Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting the B800-850 complex and its dependence on the spectral composition of the light is discussed.     

Evolution of low-light adapted peripheral light-harvesting complexes in strains of Rhodopseudomonas palustris

Purple bacteria have peripheral light-harvesting (PLH) complexes adapted to high-light (LH2) and low-light (LH3, LH4) growth conditions. The latter two have only been fully characterised in Rhodopseudomonas acidophila 7050 and Rhodopseudomonas palustris CGA009, respectively. It is known that LH4 complexes are expressed under the control of two light sensing bacteriophytochromes (BphPs). Recent genomic sequencing of a number of Rps. palustris strains has provided extensive information on PLH genes. We show that both LH3 and LH4 complexes are present in Rps. palustris and have evolved in the same operon controlled by the two adjacent BphPs. Two rare marker genes indicate that a gene cluster CL2, containing LH2 genes and the BphP RpBphP4, was internally transferred within the genome to form a new operon CL1. In CL1, RpBphP4 underwent gene duplication to RpBphP2 and RpBphP3, which evolved to sense light intensity rather than spectral red/far-red intensity ratio. We show that a second LH2 complex was acquired in CL1 belonging to a different PLH clade and these two PLH complexes co-evolved together into LH3 or LH4 complexes. The near-infrared spectra provide additional support for our conclusions on the evolution of PLH complexes based on genomic data.     

Characterization of the different peripheral light-harvesting complexes from high- and low-light grown cells from Rhodopseudomonas palustris

In this paper we demonstrate that the spectroscopically different peripheral light-harvesting complexes from Rhodopseudomonas palustris, strain 2.6.1, isolated from high- and low-light grown cells have widely differing bacteriochlorophyll a (BChl a) resonance Raman spectra in the high-frequency carbonyl region (1550-1750 cm-1). Complexes synthesized in low-light grown cells exhibit Raman spectra characteristic of B800-850 and B800-820 complexes, depending on the excitation conditions. The in vivo strategy for low-light adaptation in this bacterium is thus somewhat different from that generally encountered in the Rhodospirillaceae. In these bacteria, as typified by Rps. acidophila and Rps. cryptolactis, low-light conditions induce the synthesis of B800-820 only complexes in which the hydrogen bonds between the acetyl carbonyl and the B850 binding pocket are broken, inducing changes in the absorption properties of the monomeric bacteriochlorophylls. In the case of Rps. palustris, additional spectral effects occur due to the coupling of the electronic levels of the differently interacting dimers. The extensive use of differential alpha/beta-polypeptide expression [Tadros et al. (1993) Eur. J. Biochem. 217, 867-875] thus allows Rps. palustris to alter its BChl a binding site environments causing the observed spread of BChl a Qy transitions, ranging from 801 to 856 nm.     

Statistical considerations on the formation of circular photosynthetic light-harvesting complexes from Rhodopseudomonas palustris

Depending on growth conditions, some species of purple photosynthetic bacteria contain peripheral light-harvesting (LH2) complexes that are heterogeneous owing to the presence of different protomers (containing different αβ-apoproteins). Recent spectroscopic studies of Rhodopseudomonas palustris grown under low-light conditions suggest the presence of a C ₃-symmetric LH2 nonamer comprised of two distinct protomers. The software program Cyclaplex, which enables generation and data-mining of virtual libraries of molecular rings formed upon combinatorial reactions, has been used to delineate the possible number and type of distinct nonamers as a function of numbers of distinct protomers. The yield of the C ₃-symmetric nonamer from two protomers (A and B in varying ratios) has been studied under the following conditions: (1) statistical, (2) enriched (preclusion of the B-B sequence), and (3) seeded (pre-formation of an A-B-A block). The yield of C ₃-symmetric nonamer is at most 0.98 % under statistical conditions versus 5.6 % under enriched conditions, and can be dominant under conditions of pre-seeding with an A-B-A block. In summary, the formation of any one specific nonamer even from only two protomers is unlikely on statistical grounds but must stem from enhanced free energy of formation or a directed assembly process by as-yet unknown factors.     

