Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre.

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes3 and bacteriochlorophylls4 related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.

Growth and mortality of aerobic anoxygenic phototrophs in the North Pacific Subtropical Gyre.

Aerobic anoxygenic phototrophic (AAP) bacteria harvest light energy using bacteriochlorophyll-containing reaction centers to supplement their mostly heterotrophic metabolism. While their abundance and growth have been intensively studied in coastal environments, much less is known about their activity in oligotrophic open ocean regions. Therefore, we combined in situ sampling in the North Pacific Subtropical Gyre, north of O'ahu island, Hawaii, with two manipulation experiments. Infra-red epifluorescence microscopy documented that AAP bacteria represented approximately 2% of total bacteria in the euphotic zone with the maximum abundance in the upper 50 m. They conducted active photosynthetic electron transport with maximum rates up to 50 electrons per reaction center per second. The in situ decline of bacteriochlorophyll concentration over the daylight period, an estimate of loss rates due to predation, indicated that the AAP bacteria in the upper 50 m of the water column turned over at rates of 0.75-0.90 d-1. This corresponded well with the specific growth rate determined in dilution experiments where AAP bacteria grew at a rate 1.05 ± 0.09 d-1. An amendment of inorganic nitrogen to obtain N:P = 32 resulted in a more than 10 times increase in AAP abundance over 6 days. The presented data document that AAP bacteria are an active part of the bacterioplankton community in the oligotrophic North Pacific Subtropical Gyre and that their growth was mostly controlled by nitrogen availability and grazing pressure.IMPORTANCEMarine bacteria represent a complex assembly of species with different physiology, metabolism, and substrate preferences. We focus on a specific functional group of marine bacteria called aerobic anoxygenic phototrophs. These photoheterotrophic organisms require organic carbon substrates for growth, but they can also supplement their metabolic needs with light energy captured by bacteriochlorophyll. These bacteria have been intensively studied in coastal regions, but rather less is known about their distribution, growth, and mortality in the oligotrophic open ocean. Therefore, we conducted a suite of measurements in the North Pacific Subtropical Gyre to determine the distribution of these organisms in the water column and their growth and mortality rates. A nutrient amendment experiment showed that aerobic anoxygenic phototrophs were limited by inorganic nitrogen. Despite this, they grew more rapidly than average heterotrophic bacteria, but their growth was balanced by intense grazing pressure.

Light-Quality-Adapted Carotenoid Photoprotection in the Photosystem of Roseiflexus castenholzii.

The photosystem of filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii comprises a light-harvesting (LH) complex encircling a reaction center (RC), which intensely absorbs blue-green light by carotenoid (Car) and near-infrared light by bacteriochlorophyll (BChl). To explore the influence of light quality (color) on the photosynthetic activity, we compared the pigment compositions and triplet excitation dynamics of the LH-RCs from Rfl. castenholzii was adapted to blue-green light (bg-LH-RC) and to near-infrared light (nir-LH-RC). Both LH-RCs bind γ-carotene derivatives; however, compared to that of nir-LH-RC (12%), bg-LH-RC contains substantially higher keto-γ-carotene content (43%) and shows considerably faster BChl-to-Car triplet excitation transfer (10.9 ns vs 15.0 ns). For bg-LH-RC, but not nir-LH-RC, selective photoexcitation of Car and the 800 nm-absorbing BChl led to Car-to-Car triplet transfer and BChl-Car singlet fission reactions, respectively. The unique excitation dynamics of bg-LH-RC enhances its photoprotection, which is crucial for the survival of aquatic anoxygenic phototrophs from photooxidative stress.

A photoheterotrophic bacterium from Iceland has adapted its photosynthetic machinery to the long days of polar summer.

