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The resilience of biological systems to fluctuating environmental conditions is a crucial evolutionary advantage. In this study, we examine the thermo- and piezo-stability of the LH1-RC pigment-protein complex, the simplest photosynthetic unit, in three species of phototropic purple bacteria, each containing only this core complex. Among these species, Blastochloris viridis and Blastochloris tepida utilize bacteriochlorophyll b as the main light-harvesting pigment, while Rhodospirillum rubrum relies on bacteriochlorophyll a. Through spectroscopic analyses, we observed limited reversibility in the effects of temperature and pressure, likely due to the malleability of pigment binding sites within the light-harvesting LH1 complex. In terms of thermal robustness, LH1 complexes in a detergent environment progressively dissociate into dimeric (B820) and monomeric (B777) subunits. However, in the native membrane, degradation primarily occurs directly into B777 without the intermediate formation of B820. Interestingly, while high-pressure compression of core complexes from Blastochloris viridis and Blastochloris tepida caused significant changes in compressibility around 1.3 kbar and the formation of B777 and B820 subunits upon decompression, no such compressibility changes or pressure-induced dissociation were observed in Rhodospirillum rubrum complexes, even at pressures as high as 11 kbar. This study reveals significant differences in the piezo- and thermal properties of phototrophs containing either BChl a or BChl b, underscoring the critical role of structural factors in understanding the temperature- and pressure-induced denaturation phenomena in photosynthetic complexes. Rhodospirillum rubrum, in particular, stands out as one of the most thermodynamically stable systems among phototrophic microorganisms, capable of withstanding temperatures up to 70 °C and pressures exceeding 11 kbar., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Spectral variations of light-harvesting (LH) proteins of purple photosynthetic bacteria provide insight into the molecular mechanisms underlying spectral tuning of circular bacteriochlorophyll (BChl) arrays, which play crucial roles in photoenergy conversion in these organisms. Here we investigate spectral changes of the Q y band of B850 BChl a in LH2 protein from purple sulfur bacterium Thermochromatium tepidum (tepidum-LH2) by detergents and Ca 2+ . The tepidum-LH2 solubilized with lauryl dimethylamine N-oxide and n-octyl-β-D-glucoside (LH2 LDAO and LH2 OG , respectively) exhibited blue-shift of the B850 Q y band with hypochromism compared with the tepidum-LH2 solubilized with n-dodecyl-β-D-maltoside (LH2 DDM ), resulting in the LH3-like spectral features. Resonance Raman spectroscopy indicated that this blue-shift was ascribable to the loss of hydrogen-bonding between the C3-acetyl group in B850 BChl a and the LH2 polypeptides. Ca 2+ produced red-shift of the B850 Q y band in LH2 LDAO by forming hydrogen-bond for the C3-acetyl group in B850 BChl a, probably due to a change in the microenvironmental structure around B850. Ca 2+ -induced red-shift was also observed in LH2 OG although the B850 acetyl group is still free from hydrogen-bonding. Therefore, the Ca 2+ -induced B850 red-shift in LH2 OG would originate from an electrostatic effect of Ca 2+ . The current results suggest that the B850 Q y band in tepidum-LH2 is primarily tuned by two mechanisms, namely the hydrogen-bonding of the B850 acetyl group and the electrostatic effect., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Halorhodospira (Hlr.) halochloris is a triply extremophilic phototrophic purple sulfur bacterium, as it is thermophilic, alkaliphilic, and extremely halophilic. The light-harvesting-reaction center (LH1-RC) core complex of this bacterium displays an LH1-Q y transition at 1,016 nm, which is the lowest-energy wavelength absorption among all known phototrophs. Here we report the cryo-EM structure of the LH1-RC at 2.42 Å resolution. The LH1 complex forms a tricyclic ring structure composed of 16 αβγ-polypeptides and one αβ-heterodimer around the RC. From the cryo-EM density map, two previously unrecognized integral membrane proteins, referred to as protein G and protein Q, were identified. Both of these proteins are single transmembrane-spanning helices located between the LH1 ring and the RC L-subunit and are absent from the LH1-RC complexes of all other purple bacteria of which the structures have been determined so far. Besides bacteriochlorophyll b molecules (B1020) located on the periplasmic side of the Hlr. halochloris membrane, there are also two arrays of bacteriochlorophyll b molecules (B800 and B820) located on the cytoplasmic side. Only a single copy of a carotenoid (lycopene) was resolved in the Hlr. halochloris LH1-α3β3 and this was positioned within the complex. The potential quinone channel should be the space between the LH1-α3β3 that accommodates the single lycopene but does not contain a γ-polypeptide, B800 and B820. Our results provide a structural explanation for the unusual Q y red shift and carotenoid absorption in the Hlr. halochloris spectrum and reveal new insights into photosynthetic mechanisms employed by a species that thrives under the harshest conditions of any phototrophic microorganism known., (© 2024 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Purple bacteria possess two ring-shaped protein complexes, light-harvesting 1 (LH1) and 2 (LH2), both of which function as antennas for solar energy utilization for photosynthesis but exhibit distinct absorption properties. The two antennas have differing amounts of bacteriochlorophyll (BChl) a ; however, their significance in spectral tuning remains elusive. Here, we report a high-precision evaluation of the physicochemical factors contributing to the variation in absorption maxima between LH1 and LH2, namely, BChl a structural distortion, protein electrostatic interaction, excitonic coupling, and charge transfer (CT) effects, as derived from detailed spectral calculations using an extended version of the exciton model, in the model purple bacterium Rhodospirillum rubrum . Spectral analysis confirmed that the electronic structure of the excited state in LH1 extended to the BChl a 16-mer. Further analysis revealed that the LH1-specific redshift (∼61% in energy) is predominantly accounted for by the CT effect resulting from the closer inter-BChl distance in LH1 than in LH2. Our analysis explains how LH1 and LH2, both with chemically identical BChl a chromophores, use distinct physicochemical effects to achieve a progressive redshift from LH2 to LH1, ensuring efficient energy transfer to the reaction center special pair., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Purple phototrophic bacteria possess light-harvesting 1 and reaction center (LH1-RC) core complexes that play a key role in converting solar energy to chemical energy. High-resolution structures of LH1-RC and RC complexes have been intensively studied and have yielded critical insight into the architecture and interactions of their proteins, pigments, and cofactors. Nevertheless, a detailed picture of the structure and assembly of LH1-only complexes is lacking due to the intimate association between LH1 and the RC. To study the intrinsic properties and structure of an LH1-only complex, a genetic system was constructed to express the Thermochromatium (Tch.) tepidum LH1 complex heterologously in a modified Rhodospirillum rubrum mutant strain. The heterologously expressed Tch. tepidum LH1 complex was isolated in a pure form free of the RC and exhibited the characteristic absorption properties of Tch. tepidum. Cryo-EM structures of the LH1-only complexes revealed a closed circular ring consisting of either 14 or 15 αβ-subunits, making it the smallest completely closed LH1 complex discovered thus far. Surprisingly, the Tch. tepidum LH1-only complex displayed even higher thermostability than that of the native LH1-RC complex. These results reveal previously unsuspected plasticity of the LH1 complex, provide new insights into the structure and assembly of the LH1-RC complex, and show how molecular genetics can be exploited to study membrane proteins from phototrophic organisms whose genetic manipulation is not yet possible., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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 β-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/β1- or α1/β3-polypeptides through a different Ca 2+ -binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca 2+ -bound LH1 complexes. Two water molecules were identified as additional Ca 2+ -coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca 2+ 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 Ca 2+ . The presence of Ca 2+ in the photocomplex of Alc. vinosum suggests that Ca 2+ -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., (© 2024. The Author(s).)

