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.     

Identification of the bchP gene, encoding geranylgeranyl reductase in Chlorobaculum tepidum

The Chlorobaculum tepidum genome contains two paralogous genes, CT2256 and CT1232, whose products are members of the FixC dehydrogenase superfamily and have sequence similarity to geranylgeranyl reductases. Each gene was insertionally inactivated, and the resulting mutants were characterized. CT2256 encodes geranylgeranyl reductase (BchP); CT1232 is not involved in bacteriochlorophyll or chlorophyll biosynthesis.     

Internal structure of chlorosomes from brown-colored chlorobium species and the role of carotenoids in their assembly

Chlorosomes are the main light harvesting complexes of green photosynthetic bacteria. Recently, a lamellar model was proposed for the arrangement of pigment aggregates in Chlorobium tepidum chlorosomes, which contain bacteriochlorophyll (BChl) c as the main pigment. Here we demonstrate that the lamellar organization is also found in chlorosomes from two brown-colored species (Chl. phaeovibrioides and Chl. phaeobacteroides) containing BChl e as the main pigment. This suggests that the lamellar model is universal among green sulfur bacteria. In contrast to green-colored Chl. tepidum, chlorosomes from the brown-colored species often contain domains of lamellar aggregates that may help them to survive in extremely low light conditions. We suggest that carotenoids are localized between the lamellar planes and drive lamellar assembly by augmenting hydrophobic interactions. A model for chlorosome assembly, which accounts for the role of carotenoids and secondary BChl homologs, is presented.     

Identification of a gene essential for the first committed step in the biosynthesis of bacteriochlorophyll c

Bacteriochlorophylls (BChls) c, d, and e are the major chlorophylls in chlorosomes, which are the largest and one of the most efficient antennae produced by chlorophototrophic organisms. In the biosynthesis of these three BChls, a C-13(2)-methylcarboxyl group found in all other chlorophylls (Chls) must be removed. This reaction is postulated to be the first committed step in the synthesis of these BChls. Analyses of gene neighborhoods of (B)Chl biosynthesis genes and distribution patterns in organisms producing chlorosomes helped to identify a gene (bciC) that appeared to be a good candidate to produce the enzyme involved in this biochemical reaction. To confirm that this was the case, a deletion mutant of an open reading frame orthologous to bciC, CT1077, was constructed in Chlorobaculum tepidum, a genetically tractible green sulfur bacterium. The CT1077 deletion mutant was unable to synthesize BChl c but still synthesized BChl a and Chl a. The deletion mutant accumulated large amounts of various (bacterio)pheophorbides, all of which still had C-13(2)-methylcarboxyl groups. A C. tepidum strain in which CT1077 was replaced by an orthologous gene, Cabther_B0081 [corrected] from "Candidatus Chloracidobacterium thermophilum" was constructed. Although the product of Cabther_B0081 [corrected] was only 28% identical to the product of CT1077, this strain synthesized BChl c, BChl a, and Chl a in amounts similar to wild-type C. tepidum cells. To indicate their roles in the first committed step of BChl c, d, and e biosynthesis, open reading frames CT1077 and Cabther_B0081 [corrected] have been redesignated bciC. The potential mechanism by which BciC removes the C-13(2)-methylcarboxyl moiety of chlorophyllide a is discussed.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Isolation and pigment composition of the antenna system of four species of green sulfur bacteria

New and rapid procedures were developed for the isolation of chlorosomes and FMO-protein from the green sulfur bacteria Prosthecochloris (P.) aestuarii, Chlorobium (Cb.) phaeovibrioides, Cb. tepidum and Cb. vibrioforme. The resulting preparations were free from contaminating pigments and proteins as was shown by absorption spectroscopy, pigment analysis and SDS-PAGE. Two spectrally different types of FMO-protein were found. The first type, present in P. aestuarii and Cb. vibrioforme, has a main absorption band at 6 K at 815 nm, whereas the second type, isolated from Cb. tepidum and Cb. phaeovibrioides, has a strong band at 806 nm. In contrast to what was recently suggested (Tronrud DE and Matthews BW (1993) In: Deisenhofer J and Norris J (eds) The Photosynthetic Reaction Center, Vol 1, pp 13–21. Academic Press, San Diego, CA) the FMO-proteins contained no polar BChl a homologue. The isolated chlorosomes showed a small blue-shift of the QY absorption maximum with respect to intact cells. For the different species, grown under the same light conditions, the homologue composition of BChls c and d was approximately identical whereas for the BChl e in Cb. phaeovibrioides the relative amounts of homologues with larger alkyl substituents at position 8 were considerably larger. Baseplate BChl a was present in all chlorosomes and comprised 1–2% of the chlorosomal BChl. Its QY absorption band was located at about 802 nm and was clearly separated from the major QY absorption band at 6 K. The predominant esterifying alcohol of BChl a in the chlorosomes as well as in the FMO-proteins was phytol, but both antenna complexes also contained small amounts of BChl a esterified with the metabolic intermediates geranylgeraniol, dihydrogeranylgeraniol and tetrahydrogeranylgeraniol, like most purple bacteria. Since the esterifying alcohols of the chlorosomal BChl a and of the main chlorosomal pigments (BChls c, d and e) are different, esterification, and perhaps also the synthesis, of the BChls in the interior of the chlorosome and of the BChls in the baseplate must be spatially and genetically separated processes.     