During their long evolution, anoxygenic phototrophic bacteria have inhabited a wide variety of natural habitats and developed specific strategies to cope with the challenges of any particular environment. Expression, assembly, and safe operation of the photosynthetic apparatus must be regulated to prevent reactive oxygen species generation under illumination in the presence of oxygen. Here, we report on the photoheterotrophic Sediminicoccus sp. strain KRV36, which was isolated from a cold stream in north-western Iceland, 30 km south of the Arctic Circle. In contrast to most aerobic anoxygenic phototrophs, which stop pigment synthesis when illuminated, strain KRV36 maintained its bacteriochlorophyll synthesis even under continuous light. Its cells also contained between 100 and 180 chromatophores, each accommodating photosynthetic complexes that exhibit an unusually large carotenoid absorption spectrum. The expression of photosynthesis genes in dark-adapted cells was transiently downregulated in the first 2 hours exposed to light but recovered to the initial level within 24 hours. An excess of membrane-bound carotenoids as well as high, constitutive expression of oxidative stress response genes provided the required potential for scavenging reactive oxygen species, safeguarding bacteriochlorophyll synthesis and photosystem assembly. The unique cellular architecture and an unusual gene expression pattern represent a specific adaptation that allows the maintenance of anoxygenic phototrophy under arctic conditions characterized by long summer days with relatively low irradiance.IMPORTANCEThe photoheterotrophic bacterium Sediminicoccus sp. KRV36 was isolated from a cold stream in Iceland. It expresses its photosynthesis genes, synthesizes bacteriochlorophyll, and assembles functional photosynthetic complexes under continuous light in the presence of oxygen. Unraveling the molecular basis of this ability, which is exceptional among aerobic anoxygenic phototrophic species, will help to understand the evolution of bacterial photosynthesis in response to changing environmental conditions. It might also open new possibilities for genetic engineering of biotechnologically relevant phototrophs, with the aim of increasing photosynthetic activity and their tolerance to reactive oxygen species.

Engineering Chlorophyll, Bacteriochlorophyll, and Carotenoid Biosynthetic Pathways in Escherichia coli.

The biosynthesis of chlorophylls (Chls) and bacteriochlorophylls (BChls) represents a key aspect of photosynthesis research. Our previous work assembled the complete pathway for the synthesis of Chl a in Escherichia coli; here we engineer the more complex BChl a pathway in the same heterotrophic host. Coexpression of 18 genes enabled E. coli to produce BChl a, verifying that we have identified the minimum set of genes for the BChl a biosynthesis pathway. The protochlorophyllide reduction step was mediated by the bchNBL genes, and this same module was used to modify the Chl a pathway previously constructed in E. coli, eliminating the need for the light-dependent protochlorophyllide reductase. Furthermore, we demonstrate the feasibility of synthesizing more than one family of photosynthetic pigments in one host by engineering E. coli strains that accumulate the carotenoids neurosporene and ß-carotene in addition to BChl a.

Single-photon absorption and emission from a natural photosynthetic complex.

Photosynthesis is generally assumed to be initiated by a single photon1-3 from the Sun, which, as a weak light source, delivers at most a few tens of photons per nanometre squared per second within a chlorophyll absorption band1. Yet much experimental and theoretical work over the past 40 years has explored the events during photosynthesis subsequent to absorption of light from intense, ultrashort laser pulses2-15. Here, we use single photons to excite under ambient conditions the light-harvesting 2 (LH2) complex of the purple bacterium Rhodobacter sphaeroides, comprising B800 and B850 rings that contain 9 and 18 bacteriochlorophyll molecules, respectively. Excitation of the B800 ring leads to electronic energy transfer to the B850 ring in approximately 0.7 ps, followed by rapid B850-to-B850 energy transfer on an approximately 100-fs timescale and light emission at 850-875 nm (refs. 16-19). Using a heralded single-photon source20,21 along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. We also find that the probability distribution of the number of heralds per detected fluorescence photon supports the view that a single photon can upon absorption drive the subsequent energy transfer and fluorescence emission and hence, by extension, the primary charge separation of photosynthesis. An analytical stochastic model and a Monte Carlo numerical model capture the data, further confirming that absorption of single photons is correlated with emission of single photons in a natural light-harvesting complex.

Cryogenic Single-Molecule Fluorescence Detection of the Mid-Infrared Response of an Intrinsic Pigment in a Light-Harvesting Complex.

