Bacterium Rhodobacter Sphaeroides Research Articles

Femtosecond Dynamics of the Excited Primary Electron Donor in Reaction Centers of the Purple Bacterium Rhodobacter sphaeroides.

Femtosecond transient absorption spectroscopy was used to study the dynamics of the excited primary electron donor in the reaction centers of the purple bacterium Rhodobacter sphaeroides. Using global analysis and the interval method, we found a correlation between the vibrational coherence damping of the excited primary electron donor and the lifetime of the charge-separated stateP+BA-, indicating the reversibility of electron transfer to the primary electron acceptor, theBA molecule. In the reaction centers, the signs of superposition of two electronic states of P were found for a delay time of less than 200fs. It is suggested that the admixture value of the charge transfer statePA+PB- with the excited primary electron donorP* is about 24%. The results obtained are discussed in terms of the two-step electron transfer mechanism.

DYE 10-N-NONYL ACRIDINE ORANGE AS A FLUORESCENT INDICATOR OF THE ACTION OF THE ANTISEPTIC OCTENIDINE ON THE MEMBRANES OF RHODOBACTER SPHAEROIDES CHROMATOPHORES

Increasing volumes of produced antiseptics and disinfectants, which are widely used in medicine, veterinary medicine, the food industry and everyday life, can create a serious environmental problem after use. Previously, under the infl uence of micromolar concentrations of a number of antiseptics, we identifi ed disturbances in the functioning of photosynthetic membranes and phototransforming pigment-protein complexes isolated from them in various representatives of photosynthetic organisms. In this work, to determine the sensitivity of photosynthetic membranes to the action of the cationic antiseptic octenidine, we used chromatophores of the purple nonsulfur bacteria Rhodobacter sphaeroides, labeled with the fluorescent dye 10-N-nonylacridine orange (NAO). It was shown that the binding of NAO to chromatophores is accompanied by a shift in the dye emission maximum from 525 to 640 nm. The “red” fluorescence of NAO associated with chromatophores turned out to be sensitive to the effect of increasing concentrations of octenidine on photosynthetic membranes. We have found antiseptic concentrations which led to the degradation of chromatophore structures and change in the NAO aggregative state. It can be detected by an increase of “green” fluorescence in the emission spectra of the dye. The properties of NAO as a fluorescent indicator of the functional state of photosynthetic membranes and possible changes that can occur in such systems under the influence of a cationic antiseptic are discussed.

Generation of electric potential difference by chromatophores from photosynthetic bacteria in the presence of trehalose under continuous illumination

Measurement of the electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides, associated with the surface of a nitrocellulose membrane filter (MF), impregnated with phospholipid solution in decane, as well as immersed inside it, in the presence of exogenous mediators and disaccharide trehalose, demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The ascorbate/N,N,N′N′-tetramethyl-p-phenylenediamine pair and ubiquinone-0 were used as mediators (electron donor and acceptor), respectively. Although the stabilization of photoelectric responses under long-term continuous illumination was observed in both cases of immobilization of chromatophores, only when the samples were immersed inside the MF, the functional activity of the reaction centers was maintained for a month when they were stored in the dark at room temperature. Stable photoresponses are probably associated with the preservation of the integrity of chromatophore proteins inside MF pores. The stabilizing effect of the bioprotector trehalose may be due to both its effect on RC proteins and on the phospholipid bilayer membrane. The results obtained will expand modern ideas about the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into an electrochemical form.

Generation of Electric Potential Difference by Chromatophores from Photosynthetic Bacteria in the Presence of Trehalose under Continuous Illumination.

Measurement of electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter (MF) impregnated with a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators were the ascorbate/N,N,N'N'-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor, respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity of reaction centers (RCs) for a month when stored in the dark at room temperature, which might be due to the preservation of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into its electrochemical form.

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.

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division.

Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling.IMPORTANCEBy coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

Conversion of light into electricity in a semi-synthetic system based on photosynthetic bacterial chromatophores

Chromatophores (Chr) from photosynthetic nonsulfur purple bacterium Rhodobacter sphaeroides immobilized onto a Millipore membrane filter (MF) and sandwiched between two semiconductor indium tin oxide (ITO) electrodes (termed ITO|Chr – MF|ITO) have been used to measure voltage (ΔV) induced by continuous illumination. The maximum ΔV was detected in the presence of ascorbate / N,N,N’N'-tetramethyl-p-phenylenediamine couple, coenzyme UQ0, disaccaride trehalose and antimycin A, an inhibitor of cytochrome bc1 complex. In doing so, the light-induced electron transfer in the reaction centers was the major source of photovoltages. The stability of the voltage signal upon prolonged irradiation (>1 h) may be due to the maintenance of a conformation that is optimal for the functioning of integral protein complexes and stabilization of lipid bilayer membranes in the presence of trehalose. Retaining ∼70 % of the original photovoltage performance on the 30th day of storage at 23 °C in the dark under air was achieved after re-injection of fresh buffer (∼40 μL) containing redox mediators into the ITO|Chr – MF|ITO system. The approach we use is easy and can be extended to other biological intact systems (cells, thylakoid membranes) capable of converting energy of light.

Supramolecular Biohybrid Construct for Photoconversion Based on a Bacterial Reaction Center Covalently Bound to Cytochrome c by an Organic Light Harvesting Bridge.

A supramolecular construct for solar energy conversion is developed by covalently bridging the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides and cytochrome c (Cyt c) proteins with a tailored organic light harvesting antenna (hCy2). The RC-hCy2-Cyt c biohybrid mimics the working mechanism of biological assemblies located in the bacterial cell membrane to convert sunlight into metabolic energy. hCy2 collects visible light and transfers energy to the RC, increasing the rate of photocycle between a RC and Cyt c that are linked in such a way that enhances proximity without preventing protein mobility. The biohybrid obtained with average 1 RC/10 hCy2/1.5 Cyt c molar ratio features an almost doubled photoactivity versus the pristine RC upon illumination at 660 nm, and ∼10 times higher photocurrent versus an equimolar mixture of the unbound proteins. Our results represent an interesting insight into photoenzyme chemical manipulation, opening the way to new eco-sustainable systems for biophotovoltaics.

The Role of Electrostatic Binding Interfaces in the Performance of Bacterial Reaction Center Biophotoelectrodes.

Photosynthetic reaction centers (RCs) efficiently capture and convert solar radiation into electrochemical energy. Accordingly, RCs have the potential as components in biophotovoltaics, biofuel cells, and biosensors. Recent biophotoelectrodes containing the RC from the bacterium Rhodobacter sphaeroides utilize a natural electron donor, horse heart cytochrome c (cyt c), as an electron transfer mediator with the electrode. In this system, electrostatic interfaces largely control the protein-electrode and protein-protein interactions necessary for electron transfer. However, recent studies have revealed kinetic bottlenecks in cyt-mediated electron transfer that limit biohybrid photoelectrode efficiency. Here, we seek to understand how changing protein-protein and protein-electrode interactions influence RC turnover and biophotoelectrode efficiency. The RC-cyt c binding interaction was modified by substituting interfacial RC amino acids. Substitutions Asn-M188 to Asp and Gln-L264 to Glu, which are known to produce a higher cyt-binding affinity, led to a decrease in the RC turnover frequency (TOF) at the electrode, suggesting that a decrease in cyt c dissociation was rate-limiting in these RC variants. Conversely, an Asp-M88 to Lys substitution producing a lower binding affinity had little effect on the RC TOF, suggesting that a decrease in the cyt c association rate was not a rate-limiting factor. Modulating the electrode surface with a self-assembled monolayer that oriented the cyt c to face the electrode did not affect the RC TOF, suggesting that the orientation of cyt c was also not a rate-limiting factor. Changing the ionic strength of the electrolyte solution had the most potent impact on the RC TOF, indicating that cyt c mobility was important for effective electron donation to the photo-oxidized RC. An ultimate limitation for the RC TOF was that cyt c desorbed from the electrode at ionic strengths above 120 mM, diluting its local concentration near the electrode-adsorbed RCs and resulting in poor biophotoelectrode performance. These findings will guide further tuning of these interfaces for improved performance.

