Structure of photosynthetic LH1-RC supercomplex at 1.9 Å resolution.

Light-harvesting complex 1 (LH1) and the reaction centre (RC) form a membrane-protein supercomplex that performs the primary reactions of photosynthesis in purple photosynthetic bacteria. The structure of the LH1-RC complex can provide information on the arrangement of protein subunits and cofactors; however, so far it has been resolved only at a relatively low resolution. Here we report the crystal structure of the calcium-ion-bound LH1-RC supercomplex of Thermochromatium tepidum at a resolution of 1.9 Å. This atomic-resolution structure revealed several new features about the organization of protein subunits and cofactors. We describe the loop regions of RC in their intact states, the interaction of these loop regions with the LH1 subunits, the exchange route for the bound quinone QB with free quinone molecules, the transport of free quinones between the inside and outside of the LH1 ring structure, and the detailed calcium-ion-binding environment. This structure provides a solid basis for the detailed examination of the light reactions that occur during bacterial photosynthesis.

Charge-density analysis of an iron-sulfur protein at an ultra-high resolution of 0.48 Å.

The fine structures of proteins, such as the positions of hydrogen atoms, distributions of valence electrons and orientations of bound waters, are critical factors for determining the dynamic and chemical properties of proteins. Such information cannot be obtained by conventional protein X-ray analyses at 3.0-1.5 Å resolution, in which amino acids are fitted into atomically unresolved electron-density maps and refinement calculations are performed under strong restraints. Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with pre-existing common knowledge obtained by chemistry in small molecules. However, even now, computational calculation of such information with quantum chemistry also tends to be difficult, especially for polynuclear metalloproteins. Here we report a charge-density analysis of the high-potential iron-sulfur protein from the thermophilic purple bacterium Thermochromatium tepidum using X-ray data at an ultra-high resolution of 0.48 Å. Residual electron densities in the conventional refinement are assigned as valence electrons in the multipolar refinement. Iron 3d and sulfur 3p electron densities of the Fe4S4 cluster are visualized around the atoms. Such information provides the most detailed view of the valence electrons of the metal complex in the protein. The asymmetry of the iron-sulfur cluster and the protein environment suggests the structural basis of charge storing on electron transfer. Our charge-density analysis reveals many fine features around the metal complex for the first time, and will enable further theoretical and experimental studies of metalloproteins.

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 αß-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.

A Dual Role for Ca2+ in Expanding the Spectral Diversity and Stability of Light-Harvesting 1 Reaction Center Photocomplexes of Purple Phototrophic Bacteria.

The light-harvesting 1 reaction center (LH1-RC) complex in the purple sulfur bacterium Thiorhodovibrio ( Trv.) strain 970 cells exhibits its LH1 Q y transition at 973 nm, the lowest-energy Q y absorption among purple bacteria containing bacteriochlorophyll a (BChl a). Here we characterize the origin of this extremely red-shifted Q y transition. Growth of Trv. strain 970 did not occur in cultures free of Ca2+, and elemental analysis of Ca2+-grown cells confirmed that purified Trv. strain 970 LH1-RC complexes contained Ca2+. The LH1 Q y band of Trv. strain 970 was blue-shifted from 959 to 875 nm upon Ca2+ depletion, but the original spectral properties were restored upon Ca2+ reconstitution, which also occurs with the thermophilic purple bacterium Thermochromatium ( Tch.) tepidum. The amino acid sequences of the LH1 α- and ß-polypeptides from Trv. strain 970 closely resemble those of Tch. tepidum; however, Ca2+ binding in the Trv. strain 970 LH1-RC occurred more selectively than in Tch. tepidum LH1-RC and with a reduced affinity. Ultraviolet resonance Raman analysis indicated that the number of hydrogen-bonding interactions between BChl a and LH1 proteins of Trv. strain 970 was significantly greater than for Tch. tepidum and that Ca2+ was indispensable for maintaining these bonds. Furthermore, perfusion-induced Fourier transform infrared analyses detected Ca2+-induced conformational changes in the binding site closely related to the unique spectral properties of Trv. strain 970. Collectively, our results reveal an ecological strategy employed by Trv. strain 970 of integrating Ca2+ into its LH1-RC complex to extend its light-harvesting capacity to regions of the near-infrared spectrum unused by other purple bacteria.