The ecological success of purple sulfur bacteria (PSB) is linked to their ability to collect near-infrared solar energy by membrane-integrated, pigment-protein photocomplexes. These include a Core complex containing both light-harvesting 1 (LH1) and reaction centre (RC) components (called the LH1-RC photocomplex) present in all PSB and a peripheral light-harvesting complex present in most but not all PSB. In research to explain the unusual absorption properties of the thermophilic purple sulfur bacterium Thermochromatium tepidum, Ca 2+ was discovered bound to LH1 polypeptides in its LH1-RC; further work showed that calcium controls both the thermostability and unusual spectrum of the Core complex. Since then, Ca 2+ has been found in the LH1-RC photocomplexes of several other PSB, including mesophilic species, but not in the LH1-RC of purple non-sulfur bacteria. Here we focus on four species of PSB-two thermophilic and two mesophilic-and describe how Ca 2+ is integrated into and affects their photosynthetic machinery and why this previously overlooked divalent metal is a key nutrient for their ecological success., (© 2024 Applied Microbiology International and John Wiley & Sons Ltd.)

Phospholipid-protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1-reaction center core complexes (LH1-RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1-RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex-phospholipid interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In wild-type phototrophic organisms, carotenoids (Crts) are primarily packed into specific pigment-protein complexes along with (Bacterio)chlorophylls and play important roles in the photosynthesis. Diphenylamine (DPA) inhibits carotenogenesis but not phototrophic growth of anoxygenic phototrophs and eliminates virtually all Crts from photocomplexes. To investigate the effect of Crts on assembly of the reaction center-light-harvesting (RC-LH) complex from the filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii, we generated carotenoidless (Crt-less) RC-LH complexes by growing cells in the presence of DPA. Here, we present cryo-EM structures of the Rfl. castenholzii native and Crt-less RC-LH complexes with resolutions of 2.86 Å and 2.85 Å, respectively. From the high-quality map obtained, several important but previously unresolved details in the Rfl. castenholzii RC-LH structure were determined unambiguously including the assignment and likely function of three small polypeptides, and the content and spatial arrangement of Crts with bacteriochlorophyll molecules. The overall structures of Crt-containing and Crt-less complexes are similar. However, structural comparisons showed that only five Crts remain in complexes from DPA-treated cells and that the subunit X (TMx) flanked on the N-terminal helix of the Cyt-subunit is missing. Based on these results, the function of Crts in the assembly of the Rfl. castenholzii RC-LH complex and the molecular mechanism of quinone exchange is discussed. These structural details provide a fresh look at the photosynthetic apparatus of an evolutionary ancient phototroph as well as new insights into the importance of Crts for proper assembly and functioning of the RC-LH complex., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Although several species of purple sulfur bacteria inhabit soda lakes, Rhodobaca bogoriensis is the first purple nonsulfur bacterium cultured from such highly alkaline environments. Rhodobaca bogoriensis strain LBB1 T was isolated from Lake Bogoria, a soda lake in the African Rift Valley. The phenotype of Rhodobaca bogoriensis is unique among purple bacteria; the organism is alkaliphilic but not halophilic, produces carotenoids absent from other purple nonsulfur bacteria, and is unable to grow autotrophically or fix molecular nitrogen. Here we analyze the draft genome sequence of Rhodobaca bogoriensis to gain further insight into the biology of this extremophilic purple bacterium. The strain LBB1 T genome consists of 3.91 Mbp with no plasmids. The genome sequence supports the defining characteristics of strain LBB1 T , including its (1) production of a light-harvesting 1-reaction center (LH1-RC) complex but lack of a peripheral (LH2) complex, (2) ability to synthesize unusual carotenoids, (3) capacity for both phototrophic (anoxic/light) and chemotrophic (oxic/dark) energy metabolisms, (4) utilization of a wide variety of organic compounds (including acetate in the absence of a glyoxylate cycle), (5) ability to oxidize both sulfide and thiosulfate despite lacking the capacity for autotrophic growth, and (6) absence of a functional nitrogen-fixation system for diazotrophic growth. The assortment of properties in Rhodobaca bogoriensis has no precedent among phototrophic purple bacteria, and the results are discussed in relation to the organism's soda lake habitat and evolutionary history., (© 2023. The Author(s), under exclusive licence to Springer Nature Japan KK, part of Springer Nature.)

