Carotenoid-bacteriochlorophyll energy transfer in LH2 complexes studied with 10-fs time resolution.
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In this report, we present a study of carotenoid-bacteriochlorophyll energy transfer processes in two peripheral light-harvesting complexes (known as LH2) from purple bacteria. We use transient absorption spectroscopy with approximately 10 fs temporal resolution, which is necessary to observe the very fast energy relaxation processes. By comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it has been possible to directly evaluate their energy transfer efficiency to the bacteriochlorophylls. In the case of okenone in the LH2 complex from Chromatium purpuratum, we obtained an energy transfer efficiency of etaET2=63+/-2.5% from the optically active excited state (S2) and etaET1=61+/-2% from the optically dark state (S1); for rhodopin glucoside contained in the LH2 complex from Rhodopseudomonas acidophila these values become etaET2=49.5+/-3.5% and etaET1=5.1+/-1%. The measurements also enabled us to observe vibrational energy relaxation in the carotenoids' S1 state and real-time collective vibrational coherence initiated by the ultrashort pump pulses. Our results are important for understanding the dynamics of early events of photosynthesis and relating it to the structural arrangement of the chromophores. (+info)
Localization of chromatophore proteins of Rhodobacter sphaeroides. II. Topography of cytochrome c1 and the Rieske iron-sulfur protein as determined by proteolytic digestion of the outer and luminal membrane surfaces.
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Proteinase K was used to degrade membrane proteins exposed at the outer (cytoplasmic) and inner (periplasmic) surface of sealed, uniformly oriented chromatophore vesicles of Rhodobacter sphaeroides. Exclusive and controlled digestion of the chromatophore interior was achieved after Ca(2+)-induced fusion with large unilamellar phosphatidylglycerol liposomes containing microencapsulated enzyme. Reaction center subunit H, which served as a marker for the outer surface, was degraded to a slightly smaller product in chromatophores. This protein remained intact after liposome-chromatophore fusion, suggesting that the intermixing of lipid bilayers proceeded without significant leakage of the aqueous vesicle contents. In contrast, while cytochrome c1 was not affected in chromatophores, 70-75% was degraded within 60 min after liposome-chromatophore fusion. These results support an arrangement in which the bulk of this protein, including the mesoheme component and active site residues, faces the periplasmic side of the membrane. Although current functional models for the cytochrome bc1 complex predict that the Rieske iron-sulfur center interacts with cytochrome c1 in the periplasm, the iron-sulfur protein resisted proteolytic attack in the liposome-chromatophore fusion products under conditions that caused extensive degradation of cytochrome c1. Two cleavage products of the iron-sulfur protein were observed after the digestion of chromatophores, suggesting both a heterogeneity in the population of this protein and the exposure of at least part of its molecular mass to the cytoplasm. (+info)
A hybrid of the transhydrogenases from Rhodospirillum rubrum and Mycobacterium tuberculosis catalyses rapid hydride transfer but not the complete, proton-translocating reaction.
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All transhydrogenases appear to have three components: dI, which binds NAD(H), and dIII, which binds NADP(H), protrude from the membrane, and dII spans the membrane. However, the polypeptide composition of the enzymes varies amongst species. The transhydrogenases of Mycobacterium tuberculosis and of Rhodospirillum rubrum have three polypeptides. Sequence analysis indicates that an ancestral three-polypeptide enzyme evolved into transhydrogenases with either two polypeptides (such as the Escherichia coli enzyme) or one polypeptide (such as the mitochondrial enzyme). The fusion steps in each case probably led to the development of an additional transmembrane helix. A hybrid transhydrogenase was constructed from the dI component of the M. tuberculosis enzyme and the dII and dIII components of the R. rubrum enzyme. The hybrid catalyses cyclic transhydrogenation but not the proton-translocating, reverse reaction. This shows that nucleotide-binding/release at the NAD(H) site, and hydride transfer, are fully functional but that events associated with NADP(H) binding/release are compromised. It is concluded that sequence mismatch in the hybrid prevents a conformational change between dI and dIII which is essential for the step accompanying proton translocation. (+info)
Modulation of proton pumping efficiency in bacterial ATP synthases.
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The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled. (+info)
Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition.
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Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state. (+info)
Transmission electron microscopic study on supramolecular nanostructures of bacteriochlorophyll self-aggregates in chlorosomes of green photosynthetic bacteria.
