Chromophore-protein interactions and the function of the photosynthetic reaction center: a molecular dynamics study.
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The coupling between electron transfer and protein structure and dynamics in the photosynthetic reaction center of Rhodopseudomonas viridis is investigated. For this purpose molecular dynamics simulations of the essential portions (a segment of 5797 atoms) of this protein complex have been carried out. Electron transfer in the primary event is modeled by altering the charge distributions of the chromophores according to quantum chemical calculations. The simulations show (i) that fluctuations of the protein matrix, which are coupled electrostatically to electron transfer, play an important role in controlling the electron transfer rates and (ii) that the protein matrix stabilizes the separated electron pair state through rapid (200 fs) and temperature-independent dielectric relaxation. The photosynthetic reaction center resembles a polar liquid in that the internal motions of the whole protein complex, rather than only those of specific side groups, contribute to i and ii. The solvent reorganization energy is about 4.5 kcal/mol. The simulations indicate that rather small structural rearrangements and changes in motional amplitudes accompany the primary electron transfer. (+info)
Localization of chromatophore proteins of Rhodobacter sphaeroides. I. Rapid Ca(2+)-induced fusion of chromatophores with phosphatidylglycerol liposomes for proteinase delivery to the luminal membrane surface.
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A protease delivery system was developed for the exclusive and controlled digestion of proteins exposed at the morphological inside (periplasmic surface) of Rhodobacter sphaeroides chromatophores. In this procedure, proteinase K is encapsulated within large unilamellar liposomes which are fused to the chromatophores in the presence of Ca2+ ions. The liposomes were prepared by a detergent dialysis procedure from native phosphatidylglycerol and found to undergo rapid bilayer fusion with purified chromatophore preparations above a threshold concentration of 12.5 mM CaCl2. The fusion process was complete within 10 min at 35 mM Ca2+ with about 80% of the pigment located in the fusion products. Electron micrographs of freeze-fracture replicas confirmed the intermixing of the lipid bilayers and the unilamellar structure of the fused membrane vesicles. The procedure did not affect the labile B800 chromophore of the B800-850 antenna complex, but reduced slightly the absorption due to the B875 core antenna. Emission from both light-harvesting complexes was increased in the fused membranes, suggesting a partial dissociation of photosynthetic units in the expanded bilayer. The results, together with those presented in the following paper (Theiler, R., and Niederman, R. A. (1991) J. Biol. Chem. 266, 23163-23168), demonstrate that this new method fulfills the stringent requirements for a successful delivery of macromolecules to the chromatophore interior. (+info)
Candidatus Chloracidobacterium thermophilum: an aerobic phototrophic Acidobacterium.
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Only five bacterial phyla with members capable of chlorophyll (Chl)-based phototrophy are presently known. Metagenomic data from the phototrophic microbial mats of alkaline siliceous hot springs in Yellowstone National Park revealed the existence of a distinctive bacteriochlorophyll (BChl)-synthesizing, phototrophic bacterium. A highly enriched culture of this bacterium grew photoheterotrophically, synthesized BChls a and c under oxic conditions, and had chlorosomes and type 1 reaction centers. "Candidatus Chloracidobacterium thermophilum" is a BChl-producing member of the poorly characterized phylum Acidobacteria. (+info)
Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle.
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The photosynthetic unit (PSU) of purple photosynthetic bacteria consists of a network of bacteriochlorophyll-protein complexes that absorb solar energy for eventual conversion to ATP. Because of its remarkable simplicity, the PSU can serve as a prototype for studies of cellular organelles. In the purple bacterium Rhodobacter sphaeroides the PSU forms spherical invaginations of the inner membrane, approximately 70 nm in diameter, composed mostly of light-harvesting complexes, LH1 and LH2, and reaction centers (RCs). Atomic force microscopy studies of the intracytoplasmic membrane have revealed the overall spatial organization of the PSU. In the present study these atomic force microscopy data were used to construct three-dimensional models of an entire membrane vesicle at the atomic level by using the known structure of the LH2 complex and a structural model of the dimeric RC-LH1 complex. Two models depict vesicles consisting of 9 or 18 dimeric RC-LH1 complexes and 144 or 101 LH2 complexes, representing a total of 3,879 or 4,464 bacteriochlorophylls, respectively. The in silico reconstructions permit a detailed description of light absorption and electronic excitation migration, including computation of a 50-ps excitation lifetime and a 95% quantum efficiency for one of the model membranes, and demonstration of excitation sharing within the closely packed RC-LH1 dimer arrays. (+info)
Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: size dispersion and ATP synthase distribution.
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The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle. (+info)
Polarographic studies in presence of Triton X-100 on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum.
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Polarographic studies on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum were carried out at 24 degrees. 1. Using a carbon-paste electrode as the working electrode, polarographic waves characteristic of oxidation-reduction components were observed in the presence, but not in the absence of Triton X-100; these waves were therefore measured in the presence of the detergent. 2. At least two kinds of oxidation-reduction components were detectable, having different half-wave potentials (E1/2); at pH 7, one had an E1/2 value of +275 mV (POC+275) and the other had a value of +60 mV (POC+60). 3. POC+275 was reduced by succinate and by NADH. Both reductions were almost completely inhibited by antimycin A, which hardly affected the reductions of ubiquinone-10 by succinate and by NADH. Most POC+275 molecules were not reduced by the substrates when quinones were extracted from the chromatophores, and the reductions were mostly restored when ubiquinone-10 was re-added. This indicates that POC+275 is functional between ubiquinone-10 and cytochrome c2 in the electron transport system. 4. POC+60 was reduced by succinate, but hardly at all by NADH. The reduction of POC+60 was not influenced either by the addition of antimycin A or by the extraction of quinones. This suggests that POC+60 is functional in the process from succinate dehydrogenase [EC 1.3.99.1] to ubiquinone-10 in the electron transport system. 5. Of the POC+275 reducible by dithionite, approximately 70% could be reduced in the absence of Triton X-100, provided that the potential of the working electrode immersed in chromatophore suspensions was set at potentials of 0 mV or lower and that the electrochemical reaction was carried out at pH 7.5. When the potential of the electrode was set at +50 mV (the same as the E1/2 value of ubiquinone-10 bound with chromatophores), and the suspension was allowed to stand for various lengths in the presence of the detergent, it was found that approximately half of the electrochemically reducible POC+275 was rapidly reduced, followed by a slow reduction. The discrepancy in the oxidation-reduction equilibrium on the basis of the E1/2 values of ubiquinone-10 and POC+275 is discussed. (+info)
Purification and properties of the H(+)-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus.
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1. H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H(+)-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive. (+info)
Intrinsic curvature properties of photosynthetic proteins in chromatophores.
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