We observed the mid-infrared (MIR) response of a single pigment of bacteriochlorophyll a at the B800 binding site of a light-harvesting 2 complex. At a temperature of 1.5 K, a single complex in a spatially isolated spot in a near-infrared (NIR) fluorescence image was selected and was simultaneously irradiated with MIR and NIR light. We found that the temporal behavior of the NIR fluorescence excitation spectrum of individual pigments in a single complex was modulated by the MIR irradiation at 1650 cm-1. The MIR modulation of a single pigment was linearly proportional to the MIR intensity. The MIR linear response was detected in the range from 1580 to 1670 cm-1.

Optimized Rhodobacter sphaeroides for the Production of Antioxidants and the Pigments with Antioxidant Activity.

The photosynthetic bacterium, Rhodobacter sphaeroides, is a bacterium that can grow in a variety of environments and produces substances with antioxidant effects. In this study, we investigated the culture conditions to increase the production of antioxidants in R. sphaeroides, which can grow under both aerobic and anaerobic conditions. After incubation in the exponential phase and stationary phase under both conditions, a 2,2-diphenyl-1-picrylhydrazyl assay was used to confirm the antioxidant effect. Although the highest antioxidant effect was shown in the stationary phase under aerobic conditions, the antioxidant effect of each cell was found to be greater when cultured under anaerobic conditions. The antioxidant activity of R. sphaeroides was increased by sonication. In addition, the contents of carotenoids and bacteriochlorophyll, which are pigments with antioxidant effects, produced by R. sphaeroides were measured. We confirmed that the content of carotenoids was higher in anaerobic conditions than in aerobic conditions. However, when measuring the content of the bacterium, we found that there was more content in aerobic conditions. Therefore, we confirm that when grown in anaerobic conditions, the antioxidant effect of R. sphaeroides is higher, which suggests that this antioxidant effect comes from the effect of carotenoid.

Identification of amino acid residues in a proton release pathway near the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides.

Insight into control of proton transfer, a crucial attribute of cellular functions, can be gained from investigations of bacterial reaction centers. While the uptake of protons associated with the reduction of the quinone is well characterized, the release of protons associated with the oxidized bacteriochlorophyll dimer has been poorly understood. Optical spectroscopy and proton release/uptake measurements were used to examine the proton release characteristics of twelve mutant reaction centers, each containing a change in an amino acid residue near the bacteriochlorophyll dimer. The mutant reaction centers had optical spectra similar to wild-type and were capable of transferring electrons to the quinones after light excitation of the bacteriochlorophyll dimer. They exhibited a large range in the extent of proton release and in the slow recovery of the optical signal for the oxidized dimer upon continuous illumination. Key roles were indicated for six amino acid residues, Thr L130, Asp L155, Ser L244, Arg M164, Ser M190, and His M193. Analysis of the results points to a hydrogen-bond network that contains these residues, with several additional residues and bound water molecules, forming a proton transfer pathway. In addition to proton transfer, the properties of the pathway are proposed to be responsible for the very slow charge recombination kinetics observed after continuous illumination. The characteristics of this pathway are compared to proton transfer pathways near the secondary quinone as well as those found in photosystem II and cytochrome c oxidase.

Effects of Heterogeneous Protein Environment on Excitation Energy Transfer Dynamics in the Fenna-Matthews-Olson Complex.

