Femtosecond probe of structural analogies between chlorosomes and bacteriochlorophyll c aggregates.

Bacteriochlorophyll c pigments extracted from light harvesting chlorosomes in green photosynthetic bacteria are known to self-assemble into aggregates whose electronic spectroscopy resembles that of intact chlorosomes. Femtosecond optical experiments reveal that the chlorosomes and their reconstituted aggregates exhibit closely analogous internal energy transfer kinetics and exciton state evolution. These comparisons furnish compelling new evidence that proteins do not exert a major local role in the BChl c antenna pigment organization of intact chlorosomes.     

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.     

The functional role of protein in the organization of bacteriochlorophyll c in chlorosomes of Chloroflexus aurantiacus.

The preparation of five different fractions containing bacteriochlorophyll (Bchl) c and their absorption and circular dichroic properties have been described. The fractions investigated were purified chlorosomes, proteolytically modified chlorosomes, chlorosomes treated with lithium dodecyl sulfate (LDS) which were subsequently subjected to size-exclusion chromatography, in vitro Bchl c aggregates and, additionally, the so-called GEF chlorosomes [prepared according to Griebenow and Holzwarth (1989) Biochim. Biophys. Acta 973, 235-240]. Proteolysis of chlorosomes caused a 35-40% decrease in absorption intensity, a 6-8 nm blue shift of the 740-nm peak and, in particular, a drastic increase of rotational strength as revealed by CD spectroscopy. Although oligomeric Bchl c aggregates and LDS-treated chlorosomes had absorption characteristics similar to Bchl c in vivo, the data clearly indicated that protein, perhaps the chlorosome-specific Mr-3700 polypeptide, was involved in the organization of Bchl c in chlorosomes from C. aurantiacus. Furthermore, the results showed that the LDS-treated chlorosome fraction was most likely comprised of a micellar complex of Bchl c with LDS which represented an entity entirely different from chlorosomes.     

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.     

Atomic structure of the bacteriochlorophyll c assembly in intact chlorosomes from Chlorobium limicola determined by solid-state NMR

Green sulfur photosynthetic bacteria optimize their antennas, chlorosomes, especially for harvesting weak light by organizing bacteriochlorophyll (BChl) assembly without any support of proteins. As it is difficult to crystallize the organelles, a high-resolution structure of the light-harvesting devices in the chlorosomes has not been clarified. We have determined the structure of BChl c assembly in the intact chlorosomes from Chlorobium limicola on the basis of ¹³C dipolar spin-diffusion solid-state NMR analysis of uniformly ¹³C-labeled chlorosomes. About 90 intermolecular C–C distances were obtained by the simultaneous assignment of distance correlations and the structure optimization preceded by the polarization-transfer matrix analysis. An atomic structure was obtained, using these distance constraints. The determined structure of the chlorosomal BChl c assembly is built with the parallel layers of piggyback-dimers. This supramolecular structure would provide insights into the mechanism of weak-light capturing.     

Characterization of Chlorobium tepidum chlorosomes: a calculation of bacteriochlorophyll c per chlorosome and oligomer modeling

The bacteriochlorophyll (Bchl) c content and organization was determined for Chlorobium (Cb.) tepidum chlorosomes, the light-harvesting complexes from green photosynthetic bacteria, using fluorescence correlation spectroscopy and atomic force microscopy. Single-chlorosome fluorescence data was analyzed in terms of the correlation of the fluorescence intensity with time. Using this technique, known as fluorescence correlation spectroscopy, chlorosomes were shown to have a hydrodynamic radius (Rh) of 25 +/- 3.2 nm. This technique was also used to determine the concentration of chlorosomes in a sample, and pigment extraction and quantitation was used to determine the molar concentration of Bchl c present. From these data, a number of approximately 215,000 +/- 80,000 Bchl c per chlorosome was determined. Homogeneity of the sample was further characterized by dynamic light scattering, giving a single population of particles with a hydrodynamic radius of 26.8 +/- 3.7 nm in the sample. Tapping-mode atomic force microscopy (TMAFM) was used to determine the x,y,z dimensions of chlorosomes present in the sample. The results of the TMAFM studies indicated that the average chlorosome dimensions for Cb. tepidum was 174 +/- 8.3 x 91.4 +/- 7.7 x 10.9 +/- 2.71 nm and an overall average volume 90,800 nm(3) for the chlorosomes was determined. The data collected from these experiments as well as a model for Bchl c aggregate dimensions was used to determine possible arrangements of Bchl c oligomers in the chlorosomes. The results obtained in this study have significant implications on chlorosome structure and architecture, and will allow a more thorough investigation of the energetics of photosynthetic light harvesting in green bacteria.     

