Exciton delocalization in the B808-866 antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy.

A model of pigment organization in the B808-866 bacteriochlorophyll a antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed. The building block of the antenna was assumed to be structurally similar to that of the B800-850 light-harvesting 2 (LH2) antenna of purple bacteria and to have the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers, where N = 24 or 32. We have shown that the Qy transition dipoles of BChl808 and BChl866 molecules form the angles 43 degrees +/- 3 degrees and 8 degrees +/- 4 degrees, respectively, with the plane of the corresponding rings. Using the exciton model, we have obtained a quantitative fit of the pump-probe spectra of the B866 and B808 bands. The anomalously high bleaching value of the B866 band with respect to the B808 monomeric band provided the direct evidence for a high degree of exciton delocalization in the BChl866 ring antenna. The coherence length of the steady-state exciton wave packet corresponds to five or six BChl866 molecules at room temperature.     

Energy transfers in the B808-866 antenna from the green bacterium Chloroflexus aurantiacus.

Energy transfers within the B808-866 BChl a antenna in chlorosome-membrane complexes from the green photosynthetic bacterium Chloroflexus aurantiacus were studied in two-color pump-probe experiments at room temperature. The steady-state spectroscopy and protein sequence of the B808-866 complex are reminiscent of well-studied LH2 antennas from purple bacteria. B808-->B866 energy transfers occur with approximately 2 ps kinetics; this is slower by a factor of approximately 2 than B800-->B850 energy transfers in LH2 complexes from Rhodopseudomonas acidophila or Rhodobacter sphaeroides. Anisotropy studies show no evidence for intra-B808 energy transfers before the B808-->B866 step; intra-B866 processes are reflected in 350-550 fs anisotropy decays. Two-color anisotropies under 808 nm excitation suggest the presence of a B808-->B866 channel arising either from direct laser excitation of upper B866 exciton components that overlap the B808 absorption band or from excitation of B866 vibronic bands in nontotally symmetric modes.     

Exciton dynamics in the chlorosomal antenna of the green bacterium Chloroflexus aurantiacus: experimental and theoretical studies of femtosecond pump-probe spectra

Femtosecond absorption difference spectra were measured for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus at room temperature. Using the relative difference absorption of the oligomeric BChl c and monomeric BChl a bands, the size of a unit BChl c aggregate as well as the exciton coherence size were estimated for the chlorosomal BChl c antenna under study. A quantitative fit of the data was obtained within the framework of the exciton model proposed before [Fetisova et al. (1996) Biophys J 71: 995–1010]. The size of the antenna unit was found to be 24 exciton-coupled BChl c molecules. The anomalously high bleaching value of the oligomeric B740 band with respect to the monomeric B795 band provided the direct evidence for a high degree of exciton delocalization in the chlorosomal B740 BChl c antenna. The effective delocalization size of individual exciton wavefunctions (the thermally averaged inverse participation ratio) in the chlorosomal BChl c antenna is 9.5, whereas the steady-state wavepacket corresponds to the coherence size (the inverse participation ratio of the density matrix) of 7.4 at room temperature.This revised version was published online in October 2005 with corrections to the Cover Date.     

The complete amino acid sequence of the bacteriochlorophyll c binding polypeptide from chlorosomes of the green photosynthetic bacterium Chloroflexus aurantiacus

The BChlc polypeptide was isolated from chlorosomes of the green bacterium Chloroflexus aurantiacus on Sephadex LH-60. The complete amino acid sequence of this 5.6 kDa polypeptide (51 amino acid residues) was determined. Most probably the 5.6 kDa polypeptide forms an α-helix between Trp 5 and Ile 42 with an asymmetrical (bipolar) distribution of polar amino acid residues along the helix axis: (i) At one side of the α-helix 5 Gln and 2 Asn residues are the possible binding sites for 7 BChlc molecules, (ii) On the other side Ser, Thr, His residues seem to be polypeptide-polypeptide interaction sites within the BChlc-protein complexes. It appears that the BChl-protein complex (chlorosome subunit, 5.2 × 6 nm) composed of 12 5.6 kDa polypeptides corresponds to the 'globular units' found by electron microscopy within the chlorosomes.     

