis of In Vivo Chlorophyll Fluorescence Induction Curves in the Presence of 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea (DCMU)

A study has been made on the State 1-State 2 transitions exhibited by the unicellular green algae Chlorella pyrenoidosa. Chlorophyll fluorescence induction curves from algae adapted to State 1 or State 2 have been analyzed and a comparison made with similar curves produced by decreasing the intensity of light going to the photosystem II reaction centers. In both cases, quenching of the maximum fluorescence yield (F_m) and the initial fluorescence yield (F_o) were observed so that the F_v/F_m ratio and the area above the induction curve (A_max) remained constant. The State 1-State 2 transition also produced changes in the β_max component indicative of some alteration within photosystem II organization. The implications of these experiments on the in vivo mechanism for energy redistribution between the two photosystems are discussed in terms of changes in absorption cross-section rather than being due to spillover from photosystem II to photosystem I. These changes may reflect the phosphorylation of the light-harvesting chlorophyll a/b protein complex and its subsequent migration away from the photosystem II core leading to its closer association with photosystem I.