Mutual supply of carbon and nitrogen sources in the co-culture of aerial microalgae and nitrogen-fixing bacteria

This study investigated co-culture for supply of carbon (C) and nitrogen (N) sources by aerial microalgae and N-fixing bacteria. The aerial microalga Coelastrella sp. KGU-HN001 was isolated from the surface of steel signs in Japan. After examining monocultures of the microalga and N-fixing bacterium Rhodobacter sphaeroides, mutual nutrient supply was evaluated in co-culture using a medium without C and N sources. In co-culture under aerobic conditions, the cell number, biomass, total organic C in cells and the total N of cell suspensions increased. Carbohydrate was secreted from the microalga and nitrogenase activity was expressed by the bacterium. Addition of an N source enhanced N fixation in the co-culture. These results indicated that C and N fixation by the microalga and the bacterium occurred under aerobic conditions, and the microorganisms were able to grow by self-supplying nutrients even under nutrient deficiencies.

Rhodobacter sphaeroides as a model to study the ecotoxicity of 1-alkyl-3-methylimidazolium bromide.

The purple non-sulfur bacterium Rhodobacter sphaeroides was selected as a biological model to investigate its response to the toxicity of 1-alkyl-3-methylimidazolium bromide ([Cnmim]Br), a type of ionic liquid (IL), with different alkyl chain lengths (n describes the number of carbon atoms in the alkyl chain). The inhibition of bacterial growth by [Cnmim]Br was positively correlated with n. Morphological characterization revealed that [Cnmim]Br caused cell membrane perforation. The signal amplitude of the electrochromic absorption band shift of endogenous carotenoids showed a negatively linear correlation with n, and the amplitude of the blue-shift of the B850 band in light-harvesting complex 2 showed a positively linear correlation with n. Furthermore, an increase in blocked ATP synthesis and increase in antioxidant enzyme activity were observed in chromatophores treated with ILs containing longer alkyl chains. In summary, the purple bacterium can be developed as a model to monitor ecotoxicity and examine the mechanism of IL toxicity.

Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

Selective TLR4 Antagonism Prevents and Reverses Morphine-Induced Persistent Postoperative Cognitive Dysfunction, Dysregulation of Synaptic Elements, and Impaired BDNF Signaling in Aged Male Rats.

Perioperative neurocognitive disorders (PNDs) are characterized by confusion, difficulty with executive function, and episodic memory impairment in the hours to months following a surgical procedure. Postoperative cognitive dysfunction (POCD) represents such impairments that last beyond 30 d postsurgery and is associated with increased risk of comorbidities, progression to dementia, and higher mortality. While it is clear that neuroinflammation plays a key role in PND development, what factors underlie shorter self-resolving versus persistent PNDs remains unclear. We have previously shown that postoperative morphine treatment extends POCD from 4 d (without morphine) to at least 8 weeks (with morphine) in aged male rats, and that this effect is likely dependent on the proinflammatory capabilities of morphine via activation of toll-like receptor 4 (TLR4). Here, we extend these findings to show that TLR4 blockade, using the selective TLR4 antagonist lipopolysaccharide from the bacterium Rhodobacter sphaeroides (LPS-RS Ultrapure), ameliorates morphine-induced POCD in aged male rats. Using either a single central preoperative treatment or a 1 week postoperative central treatment regimen, we demonstrate that TLR4 antagonism (1) prevents and reverses the long-term memory impairment associated with surgery and morphine treatment, (2) ameliorates morphine-induced dysregulation of the postsynaptic proteins postsynaptic density 95 and synaptopodin, (3) mitigates reductions in mature BDNF, and (4) prevents decreased activation of the BDNF receptor TrkB (tropomyosin-related kinase B), all at 4 weeks postsurgery. We also reveal that LPS-RS Ultrapure likely exerts its beneficial effects by preventing endogenous danger signal HMGB1 (high-mobility group box 1) from activating TLR4, rather than by blocking continuous activation by morphine or its metabolites. These findings suggest TLR4 as a promising therapeutic target to prevent or treat PNDs.SIGNIFICANCE STATEMENT With humans living longer than ever, it is crucial that we identify mechanisms that contribute to aging-related vulnerability to cognitive impairment. Here, we show that the innate immune receptor toll-like receptor 4 (TLR4) is a key mediator of cognitive dysfunction in aged rodents following surgery and postoperative morphine treatment. Inhibition of TLR4 both prevented and reversed surgery plus morphine-associated memory impairment, dysregulation of synaptic elements, and reduced BDNF signaling. Together, these findings implicate TLR4 in the development of postoperative cognitive dysfunction, providing mechanistic insight and novel therapeutic targets for the treatment of cognitive impairments following immune challenges such as surgery in older individuals.