Biodegradation of 2-Chlorophenol by Rhodopseudomonas palustris

A phototrophic bacterial culture that assimilates 2-chlorophenol (2-CP) was enriched from effluents of paper and wood industry. The isolated bacterium was identified as Rhodopseudomonas palustris based on its morphological characteristics, biochemical reactions, and fatty acid methyl ester gas chromatography (FAME-GC) analysis. The bacterium R. palustris being photoheterotrophic in nutrition was also able to switch between phototropic, aerobic, and anaerobic modes of metabolism, as evidenced by modulation of the bacterial photopigments. The switching of anaerobic to aerobic metabolism was evidenced by the presence of catechol, a product of aerobic oxidation of 2-CP in the spent medium and the disappearance of photopigment-specific absorbance in the organism. R. palustris degraded about 97% of the supplemented 2-CP from the culture medium in 40 days along with production of an exo-polysaccharide, which probably provides protection from substrate toxicity and enhances its bioavailability. Thin-layer chromatography (TLC) and gas chromoatography–mass spectrometry (GC-MS) analysis of the R. palustris spent medium extracts indicated the presence of phenol. The GC-MS data also indicated that phenol is metabolized through an ortho-cleavage pathway. Further analysis of the R. palustris cell-free extracts for enzymes showed the presence of a chlorophenol dehalogenase (CD) and a chlorophenol nicotinamide adenine dinucleotide phosphate (NADPH)-oxidoreductase (CNOR) with respective specific activities of 0.114 and 0.26 μmol/min/mg, suggesting an initial reductive dechlorination step during 2-CP catabolism. The study thus highlights the important roles of phototrophic bacteria in the decontamination of environmental pollutants from the polluted sites.     

Uptake of benzoate by Rhodopseudomonas palustris grown anaerobically in light.

The uptake and anaerobic metabolism of benzoate were studied in short-term experiments with phototrophic cells of Rhodopseudomonas palustris. Cells that were preincubated and assayed anaerobically in the presence of 1 mM dithiothreitol accumulated [7-14C]benzoate at a rate of at least 0.5 nmol . min-1 . mg-1 of protein. Cells that were preincubated aerobically, or anaerobically in the absence of a reducing agent or an electron donor such as succinate, took up benzoate at reduced rates. Benzoate was removed from the external medium with remarkably high efficiency; initial uptake rates were independent of substrate concentration, and uptake remained linear down to concentrations of less than 1 microM. Uptake rates were not sensitive to external pH in the range of 6.5 to 8.1, and very little free benzoate was found associated with the cells. By contrast, benzoyl coenzyme A (CoA) was formed rapidly in cells exposed to labeled benzoate. Its appearance in such cells, together with the more gradual accumulation of other compounds tentatively identified as reduction products, is consistent with the identification of benzoyl CoA as an intermediate in the anaerobic reductive metabolism of benzoate. The very effective uptake of external benzoate can be explained by its conversion to benzoyl CoA immediately after its passage across the cell membrane by simple or facilitated diffusion. Such a chemical conversion would serve to maintain a downhill concentration gradient between the cell cytoplasm and the cell surroundings, even at very low external benzoate concentrations.     

Effects of pH on the peripheral light-harvesting antenna complex for Rhodopseudomonas palustris

In this work steady-state absorption spectroscopy, circular dichroism spectroscopy and sub-micro-second time-resolved absorption spectroscopy were used to investigate the effect of pH on the struc-tures and functions of LH2 complex for Rhodopseudomonas palustris. The results revealed that: (1) B800 Bchla was gradually transformed to free pigments absorbing around 760 nm on the minutes timescale upon the induction of strong acidic pH, and subsequently there disappeared the CD signal for Qy band of B800 in the absence of B800. In addition, Carotenoids changed with the similar tendency to B850 BChl. (2) The introduction of strong basic pH gave rise to no significant changes for B800 Bchla, while B850 BChla experienced remarkable spectral blue-shift from 852 to 837 nm. Similar phe-nomenon was seen for the CD signal for Qy band of B850. Carotenoids displayed strong and pH-independent CD signals in the visible range. (3) In the case of both physiological and basic pH, broad and asymmetrical positive Tn←T1 transient absorption appeared following the pulsed photo-excitation of Car at 532 nm. By contrast, the featureless and weak positive signal was observed on the sub-microsecond timescale in the acidic pH environment. The aforementioned experimental results indicated that acidic pH-induced removal of B800 Bchla prevented the generation of the caro-tenoid triplet state (3Car*), which is known to be essential for the photo-protection function. Neverthe-less, carotenoids can still perform this important physiological role under the basic pH condition, where the spectral blue shift of B850 exerts little effect on the overall structure of the cyclic aggregate, therefore favoring the formation of carotenoid triplet state.     

