Reaction Center Complex Research Articles

Rate-limiting steps in the dark-to-light transition of Photosystem II - revealed by chlorophyll-a fluorescence induction

Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (∆τ) between them, we found that no rise could be induced with zero or short ∆τ, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII.

Isolation and Characterization of a Novel ATPase-Photosystem I Reaction Center Complex in the Chloroplast Thylakoid Membrane

A novel 1 MDa intra-membrane ‘super-complex’ dominated by the chloroplast ATP synthase and the photosystem I (PSI) reaction-center complex has been isolated from spinach thylakoid membranes. This super-complex has been characterized by assay of ATPase activity, negative stain electron microscopy, tandem mass spectrometry, and Western assay of subunits. High-resolution Orbitrap mass spectrometry was used to quantitatively analyze the presence and relative amount of the multiple major and minor polypeptide components in the super-complex. This is the first isolation of a higher order complex involving intact ATP synthase and Photosystem I to be identified and documented by negative stain electron microscopy. The importance of the ATPase-PSI super-complex in the thylakoid domain may imply efficient channeling of substrates and cofactors utilized for localized CO2 fixation. Studies supported by NIHGMS-038323 and the Henry Koffler Distinguished Professorship (WAC), and NIHDK-063491 (JPW).

Proposal for verifying dipole properties of light-harvesting antennas

For light harvesters with a reaction center complex (LH1-RC complex) of three types, we propose an experiment to verify our analysis based upon antenna theories that automatically include the required structural information. Our analysis conforms to the current understanding of light-harvesting antennas in that we can explain known properties of these complexes. We provide an explanation for the functional roles of the notch at the light harvester, a functional role of the polypeptide called PufX or W at the opening, a functional role of the special pair, a reason that the cross section of the light harvester must not be circular, a reason that the light harvester must not be spherical, reasons for the use of dielectric bacteriochlorophylls instead of conductors to make the light harvester, a mechanism to prevent damage from excess sunlight, an advantage of the dimeric form, and reasons for the modular design of nature. Based upon our analysis we provide a mechanism for dimerization. We predict that the dimeric form of light-harvesting complexes is favored under intense sunlight. We further comment upon the classification of the dimeric or S-shape complexes. The S-shape complexes should not be considered as the third type of light harvester but simply as a composite form.

Spectrally selective fluorescence imaging of Chlorobaculum tepidum reaction centers conjugated to chelator-modified silver nanowires

A polyhistidine tag (His-tag) present on Chlorobaculum tepidum reaction centers (RCs) was used to immobilize photosynthetic complexes on a silver nanowire (AgNW) modified with nickel-chelating nitrilo-triacetic acid (Ni-NTA). The optical properties of conjugated nanostructures were studied using wide-field and confocal fluorescence microscopy. Plasmonic enhancement of RCs conjugated to AgNWs was observed as their fluorescence intensity dependence on the excitation wavelength does not follow the excitation spectrum of RC complexes in solution. The strongest effect of plasmonic interactions on the emission intensity of RCs coincides with the absorption spectrum of AgNWs and is observed for excitation into the carotenoid absorption. From the absence of fluorescence decay shortening, we attribute the emission enhancement to increase of absorption in RC complexes.

Characterization of thermotolerant phototrophic bacteria, Rhodoplanes tepidicaeni sp. nov. and Rhodoplanes azumiensis sp. nov., isolated from a geothermal spring.