The Case for Rhodobacter Sphaeroides as a probiotic: Applications for Human Health and Ulcerative Colitis

In this short review, the mechanisms through which the probiotic administration of non-pathogenic bacterium Rhodobacter Sphaeroides could contribute to human health in general, and more specifically, the treatment of ulcerative colitis, are explored. This review is built around the concept that the mitochondrion is the key player in the pathogenesis of ulcerative colitis, and proposes ways that the probiotic could contribute to a more optimal environment for mitochondrial functioning, namely through reduction of inflammation and production of beneficial compounds like ubiquinone and bacteria-derived carotenoids. It concludes with the current state of the research involving Rhodobacter Sphaeroides as a probiotic and suggests possible future directions.

Kinetics of Initial Charge Separation in the Photosynthetic Reaction Centers of Rhodobacter sphaeroides.

A stochastic kinetic study of the initial electron transfer reaction in the reaction centers of wild-type and different mutants of photosynthetic bacterium Rhodobacter sphaeroides is suggested. The present approach to the disorder-driven complex kinetics skilfully offers an alternative to the earlier dynamic analyses. Exploiting a rational description of the reaction coordinate, effected by the relaxation of the surrounding protein environment, the measured kinetics were reproduced quantitatively. Notably, comparisons of the extracted relative free energies of electron transfer for the selected mutants and the available independent electrochemical estimates show exact agreement in some cases.

Molecular insights into the role of heme in the transcriptional regulatory system AppA/PpsR

Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.

Nickel ion extracellular uptake by the phototrophic bacterium Rhodobacter sphaeroides: new insights from Langmuir modelling and X-ray photoelectron spectroscopic analysis

The presence of anionic functional groups on the surface of microbial cells makes the bacterial biomass very attractive as metal cation removal tool for environmental bioremediation. To go insight to the heavy metal sorption abilities of Gram-negative bacteria, we characterized the extracellular uptake of Ni2+ ions by the phototrophic bacterium Rhodobacter sphaeroides, whose ability to sequester toxic metal ions has been widely documented. The sorption process proved to be well described by the Langmuir model, suggesting that the cell envelope behaves as an ideal adsorbent surface, showing a layer of chemically equivalent binding sites with high affinity for Ni2+. X-ray photoelectron spectroscopic data reveal that, at binding site saturation, Ni2+ ions fully displace alkali metal ions from the bacterial surface, while they are unable to displace Ca2+ and Mg2+ from their tight binding sites. The analysis of carbon, nitrogen and oxygen speciation, as arisen from high resolution spectra fitting, highlights that the Ni2+/K+ cation exchange affects the surface distribution of main chemical components, with increased contribution by peptides and phosphatidylcholines. A role of Ni2+ ions in favoring the stacking and/or packing of lipopolysaccharide sugar chains by interaction with carboxylate groups is proposed and discussed.

Aerobes and phototrophs as microbial organic fertilizers: Exploring mineralization, fertilization and plant protection features.