The Fenna-Matthews-Olson (FMO) complex of green sulfur bacteria has been serving as a prototypical light-harvesting protein for studying excitation energy transfer (EET) dynamics in photosynthesis. The most widely used Frenkel exciton model for FMO complex assumes that each excited bacteriochlorophyll site couples to an identical and isolated harmonic bath, which does not account for the heterogeneous local protein environment. To better describe the realistic environment, we propose to use the recently developed multistate harmonic (MSH) model, which contains a globally shared bath that couples to the different pigment sites according to the atomistic quantum mechanics/molecular mechanics simulations with explicit protein scaffold and solvent. In this work, the effects of heterogeneous protein environment on EET in FMO complexes from Prosthecochloris aestuarii and Chlorobium tepidum, specifically including realistic spectral density, site-dependent reorganization energies, and system-bath couplings are investigated. Semiclassical and mixed quantum-classical mapping dynamics were applied to obtain the nonadiabatic EET dynamics in several models ranging from the Frenkel exciton model to the MSH model and their variants. The MSH model with realistic spectral density and site-dependent system-bath couplings displays slower EET dynamics than the Frenkel exciton model. Our comparative study shows that larger average reorganization energy, heterogeneity in spectral densities, and low-frequency modes could facilitate energy dissipation, which is insensitive to the static disorder in reorganization energies. The effects of the spectral densities and system-bath couplings along with the MSH model can be used to optimize EET dynamics for artificial light-harvesting systems.

Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αß-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

Effect of Dipyridamole on Membrane Energization and Energy Transfer in Chromatophores of Rba. sphaeroides.

Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of photosynthetic electron transport chain in the bacterial chromatophores of Rba. sphaeroides was investigated. DIP was found to affect efficiency of energy transfer from the light-harvesting complex LH2 to the LH1-reaction center core complex and to produce the long-wavelength ("red") shift of the absorption band of light-harvesting bacteriochlorophyll molecules in the IR spectral region at 840-900 nm. This shift is associated with the membrane transition to the energized state. It was shown that DIP is able to reduce the photooxidized bacteriochlorophyll of the reaction center, which accelerated electron flow along the electron transport chain, thereby stimulating generation of the transmembrane potential on the chromatophore membrane. The results are important for clarifying possible mechanisms of DIP influence on the activity of membrane-bound functional proteins. In particular, they might be significant for interpreting numerous therapeutic effects of DIP.

High Yield of B-Side Electron Transfer at 77 K in the Photosynthetic Reaction Center Protein from Rhodobacter sphaeroides.

The primary electron transfer (ET) processes at 295 and 77 K are compared for the Rhodobacter sphaeroides reaction center (RC) pigment-protein complex from 13 mutants including a wild-type control. The engineered RCs bear mutations in the L and M polypeptides that largely inhibit ET from the excited state P* of the primary electron donor (P, a bacteriochlorophyll dimer) to the normally photoactive A-side cofactors and enhance ET to the C2-symmetry related, and normally photoinactive, B-side cofactors. P* decay is multiexponential at both temperatures and modeled as arising from subpopulations that differ in contributions of two-step ET (e.g., P* → P+BB- → P+HB-), one-step superexchange ET (e.g., P* → P+HB-), and P* → ground state. [HB and BB are monomeric bacteriopheophytin and bacteriochlorophyll, respectively.] The relative abundances of the subpopulations and the inherent rate constants of the P* decay routes vary with temperature. Regardless, ET to produce P+HB- is generally faster at 77 K than at 295 K by about a factor of 2. A key finding is that the yield of P+HB-, which ranges from ∼5% to ∼90% among the mutant RCs, is essentially the same at 77 K as at 295 K in each case. Overall, the results show that ET from P* to the B-side cofactors in these mutants does not require thermal activation and involves combinations of ET mechanisms analogous to those operative on the A side in the native RC.

Carotenoid responds to excess energy dissipation in the LH2 complex from Rhodoblastus acidophilus.

The functions of both (bacterio) chlorophylls and carotenoids in light-harvesting complexes have been extensively studied during the past decade, yet, the involvement of BChl a high-energy Soret band in the cascade of light-harvesting processes still remains a relatively unexplored topic. Here, we present transient absorption data recorded after excitation of the Soret band in the LH2 complex from Rhodoblastus acidophilus. Comparison of obtained data to those recorded after excitation of rhodopin glucoside and B800 BChl a suggests that no Soret-to-Car energy transfer pathway is active in LH2 complex. Furthermore, a spectrally rich pattern observed in the spectral region of rhodopin glucoside ground state bleaching (420-550 nm) has been assigned to an electrochromic shift. The results of global fitting analysis demonstrate two more features. A 6 ps component obtained exclusively after excitation of the Soret band has been assigned to the response of rhodopin glucoside to excess energy dissipation in LH2. Another time component, ~ 450 ps, appearing independently of the excitation wavelength was assigned to BChl a-to-Car triplet-triplet transfer. Presented data demonstrate several new features of LH2 complex and its behavior following the excitation of the Soret band.