Two strains of thermotolerant phototrophic alphaproteobacteria, designated strains TUT3542T and TUT3581T, were isolated from sediment mud and cyanobacterial mats in a geothermal spring in Japan, respectively, and taxonomically characterized. Both the strains were budding motile rods and were able to grow at 45 °C. Phototrophically grown cells of strains TUT3542T and TUT3581T produced pink and brownish red cultures, respectively, and showed in vivo absorption maxima at 800, 858-859 and 892-895 nm in the near infrared region, indicating the presence of a core reaction centre and peripheral pigment complexes with bacteriochlorophyll a. The intracytoplasmic membrane system was of the lamellar type parallel to the cytoplasmic membrane. 16S rRNA gene sequence comparisons showed that strains TUT3542T and TUT3581T had the highest similarity level to Rhodoplanes oryazae NBRC 109406T (99.6 %) and Rhodoplaneselegans AS130T (99.3 %), respectively. Genomic DNA-DNA reassociation studies revealed that strains TUT3542T and TUT3581T had hybridization levels of less than 62 and 56 % to the type strains of all established species of the genus Rhodoplanes, respectively. The G+C contents of genomic DNA were 67.7 mol% for strain TUT3542T and 70.4 mol% for strain TUT3581T. Results of phenotypic studies showed that the two novel strains could be differentiated from any of the previously described Rhodoplanes species. Thus, the author proposes the names Rhodoplanes tepidicaeni sp. nov. for strain TUT3542T and Rhodoplanes azumiensis sp. nov. for strain TUT3581T. The type strain of Rhodoplanes tepidicaeni is TUT3542T (=KCTC 15602T=NBRC 112815T) and the type strain of Rhodoplanes azumiensis is TUT3581T (=KCTC 15603T=NBRC 112816T).

The Alleviation of Heat Damage to Photosystem II and Enzymatic Antioxidants by Exogenous Spermidine in Tall Fescue.

Tall fescue (Festuca arundinacea Schreb) is a typical cool-season grass that is widely used in turf and pasture. However, high temperature as an abiotic stress seriously affects its utilization. The objective of this study was to explore the effect of spermidine (Spd) on heat stress response of tall fescue. The samples were exposed to 22°C (normal condition) or 44°C (heat stress) for 4 h. The results showed that exogenous Spd partially improved the quality of tall fescue leaves under normal temperature conditions. Nevertheless, after heat stress treatment, exogenous Spd significantly decreased the electrolyte leakage of tall fescue leaves. Spd also profoundly reduced the H2O2 and O2⋅- content and increased antioxidant enzymes activities. In addition, PAs can also regulate antioxidant enzymes activities including SOD, POD, and APX which could help to scavenge ROS. Moreover, application of Spd could also remarkably increase the chlorophyll content and had a positive effect on the chlorophyll α fluorescence transients under high temperature. The Spd reagent enhanced the performance of photosystem II (PSII) as observed by the JIP-test. Under heat stress, the Spd profoundly improved the partial potentials at the steps of energy bifurcations (PIABS and PItotal) and the quantum yields and efficiencies (φP0, δR0, φR0, and γRC). Exogenous Spd could also reduce the specific energy fluxes per QA- reducing PSII reaction center (RC) (TP0/RC and ET0/RC). Additionally, exogenous Spd improved the expression level of psbA and psbB, which encoded the proteins of PSII core reaction center complex. We infer that PAs can stabilize the structure of nucleic acids and protect RNA from the degradation of ribonuclease. In brief, our study indicates that exogenous Spd enhances the heat tolerance of tall fescue by maintaining cell membrane stability, increasing antioxidant enzymes activities, improving PSII, and relevant gene expression.

FtsH2-Dependent Proteolysis of EXECUTER1 Is Essential in Mediating Singlet Oxygen-Triggered Retrograde Signaling in Arabidopsis thaliana.

Photosystem II reaction center (PSII RC) and light-harvesting complex inevitably generate highly reactive singlet oxygen (1O2) that can impose photo-oxidative damage, especially when the rate of generation exceeds the rate of detoxification. Besides being toxic, 1O2 has also been ascribed to trigger retrograde signaling, which leads to nuclear gene expression changes. Two distinctive molecular components appear to regulate 1O2 signaling: a volatile signaling molecule β-cyclocitral (β-CC) generated upon oxidation of β-carotene by 1O2 in PSII RC assembled in grana core, and a thylakoid membrane-bound FtsH2 metalloprotease that promotes 1O2-triggered signaling through the proteolysis of EXECUTER1 (EX1) proteins associated with PSII in grana margin. The role of FtsH2 protease in 1O2 signaling was established recently in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that generates 1O2 upon dark-to-light shift. The flu mutant lacking functional FtsH2 significantly impairs 1O2-triggered and EX1-mediated cell death. In the present study, the role of FtsH2 in the induction of 1O2 signaling was further clarified by analyzing the FtsH2-dependent nuclear gene expression changes in the flu mutant. Genome-wide transcriptome analysis showed that the inactivation of FtsH2 repressed the majority (85%) of the EX1-dependent 1O2-responsive genes (SORGs), providing direct connection between FtsH2-mediated EX1 degradation and 1O2-triggered gene expression changes. Furthermore, the overlap between β-CC-induced genes and EX1-FtsH2-dependent genes was very limited, further supporting the coexistence of two distinctive 1O2 signaling pathways.