Structural information on the circular arrangements of repeating pigment-polypeptide subunits in antenna proteins of purple photosynthetic bacteria is a clue to a better understanding of molecular mechanisms for the ring-structure formation and efficient light harvesting of such antennas. Here, we have analyzed the ring structure of light-harvesting complex 2 (LH2) from the thermophilic purple bacterium Thermochromatium tepidum (tepidum-LH2) by atomic force microscopy. The circular arrangement of the tepidum-LH2 subunits was successfully visualized in a lipid bilayer. The average top-to-top distance of the ring structure, which is correlated with the ring size, was 4.8 ± 0.3 nm. This value was close to the top-to-top distance of the octameric LH2 from Phaeospirillum molischianum (molischianum-LH2) by the previous analysis. Gaussian distribution of the angles of the segments consisting of neighboring subunits in the ring structures of tepidum-LH2 yielded a median of 44°, which corresponds to the angle for the octameric circular arrangement (45°). These results indicate that tepidum-LH2 has a ring structure consisting of eight repeating subunits. The coincidence of an octameric ring structure of tepidum-LH2 with that of molischianum-LH2 is consistent with the homology of amino acid sequences of the polypeptides between tepidum-LH2 and molischianum-LH2., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Rhodobacter (Rba.) capsulatus has been a favored model for studies of all aspects of bacterial photosynthesis. This purple phototroph contains PufX, a polypeptide crucial for dimerization of the light-harvesting 1-reaction center (LH1-RC) complex, but lacks protein-U, a U-shaped polypeptide in the LH1-RC of its close relative Rba. sphaeroides. Here we present a cryo-EM structure of the Rba. capsulatus LH1-RC purified by DEAE chromatography. The crescent-shaped LH1-RC exhibits a compact structure containing only 10 LH1 αβ-subunits. Four αβ-subunits corresponding to those adjacent to protein-U in Rba. sphaeroides were absent. PufX in Rba. capsulatus exhibits a unique conformation in its N-terminus that self-associates with amino acids in its own transmembrane domain and interacts with nearby polypeptides, preventing it from interacting with proteins in other complexes and forming dimeric structures. These features are discussed in relation to the minimal requirements for the formation of LH1-RC monomers and dimers, the spectroscopic behavior of both the LH1 and RC, and the bioenergetics of energy transfer from LH1 to the RC., (© 2023. The Author(s).)

Purple phototrophic bacteria are ancient anoxygenic phototrophs and attractive research tools because they capture light energy in the near-infrared (NIR) region of the spectrum and transform it into chemical energy by way of uphill energy transfers. The heart of this reaction occurs in light-harvesting 1-reaction center (LH1-RC) complexes, which are the simplest model systems for understanding basic photosynthetic reactions within type-II (quinone-utilizing) reaction centers. In this Perspective, we highlight structure-function relationships concerning unresolved fundamental processes in purple bacterial photosynthesis, including the diversified light-harvesting capacity of LH1-associated BChl molecules, energies necessary for photoelectric conversion in the RC special pairs, and quinone transport mechanisms. Based on recent progress in the spectroscopic and structural analysis of LH1-RC complexes from a variety of purple phototrophs, we discuss several key factors for understanding how purple bacteria resource light energy in the inherently energy-poor NIR region of the electromagnetic spectrum.

Rhodopila globiformis is the most acidophilic of anaerobic purple phototrophs, growing optimally in culture at pH 5. Here we present a cryo-EM structure of the light-harvesting 1-reaction center (LH1-RC) complex from Rhodopila globiformis at 2.24 Å resolution. All purple bacterial cytochrome (Cyt, encoded by the gene pufC) subunit-associated RCs with known structures have their N-termini truncated. By contrast, the Rhodopila globiformis RC contains a full-length tetra-heme Cyt with its N-terminus embedded in the membrane forming an α-helix as the membrane anchor. Comparison of the N-terminal regions of the Cyt with PufX polypeptides widely distributed in Rhodobacter species reveals significant structural similarities, supporting a longstanding hypothesis that PufX is phylogenetically related to the N-terminus of the RC-bound Cyt subunit and that a common ancestor of phototrophic Proteobacteria contained a full-length tetra-heme Cyt subunit that evolved independently through partial deletions of its pufC gene. Eleven copies of a novel γ-like polypeptide were also identified in the bacteriochlorophyll a-containing Rhodopila globiformis LH1 complex; γ-polypeptides have previously been found only in the LH1 of bacteriochlorophyll b-containing species. These features are discussed in relation to their predicted functions of stabilizing the LH1 structure and regulating quinone transport under the warm acidic conditions., (© 2022. The Author(s).)

Meiothermus ruber DSMZ 1279 T was isolated from a hot spring in Kamchatka and was red in color. The major carotenoid present was reported to be 1'-(β-d-glucopyranosyloxy)-3,4,3',4'-tetradehydro-1',2'-dihydro-β,ψ-caroten-2-one after saponification (Burgess et al. J. Nat. Prod. 1999, 62, 859-863). In this study, we purified the major carotenoids in this species without saponification. We then reidentified the major carotenoids present using spectroscopic data, including electronic circular dichroism (ECD), 1 H NMR, rotating-frame nuclear Overhauser effect spectroscopy (ROESY), 13 C NMR, heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and MS, and enzymatic hydrolysis of fatty acid moieties and found deinoxanthin glucoside iso fatty acid esters. The bound fatty acids present included four iso types, and their composition differed from cellular lipids. Moreover, the previously identified carotenoid glucoside was a saponification artifact of deinoxanthin glucoside esters. Ketomyxocoxanthin glucoside esters and 1'-hydroxytorulene glucoside esters were also present. On the basis of the identification of carotenoids and the whole genome sequence of M. ruber , we propose a carotenoid biosynthetic pathway and note the corresponding genes.