Exogenous quinones inhibit photosynthetic electron transfer in Chloroflexus aurantiacus by specific quenching of the excited bacteriochlorophyll c antenna.

In the photosynthetic green filamentous bacterium Chloroflexus aurantiacus, excitation energy is transferred from a large bacteriochlorophyll (BChl) c antenna via smaller BChl a antennas to the reaction center. The effects of substituted 1,4-naphthoquinones on BChl c and BChl a fluorescence and on flash-induced cytochrome c oxidation were studied in whole cells under aerobic conditions. BChl c fluorescence in a cell suspension with 5.4 microM BChl c was quenched to 50% by addition of 0.6 microM shikonin ((R)-2-(1-hydroxy-4-methyl-3-pentenyl)-5,8-dihydroxy-1, 4-naphthoquinone), 0.9 microM 5-hydroxy-1,4-naphthoquinone, or 4 microM 2-acetyl-3-methyl-1,4-naphthoquinone. Between 25 and 100 times higher quinone concentrations were needed to quench BChl a fluorescence to a similar extent. These quinones also efficiently inhibited flash-induced cytochrome c oxidation when BChl c was excited, but not when BChl a was excited. The quenching of BChl c fluorescence induced by these quinones correlated with the inhibition of flash-induced cytochrome c oxidation. We concluded that the quinones inhibited electron transfer in the reaction center by specifically quenching the excitation energy in the BChl c antenna. Our results provide a model system for studying the redox-dependent antenna quenching in green sulfur bacteria because the antennas in these bacteria inherently exhibit a sensitivity to O(2) similar to the quinone-supplemented cells of Cfx. aurantiacus.     

Polarized fluorescence of aggregated bacteriochlorophyll c and baseplate bacteriochlorophyll a in single chlorosomes isolated from Chloroflexus aurantiacus

The polarization anisotropy of fluorescence from single chlorosomes isolated from a green filamentous bacterium, Chloroflexus aurantiacus, was measured using a confocal laser microscope at 13 K. Each single chlorosome that is floating in a frozen solvent exhibited strong polarization anisotropy of fluorescence. We calculated the degrees of fluorescence polarization for 51 floating single chlorosomes. The value ranged from 0.1 to 0.76 for the BChl-c aggregate in the core chlorosomes and from 0 to 0.4 for the energy acceptor BChl-a in the baseplate protein in the outer membrane. The shifts in polarization angles between the two emission bands were distributed over all the possible values with a sharp peak around 90 degrees , suggesting the perpendicular orientation between the transition dipoles of the fluorescence emission from the BChl-c aggregate and that from BChl-a. A simulation assuming a random orientation of chlorosomes reproduced the experimental results exactly. The analysis further indicated the appreciable contribution of the transition dipole of BChl-c that has an orientation perpendicular to the major polarization axis in each chlorosome. Small values of the degrees of polarization implied the BChl-a transition dipole to be somewhat tilted with respect to the normal of the cytoplasmic membrane to which chlorosomes are attached. These conclusions can be obtained only by observing the fluorescence of single chlorosomes.     

Antenna organization in green photosynthetic bacteria. 1. Oligomeric bacteriochlorophyll c as a model for the 740 nm absorbing bacteriochlorophyll c in Chloroflexus aurantiacus chlorosomes

Bacteriochlorophyll (BChl) c was extracted from Chloroflexus aurantiacus and purified by reverse-phase high-pressure liquid chromatography. This pigment consists of a complex mixture of homologues, the major component of which is 4-ethyl-5-methylbacteriochlorophyll c stearyl ester. Unlike previously characterized BChls c, the pigment from C. aurantiacus is a racemic mixture of diastereoisomers with different configurations at the 2a chiral center. Diluting a concentrated methylene chloride solution of BChl c with hexane produces an oligomer with absorption maxima at 740-742 and at 460-462 nm. Both the absorption spectrum and the fluorescence emission spectrum (maximum at 750 nm) of this oligomer closely match those of BChl c in chlorosomes. Further support for this model comes from the ability of alcohols, which disrupt BChl c oligomers by ligating the central Mg atom, to convert BChl c in chlorosomes to a monomeric form when added in low concentrations. The lifetime of fluorescence from the 740 nm absorbing BChl c oligomer is about 80 ps. Although exciton quenching might be unusually fast in the in vitro BChl c oligomer because of its large size and/or the presence of minor impurities, this result suggests that energy transfer from the BChl c antenna in chlorosomes must be very fast if it is to be efficient.     