Calcite formation induced by Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens and Rheinheimera texasensis.

A wide range of bacterial species are able to induce calcium carbonate precipitation. Using our own laboratory-preserved strains, we have newly discovered that Ensifer sp. MY11e, Microbacterium sp. TMd9a1, Paeniglutamicibacter sp. MSa1a, Pseudomonas sp. GTc3, and Rheinheimera sp. ATWe6 can induce the formation of calcite crystals on an agar medium. Type strains of their closely related species (Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens, and Rheinheimera texasensis) could also induce calcite formation. Although the initial pH value of the agar medium was 6.1, the pH of the agar media containing calcite, induced by cultivation of the 10 bacterial strains, increased to 8.0-8.4. The ammonification (oxidative deamination) of amino acids may been responsible for this increase in pH. The crystals formed both on and around the bacterial colonies. Furthermore, when these strains (excepting two Microbacterium strains) were cultivated on a cellulose acetate membrane filter (0.20 µm pore size) resting on the surface of the agar medium (i.e., in the membrane filter culture method), the crystals formed on the agar medium separate from the bacterial cells. These results indicate that the bacterial cells did not necessarily become nucleation sites for these crystals. We also investigated whether the studied strains could be applied to the biocementation of sand, and found that only two Ensifer strains were able to form large sand lumps.

Singlet-triplet Fission of Carotenoid Excitation in the Purple Phototrophic Bacteria Thermochromatium tepidum.

Singlet fission of carotenoid excitation is studied in purple phototrophic bacterium Thermochromatium tepidum. Using time-resolved EPR and magnetic field-induced modulation of fluorescence yield it is shown that the concept of intramolecular excitation fission developed in a number of publications is not supported by the experimental results. The obtained data favor intermolecular fission mechanism involving two carotenoid molecules.

Biochemical and Spectroscopic Characterizations of a Hybrid Light-Harvesting Reaction Center Core Complex.

The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium tepidum exhibits a largely red-shifted LH1 Q y absorption at 915 nm due to binding of Ca2+, resulting in an "uphill" energy transfer from LH1 to the reaction center (RC). In a recent study, we developed a heterologous expression system (strain TS2) to construct a functional hybrid LH1-RC with LH1 from Tch. tepidum and the RC from Rhodobacter sphaeroides [Nagashima, K. V. P., et al. (2017) Proc. Natl. Acad. Sci. U. S. A. 114, 10906]. Here, we present detailed characterizations of the hybrid LH1-RC from strain TS2. Effects of metal cations on the phototrophic growth of strain TS2 revealed that Ca2+ is an indispensable element for its growth, which is also true for Tch. tepidum but not for Rba. sphaeroides. The thermal stability of the TS2 LH1-RC was strongly dependent on Ca2+ in a manner similar to that of the native Tch. tepidum, but interactions between the heterologous LH1 and RC became relatively weaker in strain TS2. A Fourier transform infrared analysis demonstrated that the Ca2+-binding site of TS2 LH1 was similar but not identical to that of Tch. tepidum. Steady-state and time-resolved fluorescence measurements revealed that the uphill energy transfer rate from LH1 to the RC was related to the energy gap in an order of Rba. sphaeroides, Tch. tepidum, and strain TS2; however, the quantum yields of LH1 fluorescence did not exhibit such a correlation. On the basis of these findings, we discuss the roles of Ca2+, interactions between LH1 and the RC from different species, and the uphill energy transfer mechanisms.

Calculation of the excited states properties of LH1 complex of Thermochromatium tepidum.