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca 2+ -dependent. However, it is unknown what structural determinants might contribute to these properties. Here, we present a cryo-EM structure of the reaction center-associated LH1 complex at 2.81 Å resolution, in which we identify multiple pigment-binding α- and β-polypeptides within an LH1 ring. Of the 16 α-polypeptides, we show that six (α1) bind Ca 2+ along with β1- or β3-polypeptides to form the Ca 2+ -binding sites. This structure differs from that of fully Ca 2+ -bound LH1 from Thermochromatium tepidum, enabling determination of the minimum structural requirements for Ca 2+ -binding. We also identified three amino acids (Trp44, Asp47, and Ile49) in the C-terminal region of the A. tepidum α1-polypeptide that ligate each Ca ion, forming a Ca 2+ -binding WxxDxI motif that is conserved in all Ca 2+ -bound LH1 α-polypeptides from other species with reported structures. The partial Ca 2+ -bound structure further explains the unusual phenotypic properties observed for this bacterium in terms of its Ca 2+ -requirements for thermostability, spectroscopy, and phototrophic growth, and supports the hypothesis that A. tepidum may represent a "transitional" species between mesophilic and thermophilic purple sulfur bacteria. The characteristic arrangement of multiple αβ-polypeptides also suggests a mechanism of molecular recognition in the expression and/or assembly of the LH1 complex that could be regulated through interactions with reaction center subunits., Competing Interests: Conflict of interest Authors K. K., N. H., I. I., and Y. O. are employees of JEOL Ltd. This does not alter the authors’ adherence to all policies on sharing data and materials. The authors declare that they have no conflicts of interest with the contents of the article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Halorhodospira ( Hlr. ) species are the most halophilic and alkaliphilic of all purple bacteria. Hlr. halochloris exhibits the lowest LH1 Q y transition energy among phototrophic organisms and is the only known triply extremophilic anoxygenic phototroph, displaying a thermophilic, halophilic, and alkaliphilic phenotype. Recently, we reported that electrostatic charges are responsible for the unusual spectroscopic properties of the Hlr. halochloris LH1 complex. In the present work, we examined the effects of salt and pH on the spectroscopic properties and thermal stability of LH1-RCs from Hlr. halochloris compared with its mesophilic counterpart, Hlr. abdelmalekii . Experiments in which the photocomplexes were subjected to different levels of salt or variable pH revealed that the thermal stability of LH1-RCs from both species was largely retained in the presence of high salt concentrations and/or at alkaline pH but was markedly reduced by lowering the salt concentration and/or pH. Based on the amino acid sequences of LH1 polypeptides and their composition of acidic/basic residues and the Hofmeister series for cation/anion species, we discuss the importance of electrostatic charge in stabilizing the Hlr. halochloris LH1-RC complex to allow it to perform photosynthesis in its warm, hypersaline, and alkaline habitat.

Carotenoid (Car) in photosynthesis plays the major roles of accessary light harvesting and photoprotection, and the underlying structure-function relationship attracts continuing research interests. We have attempted to explore the dynamics of Car triplet excitation ( 3 Car*) in the bacteriochlorophyll b (BChl b )-type light harvesting reaction center complex (LH1-RC) of photosynthetic bacterium Halorhodospira halochloris . We show that the LH1 antenna binds a single Car that was identified as a lycopene derivative. Although the Car is hardly visible in the LH1-RC stationary absorption, it shows up conspicuously in the triplet excitation profile with distinct vibronic features. This and the ultrafast formation of 3 Car* on direct photoexcitation of Car unequivocally manifest the unimolecular singlet fission reaction of the Car. Moreover, the Car with even one molecule per complex is found to be rather effective in quenching 3 BChl b *. The implications of different 3 Car* formation mechanisms are discussed, and the self-photoprotection role of BChl b are proposed for this extremophilic species.

Rhodobacter sphaeroides is a model organism in bacterial photosynthesis, and its light-harvesting-reaction center (LH1-RC) complex contains both dimeric and monomeric forms. Here we present cryo-EM structures of the native LH1-RC dimer and an LH1-RC monomer lacking protein-U (ΔU). The native dimer reveals several asymmetric features including the arrangement of its two monomeric components, the structural integrity of protein-U, the overall organization of LH1, and rigidities of the proteins and pigments. PufX plays a critical role in connecting the two monomers in a dimer, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in the other monomer. One protein-U was only partially resolved in the dimeric structure, signaling different degrees of disorder in the two monomers. The ΔU LH1-RC monomer was half-moon-shaped and contained 11 α- and 10 β-polypeptides, indicating a critical role for protein-U in controlling the number of αβ-subunits required for dimer assembly and stabilization. These features are discussed in relation to membrane topology and an assembly model proposed for the native dimeric complex., (© 2022. The Author(s).)

Calcium ions play a dual role in expanding the spectral diversity and structural stability of photocomplexes from several Ca 2+ -requiring purple sulfur phototrophic bacteria. Here, metal-sensitive structural changes in the isotopically labeled light-harvesting 1 reaction center (LH1-RC) complexes from the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were investigated by perfusion-induced attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The ATR-FTIR difference spectra induced by exchanges between native Ca 2+ and exogenous Ba 2+ exhibited interconvertible structural and/or conformational changes in the metal binding sites at the LH1 C-terminal region. Most of the characteristic Ba 2+ /Ca 2+ difference bands were detected even when only Ca ions were removed from the LH1-RC complexes, strongly indicating the pivotal roles of Ca 2+ in maintaining the LH1-RC structure of Tch. tepidum. Upon 15 N-, 13 C- or 2 H-labeling, the LH1-RC complexes exhibited characteristic 15 N/ 14 N-, 13 C/ 12 C-, or 2 H/ 1 H-isotopic shifts for the Ba 2+ /Ca 2+ difference bands. Some of the 15 N/ 14 N or 13 C/ 12 C bands were also sensitive to further 2 H-labelings. Given the band frequencies and their isotopic shifts along with the structural information of the Tch. tepidum LH1-RC complexes, metal-sensitive FTIR bands were tentatively identified to the vibrational modes of the polypeptide main chains and side chains comprising the metal binding sites. Furthermore, important new IR marker bands highly sensitive to the LH1 BChl a conformation in the Ca 2+ -bound states were revealed based on both ATR-FTIR and near-infrared Raman analyses. The present approach provides valuable insights concerning the dynamic equilibrium between the Ca 2+ - and Ba 2+ -bound states statically resolved by x-ray crystallography.