First moves towards photoautotrophic synthetic cells: In vitro study of photosynthetic reaction centre and cytochrome bc1 complex interactions

Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly “synthetic cells”) can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy.Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles. Next, the details of our ongoing research program are presented. It involves the use of the reaction centre, the coenzyme Q–cytochrome c oxidoreductase, and the ATP synthase for creating an autonomous synthetic cell. We show experimental results on the chemistry of the first two proteins showing that they can efficiently sustain light-driven chemical oscillations. Moreover, the cyclic pattern has been reproduced in silico by a minimal kinetic model.

C-terminal cleavage of the LH1 α-polypeptide in the Sr2+-cultured Thermochromatium tepidum.

The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q y absorption at 915nm and enhanced thermostability. Ca2+ can be biosynthetically replaced by Sr2+ in growing cultures of Tch. tepidum. However, the resulting Sr2+-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca2+-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca2+- and Sr2+-cultured cells. In this study, we examined the effects of the biosynthetic Sr2+-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr2+-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr2+-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr2+ substitutes for Ca2+ in the LH1 complex. Limited proteolysis of the native Ca2+-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr2+-substituted LH1-RC complexes in their Ca2+-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum.

Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein-protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the "mass tags" selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein-protein interactions in membrane protein complexes.

Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP–RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.

Spectroscopic and Thermodynamic Characterization of the Metal-Binding Sites in the LH1-RC Complex from Thermophilic Photosynthetic Bacterium Thermochromatium tepidum.

The light-harvesting 1 reaction center (LH1-RC) complex from thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits enhanced thermostability and an unusual LH1 Qy transition, both induced by Ca2+ binding. In this study, metal-binding sites and metal-protein interactions in the LH1-RC complexes from wild-type (B915) and biosynthetically Sr2+-substituted (B888) Tch. tepidum were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopies. The ITC measurements revealed stoichiometric ratios of approximately 1:1 for binding of Ca2+, Sr2+, or Ba2+ to the LH1 αβ-subunit, indicating the presence of 16 binding sites in both B915 and B888. The AA analysis provided direct evidence for Ca2+ and Sr2+ binding to B915 and B888, respectively, in their purified states. Metal-binding experiments supported that Ca2+ and Sr2+ (or Ba2+) competitively associate with the binding sites in both species. The ATR-FTIR difference spectra upon Ca2+ depletion and Sr2+ substitution demonstrated that dissociation and binding of Ca2+ are predominantly responsible for metal-dependent conformational changes of B915 and B888. The present results are largely compatible with the recent structural evidence that another binding site for Sr2+ (or Ba2+) exists in the vicinity of the Ca2+-binding site, a part of which is shared in both metal-binding sites.

Light harvesting a gold porphyrin-zinc phthalocyanine supramolecular donor-acceptor dyad.

We reported herein the spectroscopic, electrochemical and laser photolysis studies for the newly constructed light harvesting supramolecular dyad composed of gold porphyrin (AuPpy), as an electron acceptor, and zinc phthalocyanine (ZnPc), as an electron donor, to mimic the reaction centre in the photosynthetic system. For this, gold porphyrin has been functionalized by pyridine units, which axially coordinated with zinc phthalocyanine to form the stable supramolecular AuPpy:ZnPc with a rate of 2.94 × 104 M-1. Steady-state fluorescence measurements showed significant quenching of the singlet excited ZnPc emission with addition of AuPpy, suggesting an electron transfer from the singlet excited ZnPc to AuPpy. The electron transfer character was confirmed by recoding the characteristic absorption band of the zinc phthalocyanine radical cation in the NIR region by a femtosecond laser photolysis technique. The findings that the AuPpy:ZnPc supramolecular dyad exhibits relatively long-lived radical-ion pairs and absorbs light in a wide range of the solar spectrum suggest that it would be useful as a photosynthetic reaction centre.