Calculation of the excited states properties of pigment complexes is one of the key problems in the photosynthesis research. The excited states of LH1 complex of Thermochromatium tepidum were studied by means of the high-precision quantum chemistry methods. The influence of different parameters of the calculation procedure was examined. The optimal scheme of calculation was chosen by comparison of calculated results with the experimental data on absorption, electronic and magnetic circular dichroism spectra. The high importance of the account of the second excited states of bacteriochlorophylls and of site heterogeneity was shown. © 2018 Wiley Periodicals, Inc.

The Influence of the Number of Conjugated Double Bonds in Carotenoid Molecules on the Energy Transfer Efficiency to Bacteriochlorophyll in Light-Harvesting Complexes LH2 from Allochromatium vinosum Strain MSU.

Seven different carotenoids with the number of conjugated double bonds (N) from 5 to 11 were incorporated in vitro into carotenoidless complexes LH2 of the sulfur bacterium Allochromatium vinosum strain MSU. The efficiency of their incorporation varied from 4 to 99%. The influence of N in the carotenoid molecules on the energy transfer efficiency from these pigments to bacteriochlorophyll (BChl) in the modified LH2 complexes was studied for the first time. The highest level of energy transfer was recorded for rhodopin (N = 11) and neurosporene (N = 7) (37 and 51%, respectively). In the other carotenoids, this parameter ranged from 11 to 33%. In the LH2 complexes studied, we found no direct correlation between the decrease in N in carotenoids and increase in the energy transfer efficiency from these pigments to BChl.

Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting.

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880nm to 915nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and ß of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and ß polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/ß domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes.

Carotenoid Singlet Fission Reactions in Bacterial Light Harvesting Complexes As Revealed by Triplet Excitation Profiles.

Carotenoids (Cars) in bacterial photosynthesis are known as accessory light harvesters and photoprotectors. Recently, the singlet fission (SF) reaction initiated by Car photoabsorption has been recognized to be an effective excitation deactivation channel disfavoring the light harvesting function. Since the SF reaction and the triplet sensitization reaction underlying photoprotection both yield triplet excited state Cars (3Car*), their contribution to the overall 3Car* photoproduction are difficult to disentangle. To tackle this problem, we resorted to the triplet excitation profiles (TEPs), i.e., the actinic spectra of the overall 3Car* photoproduction. The TEPs combined with the conventional fluorescence excitation spectra allowed us to extract the neat SF contribution, which can serve as a spectroscopic measure for the SF reactivity. This novel spectroscopic strategy was applied to analyze the light harvesting complexes (LHs) from Tch. tepidum and Rba. sphaeroides 2.4.1. The results unambiguously showed that the SF reaction of Cars proceeds with an intramolecular scheme, even in the case of LH1-RC from Rba. sphaeroides 2.4.1 likely binding a secondary pool of Cars. Regarding the SF-reactivity, the geometric distortion in the conjugated backbone of Cars was shown to be the structural determinant, while the length of the Car conjugation was suggested to be relevant to the effective localization of the geminate triplets to avoid being annihilated. The SF reaction scheme and structure-activity relationship revealed herein will be useful not only in deepening our understanding of the roles of Cars in photosynthesis, but also in enlightening the applications of Cars in artificial light conversion systems.

Metal Cations Induced αß-BChl a Heterogeneity in LH1 as Revealed by Temperature-Dependent Fluorescence Splitting.

Two spectral forms of the core light-harvesting complex (LH1) of the purple bacterium Thermochromatium (Tch.) tepidum, the native Ca2+ -binding and the Ba2+ -substituted one, exhibit different fluorescence (FL) emission spectra at low temperature (T). While Ca-LH1 exhibits one emission band, an unusual splitting of the fluorescence is observed for Ba-LH1. These two sub-bands display the same spectral-width dependence according to T, but their intensity evolves differently with T. Based on the crystal structures, we propose that the FL splitting originates from a large αß-BChl a transition energy heterogeneity, ≈600 cm-1 , which is much larger compared with other LH1 and LH2 complexes (80-200 cm-1 ). This large heterogeneity is induced by the inhomogeneous Coulomb (and possibly hydrogen-bonding) interactions exerted by Ba2+ . The energy levels of the two LH1s were compared using exciton calculations in combination with Redfield theory. To simulate the FL splitting, an electronic transition containing two resonant bands was considered. This work shows how metal cations incorporated into the polypeptide modulate the electronic properties of BChl a aggregates.