Association of bacteriochlorophyll a with the CsmA protein in chlorosomes of the photosynthetic green filamentous bacterium Chloroflexus aurantiacus.

The protein assumed to be associated with bacteriochlorophyll (BChl) a in chlorosomes from the photosynthetic green filamentous bacterium Chloroflexus aurantiacus was investigated by alkaline treatment, proteolytic digestion and a new treatment using 1-hexanol, sodium cholate and Triton X-100. Upon alkaline treatment, only the 5.7 kDa CsmA protein was removed from the chlorosomes among six proteins detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, concomitantly with the disappearance of BChl a absorption at 795 nm. Trypsin treatment removed two proteins with molecular masses of 11 and 18 kDa (CsmN and CmsM), whereas the spectral properties of BChl a and BChl c were not changed. By the new hexanol-detergent (HD) treatment, most BChl c and all of the detected proteins except CsmA were removed from the chlorosomes without changing the BChl a spectral properties. Subsequent proteinase K treatment of these HD-treated chlorosomes caused digestion of CsmA and a simultaneous decrease of the BChl a absorption band. Based on these results, we suggest that CsmA is associated with BChl a in the chlorosomes. This suggestion was supported by the measured stoichiometric ratio of BChl a to CsmA in isolated chlorosomes, which was estimated to be between 1.2 and 2.7 by amino acid analysis of the SDS-PAGE-resolved protein bands.     

Isolation and development of chlorosomes in the green bacterium Chloroflexus aurantiacus.

Freeze-fracture electron microscopy was used to study further the changes in chlorosome structure during the development of the photosynthetic apparatus in Chloroflexus aurantiacus J-10-fl. During development, in response to decreased light intensity or lower oxygen tension, the number of chlorosomes per cell increased. The same conditions also led to a general thickening of chlorosomes but did not affect their length or width. The thickening of the chlorosomes paralleled increases in the bacteriochlorophyll c/bacteriochlorophyll a ratio. Semiaerobic induction of the photosynthetic apparatus did not produce a synchronous assembly of chlorosomes in all cells of a given culture. Even adjacent cells of a single filament showed great variations in the rate and extent of response. Parallel appearance of (i) approximately 5-nm particles (in a lattice configuration) in the membrane attachment site, (ii) the crystalline baseplate material (with a periodicity of approximately 6 nm) adjacent to the membrane attachment site, and (iii) the chlorosome envelope layer preceded addition of longitudinally oriented, rodlike elements (diameter, congruent to 6 m) to the chlorosome core. It is estimated that each chlorosome can funnel energy into approximately 100 reaction centers. Chlorosomes could be isolated by a simple density gradient procedure only from cells grown at low light intensity. A bacteriochlorophyll a species absorbing at 790 nm was associated with isolated chlorosomes. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorosomes showed only a few low-molecular-weight polypeptides (less than 15,000).     

The primary structure of cytochrome c-554 from the green photosynthetic bacterium Chloroflexus aurantiacus.

The complete nucleotide sequence of the cytochrome c-554 gene from the green photosynthetic bacterium Chloroflexus aurantiacus has been determined. The derived amino acid sequence showed that the cytochrome precursor protein consists of 414 residues and contains 4-Cys-X-X-Cys-His- heme binding motifs. The only regions of the cytochrome c-554 sequence that were found to be significantly similar to the sequences of cytochromes from other organisms were the heme binding sites. The highest similarity was found with the heme binding segments in the four-heme reaction center cytochrome subunit from the purple photosynthetic bacterium Rhodopseudomonas viridis. The importance of this similarity for the evolutionary relationship between Chloroflexus and the purple bacteria is discussed.     

Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.     

Exciton dynamics in the chlorosomal antennae of the green bacteria Chloroflexus aurantiacus and Chlorobium tepidum

The energy transfer processes in isolated chlorosomes from green bacteria Chlorobium tepidum and Chloroflexus aurantiacus have been studied at low temperatures (1.27 K) by two-pulse photon echo and one-color transient absorption techniques with approximately 100 fs resolution. The decay of the coherence in both types of chlorosomes is characterized by four different dephasing times stretching from approximately 100 fs up to 300 ps. The fastest component reflects dephasing that is due to interaction of bacteriochlorophylls with the phonon bath, whereas the other components correspond to dephasing due to different energy transfer processes such as distribution of excitation along the rod-like aggregates, energy exchange between different rods in the chlorosome, and energy transfer to the base plate. As a basis for the interpretation of the excitation dephasing and energy transfer pathways, a superlattice-like structural model is proposed based on recent experimental data and computer modeling of the Bchl c aggregates (1994. Photosynth. Res. 41:225-233.) This model predicts a fine structure of the Q(y) absorption band that is fully supported by the present photon echo data.     

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.     

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.     

The complete amino acid sequence of a bacteriochlorophyll a binding polypeptide isolated from the cytoplasmic membrane of the green photosynthetic bacterium Chloroflexus aurantiacus

A polypeptide soluble in organic solvents was isolated from whole membrane fractions of the green thermophilic bacterium Chloroflexus aurantiacus by chromatography on Sephadex LH-60, Whatman DE-32 and Bio Gel P-10. The complete amino acid sequence of this 4.9 kDa polypeptide (44 amino acid residues) was determined. The polypeptide shows a 3-domain structure, similar to the domain structure of the antenna BChI polypeptides of purple photosynthetic bacteria, and sequence homologies (27–39%) to the light-harvesting α-polypeptides of the B870 (890) antenna complexes from purple bacteria. Therefore, the 4.9 kDa polypeptide is designated B(808-866)-α. The typical His residue (conserved His residue identified in all antenna polypeptides of purple bacteria as possible BChI binding site) is found within the hydrophobic domain, which extends from Asn 10 to Leu 30.     

Model of aggregation of pigments in the chlorosomal antenna of the green bacteria Chloroflexus aurantiacus

Independent experimental and theoretical evaluation was performed for the adequacy of our previously proposed general molecular model of structural organization of light-harvesting pigments in chlorosomal bacteriochlorophyll (BChl) c/d/e-containing superantenna of different green bacteria. Simultaneous measurement of hole burning in the optical spectra of chlorosomal BChl c and temperature dependence of steady-state fluorescence spectra of BChl c was accomplished in intact cells of photosynthetic green bacterium Chloroflexus aurantiacus; this allows unambiguous determination of the structure of exciton levels of BChl c oligomers in this natural antenna, which is a fundamental criterion for adequacy of any molecular model for in vivo aggregation of antenna pigments. Experimental data were shown to confirm our model of organization of oligometric pigments in chlorosomal BChl c antenna of green bacterium Chloroflexus aurantiacus. This model, which is based on experimental data and our theory of spectroscopy of oligomeric pigments, implies that the unit building block of BChl c antenna is a cylindrical assembly containing six excitonically coupled linear pigment chains whose exciton structure with intense upper levels provides for the optimal spectral properties of the light-harvesting antenna.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

Crystal structure of auracyanin, a "blue" copper protein from the green thermophilic photosynthetic bacterium Chloroflexus aurantiacus

Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloroflexus aurantiacus, crystallizes in space group P6(4)22 (a=b=115.7 A, c=54.6 A). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was refined at 1.55 A resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H(2)O molecules, one Cl(-) and two SO(4)(2-). The final residual and estimated standard uncertainties are R=0.198, ESU=0.076 A for atomic coordinates and ESU=0.05 A for Cu---ligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.     

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.