Reaction Center Complex Research Articles

Supramolecular organization of photosynthetic complexes in membranes of Roseiflexus castenholzii.

The photosynthetic membranes of the filamentous anoxygenic phototroph Roseiflexus castenholzii have been studied with electron microscopy, atomic force microscopy, and biochemistry. Electron microscopy of the light-harvesting reaction center complex produced a 3D model that aligns with the solved crystal structure of the RC-LH1 from Thermochromatium tepidum with the H subunit removed. Atomic force microscopy of the whole membranes yielded a picture of the supramolecular organization of the major proteins in the photosynthetic electron transport chain. The results point to a loosely packed membrane without accessory antenna proteins or higher order structure.

Niveispirillum cyanobacteriorum sp. nov., a nitrogen-fixing bacterium isolated from cyanobacterial aggregates in a eutrophic lake

A Gram-stain-negative, slightly curved rod-shaped, non-spore-forming diazotrophic bacterium, designated strain TH16T, was isolated from cyanobacterial aggregates taken from eutrophic Lake Taihu, Jiangsu Province, China. The pH range for growth was 5-9 (optimum at pH 7.0), salinity range was 0-2% (w/v) NaCl (optimum 0%) and temperature range was 20-37 °C (optimum 30 °C) in nutrient broth. Phylogenetic analysis indicated that strain TH16T clusters near and is closely related to the genus Niveispirillum within the family Rhodospirillaceae of the class Alphaproteobacteria. Within the genus Niveispirillum, strain TH16T was related most closely to Niveispirillum irakense KBC1T (98.1% 16S rRNA gene sequence similarity) and Niveispirillum fermenti CC-LY736T (97.0 %). The DNA G+C content of strain TH16T was 64 mol%. DNA-DNA relatedness between strain TH16T and the type strains of N. irakense and N. fermenti was 39.6 and 30.1%, respectively. The major respiratory quinone was ubiquinone Q-10.The major fatty acids (>10%) were C18 : 1ω6c/C18 : 1ω7c, C18 : 1 2-OH and C16 : 0 3-OH. Genes in the puf operon, encoding proteins of the photosynthetic reaction centre and core light-harvesting complexes, were also present. Based on morphological, chemotaxonomic and phylogenetic data, strain TH16T represents a novel species within the genus Niveispirillum, for which the name Niveispirillum cyanobacteriorum sp. nov. is proposed. The type strain is TH16T ( = CGMCC 1.12958T = LMG 28334T).

Light-harvesting superstructures of green plant chloroplasts lacking photosystems.

The light-harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures - stacks of dozens of thylakoids or supergrana. Freeze-fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light-harvesting membranes, organized into three-dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self-assembly of the three-dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.

Expression Analysis of Salt Stress on Carotenoid Pathway Genes in Watermelon

Carotenoids, the naturally occurring isoprenoids form essential components of photosynthetic antenna and reaction centre complexes. Thus they play a significant role in absorption, dissipation and transfer of light energy for the process of photosynthesis. The expression of salt stress on carotenoid gene in watermelon leaves were studied. For that watermelon plants were subjected to different concentration of salt water. Morphological characters such as plant height, no. of fruits per plant,chlorophyll content and expression of four major carotenoid pathway genes such as phytoene synthase(PSY), phytoene desaturase(PDS), zeta carotene desaturase(ZDS) and lycopene beta cyclase(LCY-β) were analysed. The quantitative expression analysis using real time PCR has shown a decrease in the expression of all the studied genes as the salt concentration increased. Among the different concentrations of NaCl used for the experiment, it was seen that 200 mM was most detrimental for the carotenoid gene expression.Lycopene beta cyclase, the enzyme that converts lycopene to beta carotene was seen to be highly affected compared to other genes studied showing a 1.87 fold inhibition in its expression at 200 mM NaCl.