We describe a new species of purple sulfur bacteria (Chromatiaceae, anoxygenic phototrophic bacteria) isolated from a microbial mat in the sulfidic geothermal outflow of a hot spring in Rotorua, New Zealand. This phototroph, designated as strain NZ, grew optimally near 45 °C but did not show an absorption maximum at 915 nm for the light-harvesting-reaction center core complex (LH1-RC) characteristic of other thermophilic purple sulfur bacteria. Strain NZ had a similar carotenoid composition as Thermochromatium tepidum, but unlike Tch. tepidum, grew photoheterotrophically on acetate in the absence of sulfide and metabolized thiosulfate. The genome of strain NZ was significantly larger than that of Tch. tepidum but slightly smaller than that of Allochromatium vinosum. Strain NZ was phylogenetically more closely related to mesophilic purple sulfur bacteria of the genus Allochromatium than to Tch. tepidum. This conclusion was reached from phylogenetic analyses of strain NZ genes encoding 16S rRNA and the photosynthetic functional gene pufM, from phylogenetic analyses of entire genomes, and from a phylogenetic tree constructed from the concatenated sequence of 1090 orthologous proteins. Moreover, average nucleotide identities and digital DNA:DNA hybridizations of the strain NZ genome against those of related species of Chromatiaceae supported the phylogenetic analyses. From this collection of properties, we describe strain NZ here as the first thermophilic species of the genus Allochromatium, Allochromatium tepidum NZ T , sp. nov., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The complete genome sequence of the thermophilic purple sulfur bacterium Thermochromatium tepidum strain MC T (DSM 3771 T ) is described and contrasted with that of its mesophilic relative Allochromatium vinosum strain D (DSM 180 T ) and other Chromatiaceae. The Tch. tepidum genome is a single circular chromosome of 2,958,290 base pairs with no plasmids and is substantially smaller than the genome of Alc. vinosum. The Tch. tepidum genome encodes two forms of RuBisCO and contains nifHDK and several other genes encoding a molybdenum nitrogenase but lacks a gene encoding a protein that assembles the Fe-S cluster required to form a functional nitrogenase molybdenum-iron cofactor, leaving the phototroph phenotypically Nif - . Tch. tepidum contains genes necessary for oxidizing sulfide to sulfate as photosynthetic electron donor but is genetically unequipped to either oxidize thiosulfate as an electron donor or carry out assimilative sulfate reduction, both of which are physiological hallmarks of Alc. vinosum. Also unlike Alc. vinosum, Tch. tepidum is obligately phototrophic and unable to grow chemotrophically in darkness by respiration. Several genes present in the Alc. vinosum genome that are absent from the genome of Tch. tepidum likely contribute to the major physiological differences observed between these related purple sulfur bacteria that inhabit distinct ecological niches., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Rhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αβ-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general., (© 2021. The Author(s).)

Halorhodospira (Hlr.) halochloris is a unique phototrophic purple bacterium because it is a triple extremophile-the organism is thermophilic, alkalophilic, and halophilic. The most striking photosynthetic feature of Hlr. halochloris is that the bacteriochlorophyll (BChl) b-containing core light-harvesting (LH1) complex surrounding its reaction center (RC) exhibits its LH1 Q y absorption maximum at 1016 nm, which is the lowest transition energy among phototrophic organisms. Here we report that this extraordinarily red-shifted LH1 Q y band of Hlr. halochloris exhibits interconvertible spectral shifts depending on the electrostatic charge distribution around the BChl b molecules. The 1016 nm band of the Hlr. halochloris LH1-RC complex was blue-shifted to 958 nm upon desalting or pH decrease but returned to its original position when supplemented with salts or pH increase. Resonance Raman analysis demonstrated that these interconvertible spectral shifts are not associated with the strength of hydrogen-bonding interactions between BChl b and LH1 polypeptides. Furthermore, circular dichroism signals for the LH1 Q y transition of Hlr. halochloris appeared with a positive sign (as in BChl b-containing Blastochloris species) and opposite those of BChl a-containing purple bacteria, possibly due to a combined effect of slight differences in the transition dipole moments between BChl a and BChl b and in the interactions between adjacent BChls in their assembled state. Based on these findings and LH1 amino acid sequences, it is proposed that Hlr. halochloris evolved its unique and tunable light-harvesting system with electrostatic charges in order to carry out photosynthesis and thrive in its punishing hypersaline and alkaline habitat., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Light-harvesting complex 1 (LH1) of the thermophilic purple sulfur bacterium Thermochromatium tepidum can be expressed in the purple non-sulfur bacterium Rhodobacter sphaeroides and forms a functional RC-LH1 complex with the native Rba. sphaeroides reaction center (Nagashima, K. V. P., et al. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 10906-10911). Although there is a large uphill energy gap between Tch. tepidum LH1 and the Rba. sphaeroides RC in this chimeric complex, it has been shown that light energy can be transferred, consistent with that seen in the native Rba. sphaeroides RC-LH1 complex. In this study, the contribution of this chimeric complex to growth and photosynthetic energy conversion in the hybrid organism was quantified. The mutant synthesizing this chimeric complex was grown phototrophically under 940 nm light-emitting diode (LED) light preferentially absorbed by Tch. tepidum LH1 and showed faster growth at low intensities of this wavelength than both a mutant strain of Rba. sphaeroides lacking LH2 and a mutant lacking all light-harvesting complexes. When grown with 850 nm LED light, the strain containing the native Rba. sphaeroides LH1-RC grew faster than the chimeric strain. Electron transfer from the RC to the membrane-integrated cytochrome bc 1 complex was also estimated by flash-induced absorption changes in heme b . The rate of ubiquinone transport through the LH1 ring structure in the chimeric strain was virtually the same as that in native Rba. sphaeroides . We conclude that Tch. tepidum LH1 can perform the physiological functions of native LH1 in Rba. sphaeroides .