Triplet Excitation Transfer between Carotenoids in the LH2 Complex from Photosynthetic Bacterium Rhodopseudomonas palustris

We have studied, by means of sub-microsecond time-resolved absorption spectroscopy, the triplet-excited state dynamics of carotenoids (Cars) in the intermediate-light adapted LH2 complex (ML-LH2) from Rhodopseudomonas palustris containing Cars with different numbers of conjugated double bonds. Following pulsed photo-excitation at 590 nm at room temperature, rapid spectral equilibration was observed either as a red shift of the isosbestic wavelength on a time scale of 0.6-1.0 mus, or as a fast decay in the shorter-wavelength side of the T(n)<--T(1) absorption of Cars with a time constant of 0.5-0.8 mus. Two major spectral components assignable to Cars with 11 and 12 conjugated double bonds were identified. The equilibration was not observed in the ML-LH2 at 77 K, or in the LH2 complex from Rhodobacter sphaeroides G1C containing a single type of Car. The unique spectral equilibration was ascribed to temperature-dependent triplet excitation transfer among different Car compositions. The results suggest that Cars of 11 and 12 conjugated bonds, both in close proximity of BChls, may coexist in an alpha,beta-subunit of the ML-LH2 complex.     

Formation of reaction centers and light-harvesting bacteriochlorophyll-protein complexes in Rhodopseudomonas capsulata

Formation of the photosynthetic apparatus was induced in aerobically grown dark cultures of Rhodopseudomonas capsulata by lowering of the oxygen tension. Besides the wild type strain the carotenoid-less mutant strain A1a+ was investigated. Both strains exhibited initially a decrease of the molar ratio of total bacteriochlorophyll (Bchl) to reaction center (RC) Bchl, followed by an increase. Synthesis of RC-Bchl preceded the synthesis of light-harvesting (LH) Bchl. Activities of photophosphorylation in membrane preparations, isolated from cultures after different periods of incubation at low aeration, decreased on the basis of total Bchl from about 9 to 2 mumole ATP/mumole total Bchl-min, whereas the rate on the basis of RC-Bchl remained constant (about 500 mumole ATP/mumole RC-Bchl-min). Under the same conditions the membrane proteins were labelled with U-14C-protein hydrolysate. Corresponding to RC-Bchl the synthesis of RC-proteins dominated during the first 30 min of incubation at PO2 below 3 mm Hg. After 45-60 min of membrane formation at low aeration the synthesis of LH-complex proteins exceeded the synthesis of RC proteins. The correlations between protein and Bchl synthesis in the sequential formation of RC- and LH-complexes are discussed.     

Spectroscopy on individual light-harvesting 1 complexes of Rhodopseudomonas acidophila

In this paper the fluorescence-excitation spectra of individual LH1-RC complexes (Rhodopseudomonas acidophila) at 1.2 K are presented. All spectra show a limited number of broad bands with a characteristic polarization behavior, indicating that the excitations are delocalized over a large number of pigments. A significant variation in the number of bands, their bandwidths, and polarization behavior is observed. Only 30% of the spectra carry a clear signature of delocalized excited states of a circular structure of the pigments. The large spectral variety suggests that besides site heterogeneity also structural heterogeneity determines the optical spectrum of the individual LH1-RC complexes. Further research should reveal if such heterogeneity is a native property of the complex or induced during the experimental procedures.     

Dynamics of excitation energy transfer in the LH1 and LH2 light-harvesting complexes of photosynthetic bacteria.