Ultra-high resolution structure of high-potential iron-sulfur protein

It is important for understanding the electron transfer reaction to include the information about valence shell electrons and hydrogen atoms into crystal structure refinement. High-potential iron-sulfur protein (HiPIP) possesses a Fe4S4 cluster which exhibits +2/+3 redox states and acts as an electron carrier from cytochrome bc1 complex to the reaction center complex in photosynthetic purple bacteria. We have reported the X-ray crystal structure of HiPIP from Thermochtomatium tepidum at 0.72 Å resolution (1). Recently, we have successfully collected 0.48 Å resolution data of HiPIP using high-energy X-rays (31 keV) in BL41XU beamline of SPring-8. We performed multipolar refinement with the MoPro program (2) to consider valence shell electrons in the structure refinement of HiPIP. Refinement of multipolar parameters was applied to atoms of single conformational residues, water molecules with two hydrogen atoms, and the Fe4S4 cluster. After multipolar refinement, the deformation map clearly displays the distribution of valence shell electrons such as lone-pair electrons of carbonyl oxygen atoms, bonding electrons in aromatic rings, and d-orbital electrons of Fe atoms in the Fe4S4 cluster. The deformation map also indicates electrostatic interactions between the S atoms of Fe4S4-(Cys-Sγ)4 and protein environment. In addition, we performed preliminary neutron diffraction experiment at iBIX beamline of Japan Proton Accelerator Research Complex (J-PARC) and observed diffraction spots up to 1.17 Å resolution using HiPIP crystal with the size of 2.3 mm3. In the multipolar refinement, the positions of hydrogen atoms were fixed to the standard bond distances derived from neutron crystal structures of small molecules and atomic displacement parameters of hydrogen atoms were constrained to 1.2 or 1.5 fold of their root atoms. Therefore, a high resolution neutron structure of HiPIP will improve the results obtained from the multipolar refinement.

Exchange and complementation of genes coding for photosynthetic reaction center core subunits among purple bacteria.

A mutant of the phototrophic species belonging to the β-proteobacteria, Rubrivivax gelatinosus, lacking the photosynthetic growth ability was constructed by the removal of genes coding for the L, M, and cytochrome subunits of the photosynthetic reaction center complex. The L, M, and cytochrome genes derived from five other species of proteobacteria, Acidiphilium rubrum, Allochromatium vinosum, Blastochloris viridis, Pheospirillum molischianum, and Roseateles depolymerans, and the L and M subunits from two other species, Rhodobacter sphaeroides and Rhodopseudomonas palustris, respectively, have been introduced into this mutant. Introduction of the genes from three of these seven species, Rte. depolymerans, Ach. vinosum, and Psp. molischianum, restored the photosynthetic growth ability of the mutant of Rvi. gelatinosus, although the growth rates were 1.5, 9.4, and 10.7 times slower, respectively, than that of the parent strain. Flash-induced kinetic measurements for the intact cells of these three mutants showed that the photo-oxidized cytochrome c bound to the introduced reaction center complex could be rereduced by electron donor proteins of Rvi. gelatinosus with a t1/2 of less than 10 ms. The reaction center core subunits of photosynthetic proteobacteria appear to be exchangeable if the sequence identities of the LM core subunits between donor and acceptor species are high enough, i.e., 70% or more.

Structural analysis of the homodimeric reaction center complex from the photosynthetic green sulfur bacterium Chlorobaculum tepidum.

The reaction center (RC) complex of the green sulfur bacterium Chlorobaculum tepidum is composed of the Fenna–Matthews–Olson antenna protein (FMO) and the reaction center core (RCC) complex. The RCC complex has four subunits: PscA, PscB, PscC, and PscD. We studied the FMO/RCC complex by chemically cross-linking the purified sample followed by biochemical and spectroscopic analysis. Blue-native gels showed that there were two types of FMO/RCC complexes, which are consistent with complexes with one copy of FMO per RCC and two copies of FMO per RCC. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the samples after cross-linking showed that all five subunits of the RC can be linked by three different cross-linkers: bissulfosuccinimidyl suberate, disuccinimidyl suberate, and 3,3-dithiobis-sulfosuccinimidyl propionate. The interaction sites of the cross-linked complex were also studied using liquid chromatography coupled to tandem mass spectrometry. The results indicated that FMO, PscB, PscD, and part of PscA are exposed on the cytoplasmic side of the membrane. PscD helps stabilize FMO to the reaction center and may facilitate transfer of the electron from the RC to ferredoxin. The soluble domain of the heme-containing cytochrome subunit PscC and part of the core subunit PscA are located on the periplasmic side of the membrane. There is a close relationship between the periplasmic portions of PscA and PscC, which is needed for the efficient transfer of the electron between PscC and P840.