Rhodospirillum ( Rsp. ) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl a G ) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 Å resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αβ-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the Q A site in the RC. The geranylgeraniol side chains of BChl a G coordinated by LH1 β-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 α-associated BChls a G . The structure also revealed key protein-protein interactions in both N- and C-terminal regions of the LH1 αβ-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex.

Flexible color adaptation to available ecological niches is vital for the photosynthetic organisms to thrive. Hence, most purple bacteria living in the shade of green plants and algae apply bacteriochlorophyll a pigments to harvest near infra-red light around 850-875 nm. Exceptions are some Ca 2+ -containing species fit to utilize much redder quanta. The physical basis of such anomalous absorbance shift equivalent to ~5.5 kT at ambient temperature remains unsettled so far. Here, by applying several sophisticated spectroscopic techniques, we show that the Ca 2+ ions bound to the structure of LH1 core light-harvesting pigment-protein complex significantly increase the couplings between the bacteriochlorophyll pigments. We thus establish the Ca-facilitated enhancement of exciton couplings as the main mechanism of the record spectral red-shift. The changes in specific interactions such as pigment-protein hydrogen bonding, although present, turned out to be secondary in this regard. Apart from solving the two-decade-old conundrum, these results complement the list of physical principles applicable for efficient spectral tuning of photo-sensitive molecular nano-systems, native or synthetic.

The core light-harvesting complexes (LH1) in bacteriochlorophyll (BChl) b-containing purple phototrophic bacteria are characterized by a near-infrared absorption maximum around 1010 nm. The determinative cause for this ultra-redshift remains unclear. Here, we present results of circular dichroism (CD) and resonance Raman measurements on the purified LH1 complexes in a reaction center-associated form from a mesophilic and a thermophilic Blastochloris species. Both the LH1 complexes displayed purely positive CD signals for their Q y transitions, in contrast to those of BChl a-containing LH1 complexes. This may reflect differences in the conjugation system of the bacteriochlorin between BChl b and BChl a and/or the differences in the pigment organization between the BChl b- and BChl a-containing LH1 complexes. Resonance Raman spectroscopy revealed remarkably large redshifts of the Raman bands for the BChl b C3-acetyl group, indicating unusually strong hydrogen bonds formed with LH1 polypeptides, results that were verified by a published structure. A linear correlation was found between the redshift of the Raman band for the BChl C3-acetyl group and the change in LH1-Q y transition for all native BChl a- and BChl b-containing LH1 complexes examined. The strong hydrogen bonding and π-π interactions between BChl b and nearby aromatic residues in the LH1 polypeptides, along with the CD results, provide crucial insights into the spectral and structural origins for the ultra-redshift of the long-wavelength absorption maximum of BChl b-containing phototrophs.

Photosynthetic electron transfers occur through multiple components ranging from small soluble proteins to large integral membrane protein complexes. Co-crystallization of a bacterial photosynthetic electron transfer complex that employs weak hydrophobic interactions was achieved by using high-molar-ratio mixtures of a soluble donor protein (high-potential iron-sulfur protein, HiPIP) with a membrane-embedded acceptor protein (reaction center, RC) at acidic pH. The structure of the co-complex offers a snapshot of a transient bioenergetic event and revealed a molecular basis for thermodynamically unfavorable interprotein electron tunneling. HiPIP binds to the surface of the tetraheme cytochrome subunit in the light-harvesting (LH1) complex-associated RC in close proximity to the low-potential heme-1 group. The binding interface between the two proteins is primarily formed by uncharged residues and is characterized by hydrophobic features. This co-crystal structure provides a model for the detailed study of long-range trans-protein electron tunneling pathways in biological systems.

Redox-active quinones play essential roles in efficient light energy conversion in type-II reaction centers of purple phototrophic bacteria. In the light-harvesting 1 reaction center (LH1-RC) complex of purple bacteria, Q B is converted to Q B H 2 upon light-induced reduction and Q B H 2 is transported to the quinone pool in the membrane through the LH1 ring. In the purple bacterium Rhodobacter sphaeroides, the C-shaped LH1 ring contains a gap for quinone transport. In contrast, the thermophilic purple bacterium Thermochromatium (Tch.) tepidum has a closed O-shaped LH1 ring that lacks a gap, and hence the mechanism of photosynthetic quinone transport is unclear. Here we detected light-induced Fourier transform infrared (FTIR) signals responsible for changes of Q B and its binding site that accompany photosynthetic quinone reduction in Tch. tepidum and characterized Q B and Q B H 2 marker bands based on their 15 N- and 13 C-isotopic shifts. Quinone exchanges were monitored using reconstituted photosynthetic membranes comprised of solubilized photosynthetic proteins, membrane lipids, and exogenous ubiquinone (UQ) molecules. In combination with 13 C-labeling of the LH1-RC and replacement of native UQ 8 by ubiquinones of different tail lengths, we demonstrated that quinone exchanges occur efficiently within the hydrophobic environment of the lipid membrane and depend on the side chain length of UQ. These results strongly indicate that unlike the process in Rba. sphaeroides, quinone transport in Tch. tepidum occurs through the size-restricted hydrophobic channels in the closed LH1 ring and are consistent with structural studies that have revealed narrow hydrophobic channels in the Tch. tepidum LH1 transmembrane region., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αβ-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αβ-polypeptides that are hydrogen-bonded to BChl a. The Ca 2+ -facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and β-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.