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes. Received: 4 November 1996 / Accepted: 18 February 1997     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum     

Ultrafast excited-state dynamics of phenyleneethynylene oligomers in solution.

The excited-state dynamics of oligomeric phenyleneethynylenes (OPEs) of various length and substitution has been investigated by femtosecond time-resolved spectroscopy. The fluorescence lifetime of the OPEs decreases with the number of phenyleneethynylene units up to about 9. This effect is due to an increase of the oscillator strength for the S(1)-S(0) transition. Dynamic features occurring within a few tens of picoseconds and ascribed to structural relaxation directly after population of the S(1) state can be observed in non-viscous solvents. The effect of torsional disorder on the fluorescence intensity is shown to depend strongly on the nature of the substituent on the phenyl groups. All these effects are qualitatively discussed with a simple exciton model.     

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.     

Hole burning study of excited state structure and energy transfer dynamics of bacteriochlorophyll c in chlorosomes of green sulphur photosynthetic bacteria

Results of low temperature fluorescence and spectral hole burning experiments with whole cells and isolated chlorosomes of the green sulfur bacterium Chlorobium limicola containing BChl c are reported. At least two spectral forms of BChl c (short-wavelength and long-wavelength absorbing BChl c) were identified in the second derivative fluorescence spectra. The widths of persistent holes burned in the fluorescence spectrum of BChl c are determined by excited state lifetimes due to fast energy transfer. Different excited state lifetimes for both BChl c forms were observed. A site distribution function of the lowest excited state of chlorosomal BChl c was revealed. The excited state lifetimes are strongly influenced by redox conditions of the solution. At anaerobic conditions the lifetime of 5.3 ps corresponds to the rate of energy transfer between BChl c clusters. This time shortens to 2.6 ps at aerobic conditions. The shortening may be caused by introducing a quencher. Spectral bands observed in the fluorescence of isolated chlorosomes were attributed to monomeric and lower state aggregates of BChl c. These forms are not functionally connected with the chlorosome.Abbreviations BChl bacteriochlorophyll - EET electronic energy transfer - FWHM full width at half maximum - SDF site distribution function - RC reaction centre     

On the structure of bacteriochlorophyll molecular aggregates in the chlorosomes of green bacteria. A molecular modelling study

The supramolecular structure of methyl (3¹ R)-BChlided aggregation has been explored by molecular modelling in order to elucidate the unusual structure of the BChl rods in the chlorosomal antennae of green bacteria. The aggregate construction progressed from a BChlide monomer in 5c coordination which was stepwise combined to form trimeric, pentameric and decameric chlorin stacks, all incorporating Mg····O-H as a basic interaction element which links two chlorins between the 3¹-hydroxyl oxygen and the Mg. Up to the level of the trimer, the structures were optimized by both a semiempirical quantum chemical method (PM3) and a force field method, while larger structures were only modelled by the force field (MM+). Strong interactions were found by extended stacking of chlorins which are in van der Waals contact. Extended hydrogen bonding networks upon stack pairing brought about by OH····O=C bonds (bond length ca. 2.2Å, angle 139–153°) between appropriately situated chlorin pairs and by electrostatic interactions lead to very large energy stabilizations. The structural features of a modelled 40mer BChl aggregate are in full accord with all spectroscopic and low-resolution structural information on the in-vitro and chlorosomal BChl aggregates. Most important, from the rotation angle between stacks of ca. 16° and the stack-to-stack distance of 7.6 Å a tubular structure can be extrapolated to form on further extension of the aggregate. It has a predicted diameter of about 5.4 nm (Mg-Mg distance), i.e. very similar to that found for the rod elements in the chlorosomes ofChloroflexus.Dedicated to J.J. Katz, pioneer in the study of chlorophyll aggregation.Dedicated to J.J. Katz, pioneer in the study of chlorophyll aggregation.     