Dependence of the hydration status of bacterial light-harvesting complex 2 on polyol cosolvents.

Low molecular weight (MW) polyols are organic osmolytes influencing protein structure and activity. We have intended to investigate the effects of low MW polyols on the optical and the excited-state properties of the light-harvesting complex 2 (LH2) isolated from the photosynthetic bacterium Thermochromatium (Tch.) tepidum, a thermophile growing at ∼50 °C. Steady state spectroscopy demonstrated that, on increasing glycerol or sorbitol fractions up to 60% (polyol/water, v/v), the visible absorption of carotenoids (Crts) remained unchanged, while the near infrared Qy absorption of bacteriochlorophyll a (BChl) at 800 nm (B800) and 850 nm (B850) varied slightly. Further increasing the fraction of glycerol (but not sorbitol) to 80% (v/v) induced distinct changes of the near infrared absorption and fluorescence spectra. Transient absorption spectroscopy revealed that, following the fast processes of BChl-to-Crt triplet energy transfer, rather weak Qy signals of B800 and B850 remained and evolved in phase with the kinetics of triplet excited state Crt (3Crt*), which are attributed to the Qy band shift as a result of 3Crt*-BChl interaction. The steady state and the transient spectral responses of the Qy bands are found to correlate intimately with the water activity varying against polyol MW and mixing ratio, which are rationalized by the change of the hydration status of the C- and N-termini of LH2. Our results suggest that, with reference to the mesophilic purple bacterium Rhodobacter sphaeroides 2.4.1, Tch. tepidum adopts substantially more robust LH2 hydration against the osmotic effects from the low MW polyols.

Emended description of the family Chromatiaceae, phylogenetic analyses of the genera Alishewanella, Rheinheimera and Arsukibacterium, transfer of Rheinheimera longhuensis LH2-2T to the genus Alishewanella and description of Alishewanella alkalitolerans sp. nov. from Lonar Lake, India.

Phylogenetic analyses were performed for members of the family Chromatiaceae, signature nucleotides deduced and the genus Alishewanella transferred to Chromatiaceae. Phylogenetic analyses were executed for the genera Alishewanella, Arsukibacterium and Rheinheimera and the genus Rheinheimera is proposed to be split, with the creation of the Pararheinheimera gen. nov. Furthermore, the species Rheinheimera longhuensis, is transferred to the genus Alishewanella as Alishewanella longhuensis comb. nov. Besides, the genera Alishewanella and Rheinheimera are also emended. Strain LNK-7.1T was isolated from a water sample from the Lonar Lake, India. Cells were Gram-negative, motile rods, positive for catalase, oxidase, phosphatase, contained C16:0, C17:1ω8c, summed feature3 (C16:1ω6c and/or C16:1ω7c) and summed feature 8 (C18:1ω7c) as major fatty acids, PE and PG as the major lipids and Q-8 as the sole respiratory quinone. Phylogenetic analyses using NJ, ME, ML and Maximum parsimony, based on 16S rRNA gene sequences, identified Alishewanella tabrizica RCRI4T as the closely related species of strain LNK-7.1T with a 16S rRNA gene sequence similarity of 98.13%. The DNA-DNA similarity between LNK-7.1T and the closely related species (A. tabrizica) was only 12.0% and, therefore, strain LNK-7.1T was identified as a novel species of the genus Alishewanella with the proposed name Alishewanella alkalitolerans sp. nov. In addition phenotypic characteristics confirmed the species status to strain LNK-7.1T. The type strain of A. alkalitolerans is LNK-7.1T (LMG 29592T = KCTC 52279T), isolated from a water sample collected from the Lonar lake, India.

