Reaction Center Pigment-protein Complexes Research Articles

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.

Prosthecate aerobic anoxygenic phototrophs Photocaulis sulfatitolerans gen. nov. sp. nov. and Photocaulis rubescens sp. nov. isolated from alpine meromictic lakes in British Columbia, Canada.

Seven Gram-negative flagellated and subsequent prosthecate bacteria were isolated from meromictic Mahoney Lake and Blue Lake in British Columbia, Canada. Each became pink-red after 1-2weeks of incubation, containing bacteriochlorophyll a incorporated into light harvesting and reaction center pigment-protein complexes. They did not grow anaerobically under illuminated conditions, supporting their identification as obligate aerobic anoxygenic phototrophs (AAP). All isolates preferred high salinity and BL14T tolerated up to 6.5% NaCl or 16.0% Na2SO4. In addition to phenotypic differences, analysis of 16S rRNA gene sequences found both strains BL14T and ML37T were related to Alkalicaulis satelles, G-192T by 98.41 and 98.84%, respectively, and distantly associated to members of the non-phototrophic genus Glycocaulis profundi, ZYF765T (95.59 and 95.36%, respectively) within the newly recognized Maricaulales order of α-Proteobacteria. BL14T and ML37T contained photosynthetic operons of 46,143 and 46,315bp, where genes of BL14T were uniquely split into two distal operons. Furthermore, A. satelles was not originally published as an AAP, but was also found in this work to contain a similar 45,131bp fragment. The distinct morphological features, physiological traits and genomic analysis including average nucleotide identity and digital DNA:DNA hybridization of circularized genomes supported the proposal of new genus and species Photocaulis sulfatitolerans gen. nov. sp. nov., type strain BL14T and Photocaulis rubescens sp. nov. type strain ML37T.

Physiological and transcriptomic analysis highlight key metabolic pathways in relation to drought tolerance in Rhododendron delavayi.

Rhododendron delavayi is an alpine evergreen ornamental plant, but water shortage limits its growth and development in urban gardens. However, the adaptive mechanism of alpine evergreen rhododendrons to drought remains unclear. Here, a water control experiment was conducted to study the physiological and transcriptomic response of R. delavayi to drought. The drought treatment for 9days decreased photosynthetic rate, induced accumulation of reactive oxygen species (ROS), and damaged chloroplast ultrastructure of R. delavayi. However, the photosynthetic rate quickly recovered to the level before treatment when the plants were re-watered. De novo assembly of RNA-Seq data generated 86,855 unigenes with an average length of 1870bp. A total of 22,728 differentially expressed genes (DEGs) were identified between the control and drought plants. The expression of most DEGs related to photosynthesis were down-regulated during drought stress, and were up-regulated when the plants were re-watered, including the DEGs encoding subunits of light-harvesting chlorophyll-protein complex, photosystem II and photosystem I reaction center pigment-protein complexes, and photosynthetic electron transport. The expressions of many DEGs related to signal transduction, flavonoid biosynthesis and antioxidant activity were also significantly affected by drought stress. The results indicated that the response of R. delavayi to drought involved multiple physiological processes and metabolic pathways. Photosynthetic adjustment, ROS-scavenging system, abscisic acid and brassinosteroid signal transduction pathway may play important roles to improve drought tolerance of R. delavayi. Our findings provided valuable information for understanding the mechanisms of drought tolerance employed by Rhododendron species.

Enhanced Photocurrent Generation by Photosynthetic Bacterial Reaction Centers through Molecular Relays, Light-Harvesting Complexes, and Direct Protein–Gold Interactions

The utilization of proteins as nanodevices for solar cells, bioelectronics, and sensors generally necessitates the transfer of electrons to or from a conducting material. Here we report on efforts to maximize photocurrent generation by bacterial photosynthetic reaction center pigment-protein complexes (RCs) interfaced with a metal electrode. The possibility of adhering RCs to a bare gold electrode was investigated with a view to minimizing the distance for electron tunneling between the protein-embedded electron-transfer cofactors and the metal surface. Substantial photocurrents were achieved despite the absence of coating layers on the electrode or engineered linkers to achieve the oriented deposition of RCs on the surface. Comparison with SAM-covered gold electrodes indicating enhanced photocurrent densities was achieved because of the absence of an insulating layer between the photoactive pigments and the metal. Utilizing RCs surrounded by light-harvesting 1 complex resulted in higher photocurrents, surprisingly not due to enhanced photoabsorption but likely due to better surface coverage of uniformly oriented RC-LH1 complexes and the presence of a tetraheme cytochrome that could act as a connecting wire. The introduction of cytochrome-c (cyt-c) as a molecular relay also produced increases in current, probably by intercalating between the adhered RCs or RC-LH1 complexes and the electrode to mediate electron transfer. Varying the order in which components were introduced to the electrode indicated that dynamic rearrangements of RCs and cyt-c occurred at the bare metal surface. An upper limit for current generation could not be detected within the range of the illumination power available, with the maximum current density achieved by RC-LH1 complexes being on the order of 25 μA/cm(2). High currents could be generated consecutively for several hours or days under ambient conditions.

Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment–protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10 μA/cm 2 for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.

Charge stabilization in reaction center protein investigated by optical heterodyne detected transient grating spectroscopy

Photosynthetic reaction center (RC) pigment protein complex converts the free energy of light into chemical potential of charge pairs with extremely high efficiency. A transient phase in the absorption spectrum in the sub-millisecond time scale is expected to be especially important to examine the conformational gating model of the Q(A)Q(B) to Q(A)Q(B) (here Q(A )and Q(B) are the primary and secondary quinone type electron acceptors, respectively) electron transport. Essential kinetic components at few tens of microseconds scale and at around 200 microseconds have been suggested. We investigated the conformation change of RCs using heterodyne detection of the laser-induced transient grating method. An about 25 microseconds dynamics was observed, which coincides with the one described by the conformational gating model and possibly related to the nonadiabatic intrinsic Q(A)Q(B) to Q(A)Q(B) electron transport. The relative intensity of this component decreased with increasing quinone concentration indicating an initial (P+Q(A))1 or a relaxed (P+Q(A))2 conformational substate. We did not find the decay component at few hundreds of microseconds time scale indicating that there is no large displacement in the RC structure if Q(B) is present. The diffusion coefficient of the RC/LDAO detergent micelles calculated from the kinetic component was D = 3.8 x 10(-11 ) m2/s that agrees fairly well with the number estimated from the Einstein-Stokes relationship, and relates to a hydrodynamic diameter of 11.4 nm of the RC in LDAO micellar solution.

Towards a New Taxonomy of Photosynthetic Bacteria: ADMR‐Monitored Triplet Difference Spectroscopy of Reaction Center Pigment–Protein Complexes

AbstractLow‐temperature (1.2 K) triplet‐minus‐singlet absorbance difference spectra of a number of photosynthetic bacteria, including members of almost all genera with the exception of Heliobacteriaceae, have been recorded by absorbance‐detected magnetic resonance (ADMR). The spectra of the purple bacteria fall into two distinct classes. It is argued that these classes reflect the extent of delocalization of the primary donor triplet state over its two bacteriochlorophylls. The spectra from the green sulfur bacteria form a third class. In some cases, the bleaching of the long‐wavelength primary donor absorption is split, or shows a strong shoulder at lower energy. This phenomenon is attributed to an intrinsic heterogeneity of the reaction center with regard to the precise configuration of the primary donor dimer. It is argued that the zero‐field splitting parameters and the triplet‐minus‐singlet difference spectra may be used as a taxonomic tool.

Biological consequences of segmental alterations in mRNA stability: effects of deletion of the intercistronic hairpin loop region of the Rhodobacter capsulatus puf operon.

It has been proposed that intercistronic stem and loop structures located in the puf operon of the photosynthetic bacterium Rhodobacter capsulatus account for segmental differences in transcript stability and consequently, the differential expression of the B870 and reaction center (RC) proteins encoded by puf. We report here that deletion of these structures leads to a failure to detect as discrete fragments the B870-encoding 0.49 kb and 0.50 kb mRNA segments located upstream from the site of the hairpins. The absence of these stable transcript fragments is associated with altered stoichiometry of the B870 and RC pigment-protein complexes in the bacterial intracytoplasmic membrane and a decreased rate of cell growth under photosynthetic conditions. These results support the view that the hairpin loop structures of the puf intercistronic region function in vivo to impede exoribonucleolytic degradation of upstream mRNA and establish that segmental variations in mRNA stability have a biologically important role in regulating the expression of puf operon genes.

Pigment-protein complexes of purple photosynthetic bacteria: an overview.

A minireview of antenna and reaction center pigment-protein complexes of purple bacteria is presented. Advances in our knowledge of their structure and composition during the past 3 yr are emphasized and some new thoughts are introduced.

The primary charge separation, cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations. Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex. The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840 + was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed. At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a 680 and carotenoid pigments and a dampening of the sequence of O 2 evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination. Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.