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.     

Long-range organization of bacteriochlorophyll in chlorosomes of Chlorobium tepidum investigated by cryo-electron microscopy

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryo-electron microscopy to study the long-range organization of bacteriochlorophyll (BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multi-layered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed.     

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for ¹O₂ quenching by BChl molecules (k_q ^M) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the k_q ^M values coincide with the real values for ¹O₂ quenching rate constants by BChl molecules. Aggregation weakly influenced the k_q ^M values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the k_q ^M value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus ¹O₂ quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of ¹O₂ formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as ¹O₂ physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.Abbreviations BChl bacteriochlorophyll - MBpd methyl bacteriopheophorbide - Chl chlorophyll - TPP meso-tetraphenylporphyrin - TPPS meso-tetra (p-sulfophenyl) porphyrin     

Solvent polarity effect on excited-state lifetime of carotenoids and some dyes

The theory demonstrating the role of medium at the fluorescence quenching of polar compounds in solutions is briefly presented. It has been shown, that the rate of S(1) --> X(n) nonradiative conversion between the intramolecular charge transfer states depends on the permanent dipole moments in the ground (S(0)) and excited (S(1), X(n)) states as well as on solvent polarity. A relation for the rate of nonradiative excited-state energy conversion has been obtained and employed to test the known literature data for solvent effect on the S(1)-state lifetime of some biologically significant carotenoids and dyes (phthalimides). For phthalimides, the solvent isotope effect on the S(1)-state energy conversion, when hydrogen is replaced by deuterium in the OH groups of alcohols and water, has been analyzed. Based on the data for fluorescence quenching in solvents of different polarity, the dipole moments in the intermolecular charge transfer S(1) state have been obtained for carotenoids (peridinin, fucoxanthin, uriolide acetate) and for hydrogen-bonding complexes, which are formed by 4-amino-, 4-methylamino-, and 4-dimethylamino-N-methylphthalimides in alcohols and water.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

Chlorobium tepidum mutant lacking bacteriochlorophyll c made by inactivation of the bchK gene,encoding bacteriochlorophyll c synthase

The gene encoding bacteriochlorophyll (BChl) c synthase was identified by insertional inactivation in the photosynthetic green sulfur bacterium Chlorobium tepidum and was named bchK. The bchK mutant of C. tepidum was rusty-orange in color and completely lacked BChl c. Because of the absence of the BChl c antenna, the mutant grew about seven times slower than the wild type at light intensities that were limiting to the wild type (< 90 micromol m(-2) s(-1)). Various pheophorbides, which probably represent precursors of BChl c which had lost magnesium, accumulated in the mutant cells. A small fraction of these pheophorbides were apparently esterified by the remaining chlorophyll (Chl) a and BChl a synthases in cells. The amounts of BChl a, Chl a, isoprenoid quinones, carotenoids, Fenna-Matthews-Olson protein, and chlorosome envelope protein CsmA were not significantly altered on a cellular basis in the mutant compared to in the wild type. This suggests that the BChl a antennae, photosynthetic reaction centers, and remaining chlorosome components were essentially unaffected in the mutant. Electron microscopy of thin sections revealed that the mutant lacked normal chlorosomes. However, a fraction containing vestigial chlorosomes, denoted "carotenosomes," was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl a species that is probably protein associated. Because of the absence of the strong BChl c absorption found in the wild type, the bchK mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl a in photosynthesis in green bacteria. An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have evolved without chlorosomes and BChl c and instead used only BChl a-containing proteins as the major light-harvesting antennae.     

Selective protein extraction from Chlorobium tepidum chlorosomes using detergents. Evidence that CsmA forms multimers and binds bacteriochlorophyll a

Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins. Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters. When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes. Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed. These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes. Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.