Allocation of Heme Is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells.

Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.

Native Mass Spectrometry Characterizes the Photosynthetic Reaction Center Complex from the Purple Bacterium Rhodobacter sphaeroides.

Native mass spectrometry (MS) is an emerging approach to study protein complexes in their near-native states and to elucidate their stoichiometry and topology. Here, we report a native MS study of the membrane-embedded reaction center (RC) protein complex from the purple photosynthetic bacterium Rhodobacter sphaeroides. The membrane-embedded RC protein complex is stabilized by detergent micelles in aqueous solution, directly introduced into a mass spectrometer by nano-electrospray (nESI), and freed of detergents and dissociated in the gas phase by collisional activation. As the collision energy is increased, the chlorophyll pigments are gradually released from the RC complex, suggesting that native MS introduces a near-native structure that continues to bind pigments. Two bacteriochlorophyll a pigments remain tightly bound to the RC protein at the highest collision energy. The order of pigment release and their resistance to release by gas-phase activation indicates the strength of pigment interaction in the RC complex. This investigation sets the stage for future native MS studies of membrane-embedded photosynthetic pigment-protein and related complexes.Graphical Abstract.

Spectral and kinetic effects accompanying the assembly of core complexes of Rhodobacter sphaeroides

In the present work, spectral and kinetic changes accompanying the assembly of the light-harvesting 1 (LH1) complex with the reaction center (RC) complex into monomeric RC-LH1 and dimeric RC-LH1-PufX core complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides are systematically studied over the temperature range of 4.5–300K. The samples were interrogated with a combination of optical absorption, hole burning, fluorescence excitation, steady state and picosecond time resolved fluorescence spectroscopy. Fair additivity of the LH1 and RC absorption spectra suggests rather weak electronic coupling between them. A low-energy tail revealed at cryogenic temperatures in the absorption spectra of both monomeric and dimeric core complexes is proved to be due to the special pair of the RC. At selected excitation intensity and temperature, the fluorescence decay time of core complexes is shown to be a function of multiple factors, most importantly of the presence/absence of RCs, the supramolecular architecture (monomeric or dimeric) of the complexes, and whether the complexes were studied in a native membrane environment or in a detergent - purified state.

Evidence for a cysteine-mediated mechanism of excitation energy regulation in a photosynthetic antenna complex

Light-harvesting antenna complexes not only aid in the capture of solar energy for photosynthesis, but regulate the quantity of transferred energy as well. Light-harvesting regulation is important for protecting reaction center complexes from overexcitation, generation of reactive oxygen species, and metabolic overload. Usually, this regulation is controlled by the association of light-harvesting antennas with accessory quenchers such as carotenoids. One antenna complex, the Fenna-Matthews-Olson (FMO) antenna protein from green sulfur bacteria, completely lacks carotenoids and other known accessory quenchers. Nonetheless, the FMO protein is able to quench energy transfer in aerobic conditions effectively, indicating a previously unidentified type of regulatory mechanism. Through de novo sequencing MS, chemical modification, and mutagenesis, we have pinpointed the source of the quenching action to cysteine residues (Cys49 and Cys353) situated near two low-energy bacteriochlorophylls in the FMO protein from Chlorobaculum tepidum Removal of these cysteines (particularly removal of the completely conserved Cys353) through N-ethylmaleimide modification or mutagenesis to alanine abolishes the aerobic quenching effect. Electrochemical analysis and electron paramagnetic resonance spectra suggest that in aerobic conditions the cysteine thiols are converted to thiyl radicals which then are capable of quenching bacteriochlorophyll excited states through electron transfer photochemistry. This simple mechanism has implications for the design of bio-inspired light-harvesting antennas and the redesign of natural photosynthetic systems.

Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial Photosynthetic Reaction Center from Rhodobacter sphaeroides.