Carotenoid Single-Molecular Singlet Fission and the Photoprotection of a Bacteriochlorophyll b-Type Core Light-Harvesting Antenna.

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 (3Car*) 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 3Car* 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 3BChl b*. The implications of different 3Car* formation mechanisms are discussed, and the self-photoprotection role of BChl b are proposed for this extremophilic species.

Ultrafast energy transfer between self-assembled fluorophore and photosynthetic light-harvesting complex 2 (LH2) in lipid bilayer.

Photosynthetic light-harvesting (LH) systems consist of photosynthetic pigments, which are non-covalently self-assembled with protein scaffolds in many phototrophs and attain highly efficient excitation energy transfer via ultrafast dynamics. In this study, we constructed a biohybrid LH system composed of an LH complex (LH2) from Rhodoblastus acidophilus strain 10050 and a hydrophobic fluorophore ATTO647N (ATTO) as an extrinsic antenna in the lipid bilayer. Through the addition of ATTOs into a solution of LH2-reconstituted lipid vesicles, ATTOs were incorporated into the hydrophobic interior of the lipid bilayer to configure the non-covalently self-assembled biohybrid LH. Steady-state fluorescence spectroscopy clearly showed efficient energy transfer from ATTO to B850 bacteriochlorophylls in LH2. Femtosecond transient absorption spectroscopy revealed that the energy transfer took place in the time range of 3-13 ps, comparable to that of the covalently linked LH2-ATTO that we previously reported. In addition, the biohybrid LH system exhibited a much higher antenna effect than the LH2-ATTO system because of the higher loading level of ATTO in the membrane. These findings suggest that the facile self-assembled biohybrid LH system is a promising system for constructing LH for solar-energy conversion.

Disproportionate effect of cationic antiseptics on the quantum yield and fluorescence lifetime of bacteriochlorophyll molecules in the LH1-RC complex of R. rubrum chromatophores.

Photosynthetic membrane complexes of purple bacteria are convenient and informative macromolecular systems for studying the mechanisms of action of various physicochemical factors on the functioning of catalytic proteins both in an isolated state and as part of functional membranes. In this work, we studied the effect of cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine) on the fluorescence intensity and the efficiency of energy transfer from the light-harvesting LH1 complex to the reaction center (RC) of Rhodospirillum rubrum chromatophores. The effect of antiseptics on the fluorescence intensity and the energy transfer increased in the following order: chlorhexidine, picloxydine, miramistin, octenidine. The most pronounced changes in the intensity and lifetime of fluorescence were observed with the addition of miramistin and octenidine. At the same concentration of antiseptics, the increase in fluorescence intensity was 2-3 times higher than the increase in its lifetime. It is concluded that the addition of antiseptics decreases the efficiency of the energy migration LH1 → RC and increases the fluorescence rate constant kfl. We associate the latter with a change in the polarization of the microenvironment of bacteriochlorophyll molecules upon the addition of charged antiseptic molecules. A possible mechanism of antiseptic action on R. rubrum chromatophores is considered. This work is a continuation of the study of the effect of antiseptics on the energy transfer and fluorescence intensity in chromatophores of purple bacteria published earlier in Photosynthesis Research (Strakhovskaya et al. in Photosyn Res 147:197-209, 2021).

A bound iron porphyrin is redox active in hybrid bacterial reaction centers modified to possess a four-helix bundle domain.