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Supramolecular nanostructures of bacteriochlorophyll (BChl) self-aggregates in major light-harvesting complexes (chlorosomes) of green photosynthetic bacteria were successfully observed by freeze-fracture transmission electron microscope. Rod-shaped nanostructures with approximately 10 nm in diameter could be visualized in three types of green sulfur bacteria (Chlorobium). Diameter of the rod-shaped nanostructures in Chlorobium chlorosomes was independent of the molecular structures of their light-harvesting pigments, namely BChl-c or d. In contrast, chlorosomes of the green filamentous bacterium Chloroflexus aurantiacus had rod-shaped nanostructures with approximately 5 nm in diameter. The present results support that BChl self-aggregates in chlorosomes form rod-shaped nanostructures called rod-elements with approximately 10- and 5-nm diameters for Chlorobium and Chloroflexus, respectively. (+info)
Role of ubiquinone-10 in electron transport system of chromatophores from Rhodospirillum rubrum.
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The role of ubiquinone-10 in the activities for the reduction of free cytochrome c2 and bound cytochrome cc' by succinate was studied with chromatophores from a blue-green mutant (G-9) of Rhodospirillum rubrum. 1. By a single extraction with isooctane, approximately 90% of ubiquinone-10 was easily removed from the chromatophores. In the extracted chromatophores, the activity for succinate-cytochrome c2 reduction decreased to 5-10% of the original activity. This depressed activity was mostly restored by adding ubiquinone-10. The remaining quinone was hardly extractable, even by repeated extractions. With repeatedly extracted chromatophores, the activity for succinate-cytochrome c2 reduction was mostly restored to the same extent as with once-extracted chromatophores, whereas the extent of inhibtion of the activity by antimycin A gradually fell. 2. In isooctane-extracted chromatophores, the activity for the reduction of bound cytochrome cc' by succinate under anaerobic conditions decreased to 35 to 95% of the original level. With chromatophores in which the remaining activity was as low as 40% of the original level, the activity was partially restored by adding ubiquinone-10, but this was not the case with chromatophores in which the remaining activity was higher than approximately 50% of the original level. (+info)
Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum.
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In an earlier investigation (Shanmugam, K. T., Buchanan, B. B., and Arnon, D. I. (1972) Biochim. Biophys. Acta 256, 477-486) the extraction of ferredoxin from Rhodospirillum rubrum cells with the aid of a detergent (Triton X-100) and acetone revealed the existence of two types of ferredoxin (I and II) and led to the conclusion that both are membrane-bound. In the present investigation, ferredoxin and acid-labile sulfur analyses of photosynthetic membranes (chromatophores) and soluble protein extracts of the photosynthetic bacteria R. rubrum and Rhodopseudomonas spheroides showed that ferredoxins I and II are primarily components of the soluble protein fraction. After their removal, washed R. rubrum chromatophores were found to contain a considerable amount of tightly bound iron-sulfur protein(s), as evidenced by acid-labile sulfur and electron paramagnetic resonance analyses. Thus, like all other photosynthetic cells examined to date, R. rubrum cells contain both soluble ferredoxins and iron-sulfur proteins tightly bound to photosynthetic membranes. The molecular weights of ferredoxins I and II from photosynthetically grown R. rubrum cells were found to be 8,800 and 14,500, respectively. Using these molecular weights, the molar extinction coefficients at 390 nm for ferredoxins I and II were determined to be 30.3 and 17.2 mM-1 CM-1, respectively. Ferredoxin I contains 8 non-heme iron and 8 acid-labile sulfur atoms per molecule; ferredoxin II contains 4 non-heme iron and 4 acid-labile sulfur atoms per molecule. Ferredoxin I was found only in photosynthetically grown cells whereas ferredoxin II was present in both light- and dark-grown cells. Ferredoxin II from both light- and dark-grown cells has the same molecular weight (14,500) and absorption spectrum and has 4 iron and 4 acid-labile sulfur atoms per molecule. Low temperature electron paramagnetic resonance spectra of oxidized and photoreduced ferredoxins I and II from R. rubrum were recorded. The EPR spectrum of oxidized ferredoxin II exhibited a single resonance line at g = 2.012. Oxidized ferredoxin I, however, exhibited a spectrum that may arise from the superimposition of two resonance lines near g = 2.012. Photoreduced ferredoxin II displayed a rhombic EPR spectrum with a g value of 1.94. Photoreduced ferredoxin I exhibited a similar EPR spectrum at a temperature of 16 K, but when the temperature was lowered to 4.5 K the spectrum of ferredoxin I changed. This temperature-dependent spectrum may result from a weak spin-spin interaction between two iron-sulfur clusters. These results are consistent with the conclusion that R. rubrum ferredoxins I and II are, respectively, 8 iron/8 sulfur and 4 iron/4sulfur proteins. (+info)