Organic fertilizers and especially microbial biomass, also known as microbial fertilizer, can enable a paradigm shift to the conventional fertilizer-to-food chain, particularly when produced on secondary resources. Microbial fertilizers are already common practice (e.g. Bloom® and Synagro); yet microbial fertilizer blends to align the nutrient release profile to the plant’s needs are, thus far, unexplored. Moreover, most research only focuses on direct fertilization effects without considering added value properties, such as disease prevention. This study has explored three promising types of microbial fertilizers, namely dried biomass from a consortium of aerobic heterotrophic bacteria, a microalga (Arthrospira platensis) and a purple non-sulfur bacterium (Rhodobacter sphaeroides). Mineralization and nitrification experiments showed that the nitrogen mineralization profile can be tuned to the plant’s needs by blending microbial fertilizers, without having toxic ammonium peaks. In a pot trial with perennial ryegrass (Lolium perenne L.), the performance of microbial fertilizers was similar to the reference organic fertilizer, with cumulative dry matter yields of 5.6–6.7 g per pot. This was confirmed in a pot trial with tomato (Solanum lycopersicum L.), showing an average total plant length of 90–99 cm after a growing period of 62 days for the reference organic fertilizer and the microbial fertilizers. Moreover, tomato plants artificially infected with powdery mildew (Oidium neolycopersici), a devastating disease for the horticultural industry, showed reduced disease symptoms when A. platensis was present in the growing medium. These findings strengthen the application potential of this novel class of organic fertilizers in the bioeconomy, with a promising match between nutrient mineralization and plant requirements as well as added value in crop protection.

X-ray structure of the Rhodobacter sphaeroides reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex.

The first steps of the global process of photosynthesis take place in specialized membrane pigment-protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium Rhodobacter sphaeroides, a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment-protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC. It has previously been shown that the replacement of Phe M197 by His strongly affects the oxidation potential of P (E m P/P+), increasing its value by 125 mV, as well as increasing the thermal stability of RC and its stability in response to external pressure. The crystal structures of F(M197)H RC at high resolution obtained using various techniques presented in this report clarify the optical and electrochemical properties of the primary electron donor and the increased resistance of the mutant complex to denaturation. The electron-density maps are consistent with the donation of a hydrogen bond from the imidazole group of His M197 to the C2-acetyl carbonyl group of BChl PB. The formation of this hydrogen bond leads to a considerable out-of-plane rotation of the acetyl carbonyl group and results in a 1.2 Å shift of the O atom of this group relative to the wild-type structure. Besides, the distance between BChl PA and PB in the area of pyrrole ring I was found to be increased by up to 0.17 Å. These structural changes are discussed in association with the spectral properties of BChl dimer P. The electron-density maps strongly suggest that the imidazole group of His M197 accepts another hydrogen bond from the nearest water molecule, which in turn appears to form two more hydrogen bonds to Asn M195 and Asp L155. As a result of the F(M197)H mutation, BChl PB finds itself connected to the extensive hydrogen-bonding network that pre-existed in wild-type RC. Dissimilarities in the two hydrogen-bonding networks near the M197 and L168 sites may account for the different changes of the E m P/P+ in F(M197)H and H(L168)F RCs. The involvement of His M197 in the hydrogen-bonding network also appears to be related to stabilization of the F(M197)H RC structure. Analysis of the experimental data presented here and of the data available in the literature points to the fact that the hydrogen-bonding networks in the vicinity of BChl dimer P may play an important role in fine-tuning the redox properties of the primary electron donor.

Engineering purple bacterial carotenoid biosynthesis to study the roles of carotenoids in light-harvesting complexes.

Carotenoids are important photosynthetic pigments that play key roles in light harvesting and energy transfer, photoprotection, and in the folding, assembly, and stabilization of light-harvesting pigment-protein complexes. The genetically tractable purple phototrophic bacteria have been useful for investigating the biosynthesis and function of photosynthetic pigments and cofactors, including carotenoids. Here, we give an overview of the roles of carotenoids in photosynthesis and of their biosynthesis, focusing on the extensively studied purple bacterium Rhodobacter sphaeroides as a model organism. We provide detailed procedures for manipulating carotenoid biosynthesis, and for the preparation and analysis of the light-harvesting and photosynthetic reaction center complexes that bind them. Using appropriate examples from the literature, we discuss how such approaches have enhanced our understanding of the biosynthesis of carotenoids and the photosynthesis-related functions of these fascinating molecules.