An increased robustness against high temperature and the much red-shifted near-infrared absorption spectrum of excitons in the LH1-RC core pigment-protein complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted much interest. In the present work, thermal and hydrostatic pressure stability of the peripheral LH2 and core LH1-RC complexes from this bacterium were in parallel investigated by various optical spectroscopy techniques applied over a wide spectral range from far-ultraviolet to near-infrared. In contrast to expectations, very distinct robustness of the complexes was established, while the sturdiness of LH2 surpassed that of LH1-RC both with respect to temperatures between 288 and 360 K, and pressures between 1 bar and 14 kbar. Subtle structural variances related to the hydrogen bond network are likely responsible for the extra stability of LH2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Blastochloris tepida is a newly described thermophilic purple bacterium containing bacteriochlorophyll b . Using purified light-harvesting 1 reaction center (LH1-RC) core complexes from Blc . tepida , we compared the biochemical, spectroscopic, and thermal denaturation properties of these complexes with those of its mesophilic counterpart, Blc. viridis . Besides their growth temperature optima, a striking difference between the two species was seen in the carotenoid composition of their LH1-RC complexes. The more thermostable Blc . tepida complex contained more carotenoids with longer conjugation lengths ( n > 9), such as lycopenes ( n = 11), and had a total carotenoid content significantly higher than that of the Blc. viridis complex, irrespective of the light intensity used for growth. The thermostability of LH1-RCs from both Blc. tepida and Blc. viridis decreased significantly in cells grown in the presence of diphenylamine, a compound that inhibits the formation of highly conjugated carotenoids. In contrast to the thermophilic purple bacterium Thermochromatium tepidum , where Ca 2+ is essential for LH1-RC thermostability, Ca 2+ neither was present in nor had any effect on the thermostability of the Blc . tepida LH1-RC. These results point to a mechanism that carotenoids with elongated conjugations enhance hydrophobic interactions with proteins in the Blc. tepida LH1-RC, thereby allowing the complexes to withstand thermal denaturation. This conclusion is bolstered by a structural model of the Blc. tepida LH1-RC and is the first example of photocomplex thermostability being linked to a carotenoid-based mechanism.

A new taxon is created for the thermophilic purple nonsulfur bacterium previously designated as Rhodopseudomonas strain GI. Strain GI was isolated from a New Mexico (USA) hot spring microbial mat and grows optimally above 40 °C and to a maximum of 47 °C. Strain GI is a bacteriochlorophyll b-containing species of purple nonsulfur bacteria and displays a budding morphology, typical of species of the genus Blastochloris. Although resembling the species Blc. viridis in many respects, the absorption spectrum, carotenoid content, and lipid fatty acid profile of strain GI is distinct from that of Blc. viridis strain DSM133 T and other recognized Blastochloris species. Strain GI forms its own subclade within the Blastochloris clade of purple nonsulfur bacteria based on comparative 16S rRNA gene sequences, and its genome is significantly larger than that of strain DSM133 T ; average nucleotide identity between the genomes of Blc. viridis and strain GI was below 85%. Moreover, concatenated sequence analyses of PufLM and DnaK clearly showed strain GI to be distinct from both Blc. viridis and Blc. sulfoviridis. Because of its unique assortment of properties, it is proposed to classify strain GI as a new species of the genus Blastochloris, as Blc. tepida, sp.n., with strain GI T designated as the type strain (= ATCC TSD-138 = DSM 106918).

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 Ca 2+ , and elemental analysis of Ca 2+ -grown cells confirmed that purified Trv. strain 970 LH1-RC complexes contained Ca 2+ . The LH1 Q y band of Trv. strain 970 was blue-shifted from 959 to 875 nm upon Ca 2+ depletion, but the original spectral properties were restored upon Ca 2+ reconstitution, which also occurs with the thermophilic purple bacterium Thermochromatium ( Tch.) tepidum. The amino acid sequences of the LH1 α- and β-polypeptides from Trv. strain 970 closely resemble those of Tch. tepidum; however, Ca 2+ 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 Ca 2+ was indispensable for maintaining these bonds. Furthermore, perfusion-induced Fourier transform infrared analyses detected Ca 2+ -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 Ca 2+ into its LH1-RC complex to extend its light-harvesting capacity to regions of the near-infrared spectrum unused by other purple bacteria.

In contrast to plants, algae and cyanobacteria that contain glycolipids as the major lipid components in their photosynthetic membranes, phospholipids are the dominant lipids in the membranes of anoxygenic purple phototrophic bacteria. Although the phospholipid compositions in whole cells or membranes are known for a limited number of the purple bacteria, little is known about the phospholipids associated with individual photosynthetic complexes. In this study, we investigated the phospholipid distributions in both membranes and the light-harvesting 1-reaction center (LH1-RC) complexes purified from several purple sulfur and nonsulfur bacteria. 31 P NMR was used for determining the phospholipid compositions and inductively coupled plasma atomic emission spectroscopy was used for measuring the total phosphorous contents. Combining these two techniques, we could determine the numbers of specific phospholipids in the purified LH1-RC complexes. A total of approximate 20-30 phospholipids per LH1-RC were detected as the tightly bound lipids in all species. The results revealed that while cardiolipin (CL) exists as a minor component in the membranes, it became the most abundant phospholipid in the purified core complexes and the sum of CL and phosphatidylglycerol accounted for more than two thirds of the total phospholipids for most species. Preferential association of these anionic phospholipids with the LH1-RC is discussed in the context of the recent high-resolution structure of this complex from Thermochromatium (Tch.) tepidum. The detergent lauryldimethylamine N-oxide was demonstrated to selectively remove phosphatidylethanolamine from the membrane of Tch. tepidum., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c 552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c 552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