Photosynthetic light harvesting is a unique life process that occurs with amazing efficiency. Since the discovery of the structure of the bacterial peripheral light-harvesting complex (LH2), this process has been studied using a variety of advanced laser spectroscopic methods. We are now in a position to discuss the physical origins of excitation energy transfer and trapping in the LH2 and LH1 antennae of photosynthetic purple bacteria. We demonstrate that the time evolution of the state created by the light is determined by the combined action of excitonic pigment-pitment interactions, energetic disorder, and coupling to nuclear motion in a pigment-protein complex. A quantitative fit of experimental data using Redfield theory allowed us to determine the pathways and time scales of exciton and vibrational relaxation and analyze separately different contributions to the measured transient absorption dynamics. Furthermore, these dynamics were observed to be strongly dependent on the excitation wavelength. A numerical fit of this dependence turns out to be extremely critical to a variation of the structure and disorder parameters and, therefore, can be used as a test for different antenna models (disordered ring, elliptical deformations, correlated disorder, etc.). The calculated equilibration dynamics in the exciton basis allow a visualization of the exciton motion using a density matrix picture in real space.     

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.     

Singlet-triplet fission of carotenoid excitation in light-harvesting LH2 complexes of purple phototrophic bacteria

The current generally accepted structure of light-harvesting LH2 complexes from purple phototrophic bacteria conflicts with the observation of singlet-triplet carotenoid excitation fission in these complexes. In LH2 complexes from the purple bacterium Allochromatium minutissimum, a drop in the efficiency of carotenoid triplet generation is demonstrated, which correlates with the extent of selective photooxidation of bacteriochlorophylls absorbing at ～850 nm. We conclude that singlet-triplet fission of carotenoid excitation proceeds with participation of these excitonically coupled bacteriochlorophylls. In the framework of the proposed mechanism, the contradiction between LH2 structure and photophysical properties of carotenoids is eliminated. The possibility of singlet-triplet excitation fission involving a third mediator molecule was not considered earlier.     

沼泽红假单胞菌光合色素的分离、组成分析与光稳定性

【目的】探求光对不产氧光合细菌类胡萝卜素(Car)和细菌叶绿素a(BChl a)稳定性的影响规律。【方法】以沼泽红假单胞菌CQV97为材料,采用硅胶柱层析和HPLC方法进行Car和BChl a组分的纯化和成分分析,采用吸收光谱法研究Car和BChl a组分的光稳定性。【结果】在Car和BChl a组分分离过程中,Car组分回收率高且稳定,而BChl a回收率波动性较大。Car组分中含有6种螺菌黄质系Car和极少量(0.25%)的细菌脱镁叶绿素a。BChl a组分中包含BChl aGG、BChl aDHGG、BChl aTHGG和BChl aP4种成分。Car和BChl a组分在黑暗条件下非常稳定。2 000 lx白炽灯、日光灯和自然光照射时,Car在70 min内非常稳定,但对紫外光敏感,半衰期为11.15 min,BChl a组分对白炽灯、日光灯、自然光和紫外灯的光降解速率常数(min–1)分别为0.169 8、0.028 9、0.213 9和0.026 4,半衰期(min)分别为4.47、29.68、4.20和26.19。【结论】一步硅胶柱层析可同时得到Car和BChl a纯组分。Car对白光相对稳定,对紫外光不稳定。BChl光稳定性很差,分离过程中短期见光是导致BChl a回收率波动性较大的原因,光降解过程中产生了相对稳定的中间产物。该研究结果为光合色素的精制、功能研究和应用提供了理论依据。     

Production of hydrogen gas from light and the inorganic electron donor thiosulfate by Rhodopseudomonas palustris

A challenge for photobiological production of hydrogen gas (H(2)) as a potential biofuel is to find suitable electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron donor for nitrogenase-catalyzed H(2) production by the purple nonsulfur phototrophic bacterium (PNSB) Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate and bicarbonate and produced H(2) when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions). In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate to H(2). H(2) production with acetate and succinate as electron donors was less efficient (40 to 60%), partly because nongrowing cells excreted the intermediary metabolite α-ketoglutarate into the culture medium. The fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of H(2) production. The possibility to use thiosulfate expands the range of electron-donating compounds for H(2) production by PNSBs beyond biomass-based electron donors.     

A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes

Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment-protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption-fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption-fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, -carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity. Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity. Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.