In this paper we report the design of hybrid reaction centers with a novel redox-active cofactor. Reaction centers perform the primary photochemistry of photosynthesis, namely the light-induced transfer of an electron from the bacteriochlorophyll dimer to a series of electron acceptors. Hybrid complexes were created by the fusion of an artificial four-helix bundle to the M-subunit of the reaction center. Despite the large modification, optical spectra show that the purified hybrid reaction centers assemble as active complexes that retain the characteristic cofactor absorption peaks and are capable of light-induced charge separation. The four-helix bundle could bind iron-protoporphyrin in either a reduced and oxidized state. After binding iron-protoporphyrin to the hybrid reaction centers, light excitation results in a new derivative signal with a maximum at 402 nm and minimum at 429 nm. This signal increases in amplitude with longer light durations and persists in the dark. No signal is observed when iron-protoporphyrin is added to reaction centers without the four-helix bundle domain or when a redox-inactive zinc-protoporphyrin is bound. The results are consistent with the signal arising from a new redox reaction, electron transfer from the iron-protoporphyrin to the oxidized bacteriochlorophyll dimer. These outcomes demonstrate the feasibility of binding porphyrins to the hybrid reaction centers to gain new light-driven functions.

Carotenoids Do Not Protect Bacteriochlorophylls in Isolated Light-Harvesting LH2 Complexes of Photosynthetic Bacteria from Destructive Interactions with Singlet Oxygen.

The effect of singlet oxygen on light-harvesting (LH) complexes has been studied for a number of sulfur (S+) and nonsulfur (S-) photosynthetic bacteria. The visible/near-IR absorption spectra of the standard LH2 complexes (B800-850) of Allochromatium (Alc.) vinosum (S+), Rhodobacter (Rba.) sphaeroides (S-), Rhodoblastus (Rbl.) acidophilus (S-), and Rhodopseudomonas (Rps.) palustris (S-), two types LH2/LH3 (B800-850 and B800-830) of Thiorhodospira (T.) sibirica (S+), and an unusual LH2 complex (B800-827) of Marichromatium (Mch.) purpuratum (S+) or the LH1 complex from Rhodospirillum (Rsp.) rubrum (S-) were measured in aqueous buffer suspensions in the presence of singlet oxygen generated by the illumination of the dye Rose Bengal (RB). The content of carotenoids in the samples was determined using HPLC analysis. The LH2 complex of Alc. vinosum and T. sibirica with a reduced content of carotenoids was obtained from cells grown in the presence of diphenylamine (DPA), and LH complexes were obtained from the carotenoidless mutant of Rba. sphaeroides R26.1 and Rps. rubrum G9. We found that LH2 complexes containing a complete set of carotenoids were quite resistant to the destructive action of singlet oxygen in the case of Rba. sphaeroides and Mch. purpuratum. Complexes of other bacteria were much less stable, which can be judged by a strong irreversible decrease in the bacteriochlorophyll (BChl) absorption bands (at 850 or 830 nm, respectively) for sulfur bacteria and absorption bands (at 850 and 800 nm) for nonsulfur bacteria. Simultaneously, we observe the appearance of the oxidized product 3-acetyl-chlorophyll (AcChl) absorbing near 700 nm. Moreover, a decrease in the amount of carotenoids enhanced the spectral stability to the action of singlet oxygen of the LH2 and LH3 complexes from sulfur bacteria and kept it at the same level as in the control samples for carotenoidless mutants of nonsulfur bacteria. These results are discussed in terms of the current hypothesis on the protective functions of carotenoids in bacterial photosynthesis. We suggest that the ability of carotenoids to quench singlet oxygen (well-established in vitro) is not well realized in photosynthetic bacteria. We compared the oxidation of BChl850 in LH2 complexes of sulfur bacteria under the action of singlet oxygen (in the presence of 50 µM RB) or blue light absorbed by carotenoids. These processes are very similar: {[BChl + (RB or carotenoid) + light] + O2} → AcChl. We speculate that carotenoids are capable of generating singlet oxygen when illuminated. The mechanism of this process is not yet clear.

Cryo-EM structure of the monomeric Rhodobacter sphaeroides RC-LH1 core complex at 2.5†Å.

Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5 Šresolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αß heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could impede passage of quinones through small, transient pores in the LH1 ring, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilising an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered quinone diffusion.