Assembly of photosynthetic apparatus in Rhodobacter sphaeroides as revealed by functional assessments at different growth phases and in synchronized and greening cells.

The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0-4h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10(4)s(-1)) and yield (F v/F max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10(4)s(-1)). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.

Photoactive Electrodes Incorporating Electrosprayed Bacterial Reaction Centers

Highly efficient light absorption and charge separation within the photosystem and reaction center (RC) complexes of photosynthetic plants and bacteria are of great interest for solar cell and photo detector applications, since they offer almost unity quantum yield and expected ultimate power conversion efficiencies of more than 18% and 12%, respectively. In addition, the charge separated states created by these protein complexes are very long lived compared to conventional semiconductor solar cells. In this work, a novel technique is presented for the deposition of photosynthetic protein complexes, by electrospraying RCs of Rhodobacter sphaeroides onto highly ordered pyrolytic graphite (HOPG) electrodes. Remarkably, it is shown that the RCs not only survive exposure to the high electric fields but also yield peak photocurrent densities of up to 7 μA cm−2, which is equal to the highest value reported to date.

The purple heart of photosynthesis

The structure of a photosynthetic complex from a purple bacterium reveals a new class of light-harvesting protein and the channels that might allow electron-transporting molecules to escape this otherwise closed system. See Article p.228 Photosynthetic organisms convert sunlight energy into a form that can be used by cells. Kunio Miki and colleagues have now solved the first near-atomic-level structure of a complete bacterial light-harvesting antenna–reaction centre (LH1–RC) complex, bound to Ca2+ and various cofactors. The LH1 subunits form a ring surrounding the reaction centre, with bacteriochlorophyll and spirilloxanthin cofactors positioned within the ring and Ca2+ ions on the periplasmic side of LH1. The structure reveals how energy is transferred from the antenna to the reaction centre in a highly efficient way and suggests how ubiquinone might cross a closed LH1 barrier.

Structure of the LH1–RC complex from Thermochromatium tepidum at 3.0 Å

The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca(2+)-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 αβ-subunit that form a completely closed structure. The Ca(2+) ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure.

Singlet Oxygen Production by PSII Under Light Stress: Mechanism, Detection and the Protective role of β-Carotene

In this review, I outline the indirect evidence for the formation of singlet oxygen (1O2) obtained from experiments with the isolated PSII reaction center complex. I also review the methods we used to measure singlet oxygen directly, including luminescence at 1,270 nm, both steady state and time resolved. Other methods we used were histidine-catalyzed molecular oxygen uptake (enabling 1O2 yield measurements), and dye bleaching and difference absorption spectroscopy to identify where quenchers of 1O2 can access this toxic species. We also demonstrated the protective behavior of carotenoids bound within Chl–protein complexes which bring about a substantial amount of 1O2 quenching within the reaction center complex. Finally, I describe how these techniques have been used and expanded in research on photoinhibition and on the role of 1O2 as a signaling molecule in instigating cellular responses to various stress factors. I also discuss the current views on the role of 1O2 as a signaling molecule and the distance it might be able to travel within cells.

Primary radical ion pairs in photosystem II core complexes.

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700-710 nm at 278 K. It was found that the initial charge separation between P680* and ChlD1 (Chl-670) takes place with a time constant of ~1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680*((1-δ)) (P680(δ+)ChlD1(δ-)), where δ ~ 0.5. The subsequent electron transfer from P680(δ+)ChlD1(δ-) to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (ChlD1(δ-)) and bleaching of the PheoD1 bands at 420, 545, and 680 nm with development of the Pheo(-) band at 460 nm. Further electron transfer to QA occurs within 250 ps in accordance with earlier data. The spectra of P680(+) and Pheo(-) formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ~1 and ~13 ps due to the formation of P680(δ+)ChlD1(δ-) and P680(+)PheoD1(-), respectively.