Antibacterial low-molecular-weight compounds produced by the marine bacterium Rheinheimera japonica KMM 9513T.

Strain KMM 9513T was isolated from a sediment sample collected from the Sea of Japan seashore and selected due to its ability to inhibit indicator bacterial growth. The strain KMM 9513T has been recently described as a novel species Rheinheimera japonica. This study was undertaken to determine which substances produced by strain KMM 9513T could be responsible for its antimicrobial activity. Eight compounds were obtained from an ethyl acetate extract of R. japonica KMM 9513T. The structures of five diketopiperazines (4-8) and diisobutyl-, dibutyl- and bis(2-ethylhexyl) phthalates (1-3) were established on the basis of detailed interpretation of NMR data, by Marfey method and optical rotation data. The structures of diketopiperazines were determined as cyclo-(L-valyl-L-proline), cyclo-(L-valyl-D-proline), cyclo-(L-phenylalanyl-L-proline), cyclo-(L-leucyl-L-proline), and cyclo-(L-phenylalanyl-D-proline). Compounds 1-3, 5 and 8 revealed antimicrobial activities against Bacillus subtilis and/or Enterococcus faecium and Staphylococcus aureus. In this paper, we describe the isolation and structural elucidation of the isolated compounds 1-8. This is the first report of the characterisation of low molecular weight antibacterial metabolites produced by a member of the genus Rheinheimera.

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.

Embedding carotenoids of spheroidene-branch biosynthesis into antenna complexes of sulfur photosynthetic bacteria.

The possibility of embedding the carotenoids of spheroidene-branch biosynthesis (spheroidene and spheroidenone) from non-sulfur bacteria into the diphenylamine antenna complexes (DPA-complexes) from the sulfur bacteria Allochromatium minutissimum and Ectothiorhodospira haloalkaliphila with carotenoid synthesis inhibited by diphenylamine (DPA) was studied for the first time. It was found that spheroidene was embedded into the DPA-complexes from these bacteria at a level of 75-87%, with spheroidene embedding efficiency being 41-68% for the LH1-RC DPA-complexes and 71-89% for the LH2 DPA-complexes. The energy transfer efficiency from carotenoids to bacteriochlorophyll was shown to depend not only on the type of carotenoid but also on the very structure on the antenna complex.

A comparative quantitative proteomic study identifies new proteins relevant for sulfur oxidation in the purple sulfur bacterium Allochromatium vinosum.

In the present study, we compared the proteome response of Allochromatium vinosum when growing photoautotrophically in the presence of sulfide, thiosulfate, and elemental sulfur with the proteome response when the organism was growing photoheterotrophically on malate. Applying tandem mass tag analysis as well as two-dimensional (2D) PAGE, we detected 1,955 of the 3,302 predicted proteins by identification of at least two peptides (59.2%) and quantified 1,848 of the identified proteins. Altered relative protein amounts (≥1.5-fold) were observed for 385 proteins, corresponding to 20.8% of the quantified A. vinosum proteome. A significant number of the proteins exhibiting strongly enhanced relative protein levels in the presence of reduced sulfur compounds are well documented essential players during oxidative sulfur metabolism, e.g., the dissimilatory sulfite reductase DsrAB. Changes in protein levels generally matched those observed for the respective relative mRNA levels in a previous study and allowed identification of new genes/proteins participating in oxidative sulfur metabolism. One gene cluster (hyd; Alvin_2036-Alvin_2040) and one hypothetical protein (Alvin_2107) exhibiting strong responses on both the transcriptome and proteome levels were chosen for gene inactivation and phenotypic analyses of the respective mutant strains, which verified the importance of the so-called Isp hydrogenase supercomplex for efficient oxidation of sulfide and a crucial role of Alvin_2107 for the oxidation of sulfur stored in sulfur globules to sulfite. In addition, we analyzed the sulfur globule proteome and identified a new sulfur globule protein (SgpD; Alvin_2515).

High-resolution structure and biochemical properties of the LH1-RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum.

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and ß-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/ß1- or α1/ß3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1-RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.