Light-harvesting-1 (LH1)-reaction center (RC) super-complex is a membrane protein-pigment complex existing in purple photosynthetic bacteria, where LH1 absorbs light energy and transfers them rapidly and efficiently to RC to initiate the charge separation and electron transfer reactions. The structure of LH1-RC has been reported at relatively low resolutions from several different species of bacteria previously, but was solved at an atomic resolution recently from a thermophilic photosynthetic bacterium Thermochromatium tepidum. This high-resolution structure revealed the detailed organization of the super-complex including a number of unique features that are important for its functioning, such as a more intact RC structure, transporting routes for quinones to replace the bound Q B as well as for the in-and-out of the closed LH1 ring, detailed coordinating environment of the Ca 2+ ions in LH1 important for the remarkable red shift of the absorption spectrum, as well as for the enhanced thermostability. These results thus greatly advance our understanding on the mechanisms of energy transfer, quinone exchange, the red shift in the LH1-Qy transition and the enhanced thermal stability, in this super-complex., (© 2018 Federation of European Biochemical Societies.)

To explore the photoprotection role of multicompositional carotenoid (Car) in photosynthetic purple bacteria, we investigated, by means of triplet excitation profile (TEP) combined with steady-state optical spectroscopies, the core light-harvesting complex-reaction center of a mutant strain of Rhodobacter sphaeroides (m-LH1-RC) at room temperature. TEP spectra revealed that spheroidene and derivative (Spe) preferentially protect bacteriochlorophylls (BChls) of relatively lower site energy by quenching the triplet excitation ( 3 BChl*); however, spirilloxanthin (Spx) does so irrespective to the site energy of BChls. Triplet excitation results showed the triplet excitation energy-transfer (EET) reaction in a timescale of ∼0.5 μs from Spe and derivatives as a major component (∼85%) to Spx as a minor component (∼8%), suggesting the coexistence of different kinds of Cars in the individual LH1 complex. The nonequivalent quenching potency and the triplet EET reaction between Cars constitute the cooperative photoprotection by multicompositional Cars in bacterial photosynthesis.

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 Ca 2+ , 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ -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 Ca 2+ , interactions between LH1 and the RC from different species, and the uphill energy transfer mechanisms.

The uphill excitation energy transfer (EET) from the core antenna (LH1) to the reaction center (RC) of purple photosynthetic bacteria was investigated at room temperature by comparing the native LH1-RC from Thermochromatium ( Tch.) tepidum with the hybrid LH1-RC from a mutant strain of Rhodobacter ( Rba.) sphaeroides. The latter protein with chimeric Tch-LH1 and Rba-RC exhibits a substantially larger RC-to-LH1 energy difference (Δ E = 630 cm -1 ) of 3-fold thermal energy (3 k B T). The spectroscopic and kinetics results are discussed on the basis of the newly reported high-resolution structures of Tch. tepidum LH1-RC, which allow us to propose the existence of a passage formed by LH1 BChls that facilitates the LH1 → RC EET. The semilogarithmic plot of the EET rate against Δ E was found to be linear over a broad range of Δ E, which consolidates the mechanism of thermal activation as promoted by the spectral overlap between the LH1 fluorescence and the special pair absorption of RC.

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 Q B 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.

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 915 nm and enhanced thermostability. Ca 2+ can be biosynthetically replaced by Sr 2+ in growing cultures of Tch. tepidum. However, the resulting Sr 2+ -substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca 2+ -grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca 2+ - and Sr 2+ -cultured cells. In this study, we examined the effects of the biosynthetic Sr 2+ -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 Sr 2+ -cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr 2+ -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 Sr 2+ substitutes for Ca 2+ in the LH1 complex. Limited proteolysis of the native Ca 2+ -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 Sr 2+ -substituted LH1-RC complexes in their Ca 2+ -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.

The LH1-RC core complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted interest of many researchers because of its several unique properties, such as increased robustness against environmental hardships and the much red-shifted near-infrared absorption spectrum of the LH1 antenna exciton polarons. The known near-atomic-resolution crystal structure of the complex well supported this attention. Yet several mechanistic aspects of the complex prominence remained to be understood. In this work, samples of the native, Ca 2+ -containing core complexes were investigated along with those destabilized by Ba 2+ substitution, using various spectrally selective steady-state and picosecond time-resolved spectroscopic techniques at physiological and cryogenic temperatures. As a result, the current interpretation of exciton spectra of the complex was significantly clarified. Specifically, by evaluating the homogeneous and inhomogeneous compositions of the spectra, we showed that there is little to no effect of cation substitution on the dynamic or kinetic properties of antenna excitons. Reasons of the extra red shift of absorption/fluorescence spectra observed in the Ca-LH1-RC and not in the Ba-LH1-RC complex should thus be searched in subtle structural differences following the inclusion of different cations into the core complex scaffold.

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 ( 3 Car*), their contribution to the overall 3 Car* 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 3 Car* 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.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca 2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster ( pufBA from Tch. tepidum and pufLM from Rba. sphaeroides ) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides ) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca 2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca 2+ within this key antenna complex., Competing Interests: The authors declare no conflict of interest.

Two spectral forms of the core light-harvesting complex (LH1) of the purple bacterium Thermochromatium (Tch.) tepidum, the native Ca 2+ -binding and the Ba 2+ -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 αβ-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 Ba 2+ . 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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ultrafast excitation relaxation within a manifold exciton state and long-lived vibrational coherence are two universal characteristics of photosynthetic antenna complexes. In this work, we studied the two-dimensional electronic spectra of two core light-harvesting (LH1) complexes of Thermochromatium (Tch.) tepidum, native Ca 2+ -LH1 and modified Ba 2+ -LH1. The role of the vibrational coherence in the exciton relaxation was revealed by comparing the two LH1 with similar structures but different electronic properties and by the evolution of the exciton and vibrational coherence as a function of temperature.