Role of Electronic-Vibrational Mixing in Enhancing Vibrational Coherences in the Ground Electronic States of Photosynthetic Bacterial Reaction Center

We describe polarization controlled two-color coherence photon echo studies of the reaction center complex from a purple bacterium Rhodobacter sphaeroides. Long-lived oscillatory signals that persist up to 2 ps are observed in neutral, oxidized, and mutant (lacking the special pair) reaction centers, for both (0°,0°,0°,0°) and (45°,-45°,90°,0°) polarization sequences. We show that the long-lived signals arise via vibronic coupling of the bacteriopheophytin (H) and accessory bacteriochlorophyll (B) pigments that leads to vibrational wavepackets in the B ground electronic state. Fourier analysis of the data suggests that the 685 cm(-1) mode of B may play a key role in the H to B energy transfer.

1P255 紅色硫黄細菌由来光捕集1反応中心複合体における金属-タンパク質間相互作用の熱力学的解析(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

1P255 紅色硫黄細菌由来光捕集1反応中心複合体における金属-タンパク質間相互作用の熱力学的解析(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

1P253 同位体標識された好熱性紅色光合成細菌における膜タンパク質耐熱化の分子機構解析(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

1P253 同位体標識された好熱性紅色光合成細菌における膜タンパク質耐熱化の分子機構解析(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

Crystal Structure of Synechocystis Mutants and Plant Photosystem I

Plant Photosystem I (PSI) is one of the most intricate membrane complexes in Nature. It comprises two loosely bound reaction center and light-harvesting (LHC) complexes containing 18 subunits 174 chlorophylls, 32 carotenoids 2 quinines and 3 iron-sulfur clusters. The crystal structure at 3.1 A resolution reported her add several new features that were not detected in the previous structures. The location of an additional ten β-carotenes as well as five chlorophylls and several loop regions are now modeled. This represents the most complete plant Photosystem I structure obtained thus far, revealing the locations of and interactions among 18 protein subunits and 211 non-covalently bound photochemical cofactors. We also detected 12 additional carotenoids especially in the LHC complex as well as structural lipids. The latter suggest evolutionary scenario where Lhca1 and Lhca2 coevolved with LHCII and Lhca4 coevolved with CP29.Recently an operon encoding PSI was identified in cyanobacterial marine viruses. A PSI that mimics the most important feature was generated in Synechocystis 6803. This PSI is promiscuous for its electron donor and can accept electrons from respiratory cytochromes. The wild type and promiscuous PSI complexes were isolated, crystallized and their structure was solved to 4 and 3.8 A resolution, respectively. In addition we solved the virus-like structure of PsaL and PsaI minus PSI mutant at 2.8 A resolution. The novel structure of PSI from mesophilic cyanobacterium is different in several aspects in comparison with the previously published structure of PSI from Thermosynechococcus elongatus. The common properties of mesophilic cyanobacterial and plant PSI will be discussed.

Interdependence of carotenoid biosynthesis and assembly of pigment-protein complexes in Ectothiorhodospira haloalkaliphila cells

In the majority of species of photosynthetic bacteria, the lightharvesting antenna consists of two typesof pigment–protein complexes—peripheral LH2 andnearcentral LH1—which are located in the innercytoplasmic membrane [1]. The LH1 complex surrounds the reaction center (RC), forming a “core”complex with it (or the LH1–RC complex). Bothlightharvesting complexes were obtained in the pureform and are well studied [2–6]. Depending on thespecies of bacteria, the LH1 complex has the majorabsorption maximum at ~865–890 nm in the nearinfrared region of the spectrum, whereas the LH2 complex has two maxima (at ~800 and ~850 nm) [2, 3]. Inthe purple bacteria, these complexes are constructedaccording to a similar modular principle and consist oftwo types of polypeptides (α and β), bacteriochlorophyll (BChl), and carotenoids [4–6]. Carotenoids areadditional pigments. They perform several importantfunctions in vivo (lightharvesting, protecting, andstructural functions) [7, 8]. It was stated that the LH2complexes are not assembled in the cells of carotenoidless mutants of purple nonsulfur bacteria [9].There is another approach to obtaining carotenoidless LH2 complexes—the use of an inhibitor ofcarotenogenesis during cell culturing. Using theinhibitor diphenylamine (DPA), we obtained carotenoidless cells (DPA cells) of the purple sulfur bacterium