It has become increasingly clear that dynamics plays a major role in the function of many protein systems. One system that has proven particularly facile for studying the effects of dynamics on protein-mediated chemistry is the bacterial photosynthetic reaction center from Rhodobacter sphaeroides. Previous experimental and computational analysis have suggested that the dynamics of the protein matrix surrounding the primary quinone acceptor, QA, may be particularly important in electron transfer involving this cofactor. One can substantially increase the flexibility of this region by removing one of the reaction center subunits, the H-subunit. Even with this large change in structure, photoinduced electron transfer to the quinone still takes place. To evaluate the effect of H-subunit removal on electron transfer to QA, we have compared the kinetics of electron transfer and associated spectral evolution for the LM dimer with that of the intact reaction center complex on picosecond to millisecond time scales. The transient absorption spectra associated with all measured electron transfer reactions are similar, with the exception of a broadening in the QX transition and a blue-shift in the QY transition bands of the special pair of bacteriochlorophylls (P) in the LM dimer. The kinetics of the electron transfer reactions not involving quinones are unaffected. There is, however, a 4-fold decrease in the electron transfer rate from the reduced bacteriopheophytin to QA in the LM dimer compared to the intact reaction center and a similar decrease in the recombination rate of the resulting charge-separated state (P(+)QA(-)). These results are consistent with the concept that the removal of the H-subunit results in increased flexibility in the region around the quinone and an associated shift in the reorganization energy associated with charge separation and recombination.

Diversity of the aerobic anoxygenic phototrophy gene pufM in Arctic and Antarctic coastal seawaters

Aerobic anoxygenic phototrophic (AAP) bacteria serve important functions in marine carbon and energy cycling because of their capability to utilize dissolved organic substrates and harvest light energy. AAP bacteria are widely distributed in marine environments, and their diversity has been examined in marine habitats. However, information about AAP bacteria at high latitudes remains insufficient to date. Therefore, this study determined the summer AAP bacterial diversity in Arctic Kongsfjorden and in the Antarctic coastal seawater of King George Island on the basis of pufM, a gene that encodes a pigment-binding protein subunit of the reaction center complex. Four pufM clone libraries were constructed, and 674 positive clones were obtained from four investigated stations (two in Kongsfjorden and two in the Antarctic Maxwell Bay). Arctic clones were clustered within the Alphaproteobacteria, whereas Antarctic clones were classified into the Alphaproteobacteria and Betaproteobacteria classes. Rhodobacteraceae-like pufM genes dominated in all samples. In addition, sequences closely related to pufM encoded on a plasmid in Sulfitobacter guttiformis were predominant in both Arctic and Antarctic samples. This result indicates the transpolar or even global distribution of pufM genes in marine environments. Meanwhile, differences between the Arctic and Antarctic sequences may prove polar endemism. These results indicate the important role of Rhodobacteraceae as AAP bacteria in bipolar coastal waters.

Structure-Based Modeling of Fluorescence Kinetics of Photosystem II: Relation between Its Dimeric Form and Photoregulation.

A photosystem II-enriched membrane (PSII-em) consists of the PSII core complex (PSII-cc) which is surrounded by peripheral antenna complexes. PSII-cc consists of two core antenna (CP43 and CP47) and the reaction center (RC) complex. Time-resolved fluorescence spectra of a PSII-em were measured at 77 K. The data were globally analyzed with a new compartment model, which has a minimum number of compartments and is consistent with the structure of PSII-cc. The reliability of the model was investigated by fitting the data of different experimental conditions. From the analysis, the energy-transfer time constants from the peripheral antenna to CP47 and CP43 were estimated to be 20 and 35 ps, respectively. With an exponential time constant of 320 ps, the excitation energy was estimated to accumulate in the reddest chlorophyll (Red Chl), giving a 692 nm fluorescence peak. The excited state on the Red Chl was confirmed to be quenched upon the addition of an oxidant, as reported previously. The calculations based on the Förster theory predicted that the excitation energy on Chl29 is quenched by ChlZD1(+), which is a redox active but not involved in the electron-transfer chain, located in the D1 subunit of RC, in the other monomer with an exponential time constant of 75 ps. This quenching pathway is consistent with our structure-based simulation of PSII-cc, which assigned Chl29 as the Red Chl. On the other hand, the alternative interpretation assigning Chl26 as the Red Chl was not excluded. The excited Chl26 was predicted to be quenched by another redox active ChlZD2(+) in the D2 subunit of RC in the same monomer unit with an exponential time constant of 88 ps.