The cry-DASH cryptochrome encoded by the sll1629 gene in the cyanobacterium Synechocystis PCC 6803 is required for Photosystem II repair

The role of the Syn-CRY cryptochrome from the cyanobacterium Synechocystis sp. PCC 6803 has been a subject of research for more than a decade. Recently we have shown that photolyase, showing strong homology with Syn-CRY is required for Photosystem II repair by preventing accumulation of DNA lesions under UV-B (Vass et al. 2013). Here we investigated if Syn-CRY is also involved in PSII repair, either via removal of DNA lesions or other mechanism? The Δsll1629 mutant lacking Syn-CRY lost faster the PSII activity and D1 protein during UV-B or PAR than the WT. However, no detectable damages in the genomic DNA were observed. The transcript levels of the UV-B and light stress indicator gene psbA3, encoding D1, are comparable in the two strains showing that Δsll1629 cells are not defective at the transcriptional level. Nevertheless 2D protein analysis in combination with mass spectrometry showed a decreased accumulation of several, mostly cytoplasmic, proteins including PilA1 and bicarbonate transporter SbtA. Δsll1629 cells exposed to high light also showed a limitation in de novo assembly of PSII. It is concluded that Syn-CRY is required for efficient restoration of Photosystem II activity following UV-B and PAR induced photodamage. This effect is not caused by retardation of DNA repair, instead the synthesis of new D1 (and D2) subunit(s) and/or the assembly of the Photosystem II reaction center complex is likely affected due to the lack of intracellular CO2, or via a so far unidentified pathway that possibly includes the PilA1 protein.

ATR–FTIR Detection of Metal-Sensitive Structural Changes in the Light-Harvesting 1 Reaction Center Complex from the Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Thermochromatium tepidum grows at the highest temperature among purple bacteria, and the light-harvesting 1 reaction center (LH1-RC) complex enhances the thermal stability by utilizing Ca(2+), although the molecular mechanism has yet to be resolved. Here, we applied perfusion-induced attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy to highly purified LH1-RC complexes from Tch. tepidum and detected for the first time metal-sensitive fine structural changes involved in the enhanced thermal stability of this complex. The Tch. tepidum LH1-RC complex exhibited Sr(2+)/Ca(2+) ATR-FTIR difference bands that reflect changes in the polypeptide backbones and amino acid residues upon the replacement of native Ca(2+) with Sr(2+). The difference bands also appeared in the following Ca(2+)/Sr(2+) difference spectra with almost identical intensities but inverse signs, demonstrating that the structural changes induced by the metal exchange are fully reversible. In contrast, these ATR-FTIR signals were faintly detected in the mesophilic counterpart Allochromatium vinosum . A comparative analysis using LH1 complexes lacking the RCs strongly indicated that the metal-sensitive bands originate from polypeptide backbones and amino acid residues near the putative Ca(2+)-binding site at the C-terminal region of the Tch. tepidum LH1 complexes. Structural changes induced by Sr(2+) and Ba(2+) substitutions were essentially identical. However, Cd(2+) substitution exhibited unique structural modifications, which may be responsible for the severely deteriorated thermal stability of Cd(2+)-substituted complexes. Possible assignments for the present ATR-FTIR signals and their relation with the molecular mechanism of enhancing the thermal stability of Tch. tepidum LH1-RC proteins are discussed on the basis of the recent structural information on the Ca(2+)-binding site.