Identification of the bacteriochlorophylls, carotenoids, quinones, lipids, and hopanoids of "Candidatus Chloracidobacterium thermophilum"

"Candidatus Chloracidobacterium thermophilum" is a recently discovered chlorophototroph from the bacterial phylum Acidobacteria, which synthesizes bacteriochlorophyll (BChl) c and chlorosomes like members of the green sulfur bacteria (GSB) and the green filamentous anoxygenic phototrophs (FAPs). The pigments (BChl c homologs and carotenoids), quinones, lipids, and hopanoids of cells and chlorosomes of this new chlorophototroph were characterized in this study. "Ca. Chloracidobacterium thermophilum" methylates its antenna BChls at the C-8(2) and C-12(1) positions like GSB, but these BChls were esterified with a variety of isoprenoid and straight-chain alkyl alcohols as in FAPs. Unlike the chlorosomes of other green bacteria, "Ca. Chloracidobacterium thermophilum" chlorosomes contained two major xanthophyll carotenoids, echinenone and canthaxanthin. These carotenoids may confer enhanced protection against reactive oxygen species and could represent a specific adaptation to the highly oxic natural environment in which "Ca. Chloracidobacterium thermophilum" occurs. Dihydrogenated menaquinone-8 [menaquinone-8(H(2))], which probably acts as a quencher of energy transfer under oxic conditions, was an abundant component of both cells and chlorosomes of "Ca. Chloracidobacterium thermophilum." The betaine lipid diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine, esterified with 13-methyl-tetradecanoic (isopentadecanoic) acid, was a prominent polar lipid in the membranes of both "Ca. Chloracidobacterium thermophilum" cells and chlorosomes. This lipid may represent a specific adaptive response to chronic phosphorus limitation in the mats. Finally, three hopanoids, diploptene, bacteriohopanetetrol, and bacteriohopanetetrol cyclitol ether, which may help to stabilize membranes during diel shifts in pH and other physicochemical conditions in the mats, were detected in the membranes of "Ca. Chloracidobacterium thermophilum."     

Evidence for spatially separate bacteriochlorophyll c and bacteriochlorophyll d pools within the chlorosomal aggregate of the green sulfur bacterium Chlorobium limicola

We have studied the organization of the bacteriochlorophylls (BChl) in isolated chlorosomes of the green sulfur bacterium Chlorobium limicola UdG6040 containing about 50% BChl d and BChl c each. When the chlorosomes are treated in acidic buffer (pH 3.0) two phases in the conversion from BChl to bacteriopheophytin (BPhe) are observed as evidenced by the changes in the absorption spectrum. In the early phase the pheophytinization of BChl d occurs much faster than that of BChl c. In the later phase BChl c and BChl d are converted at similar rates. The delayed BChl c conversion observed in intact chlorosomes is interpreted in terms of spatial separation within the same chlorosome that makes BChl d more accessible to reaction with acid than BChl c. This was supported by acid treatment of in vitro pigment-lipid aggregates which showed that the pheophytinization of aggregates consisting of only BChl c or BChl d takes place with the same rate. Moreover in mixed in vitro aggrega tes where BChl d and BChl c are supposed to be scrambled the two pigments are converted to BPhe simultaneously. Acid treatment of hexanol exposed chlorosomes indicates that the spatial separation of BChl d and BChl c within the chlorosomes is maintained even if the excitonic interaction between BChls has been disturbed by hexanol. Based on these findings it is suggested that BChl d and BChl c in the chlorosome are located distal and proximal, respectively, relative to the chlorosome baseplate.     

In vitro demethoxycarbonylation of various chlorophyll analogs by a BciC enzyme

Unique light-harvesting antennas in the green sulfur bacterium Chlorobaculum tepidum, called chlorosomes, consist of self-aggregates of bacteriochlorophyll (BChl) c. In the biosynthesis of BChl c, BciC demethoxycarbonylase removes the C13²-methoxycarbonyl group to facilitate the self-aggregation of BChl c. We previously reported the in vitro BciC-enzymatic reactions and discussed the function of this enzyme in the biosynthesis of BChl c. This study aims to examine the substrate specificity of BciC in detail using several semi-synthetic (bacterio)chlorophyll derivatives. The results indicate that the substrate specificity of BciC is measurably affected by structural changes on the A/B rings including the bacteriochlorin π-systems. Moreover, BciC showed its activity on a Zn-chelated chlorophyll derivative. On the contrary, BciC recognized structural modifications on the D/E rings, including porphyrin pigments, which resulted in the significant decrease in the enzymatic activity. The utilization of BciC provides mild conditions that may be useful for the in vitro preparation of various chemically (un)stable chlorophyllous pigments.

     

Lamellar organization of pigments in chlorosomes, the light harvesting complexes of green photosynthetic bacteria

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations approximately 20 A apart. X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.     

Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes

Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA. All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain. Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents. However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome. These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.     

The molar extinction coefficient of bacteriochlorophyll e and the pigment stoichiometry in Chlorobium phaeobacteroides

We have determined the molar extinction coefficient of bacteriochlorophyll (BChl) e, the main light-harvesting pigment from brown-coloured photosynthetic sulfur bacteria. The extinction coefficient was determined using pure [Pr,E]BChl e_F isolated by reversed-phase HPLC from crude pigment extracts of Chlorobium (Chl.) phaeobacteroides strain CL1401. The extinction coefficients at the Soret and Q_y bands were determined in four organic solvents. The extinction coefficient of BChl e differs from those of other related Chlorobium chlorophylls (BChl c and BChl d) but is similar to that of chlorophyll b. The determined extinction coefficient was used to calculate the stoichiometric BChl e to BChl a and BChl e to carotenoids ratios in whole cells and isolated chlorosomes from Chl. phaeobacteroides strain CL1401 using the spectrum-reconstruction method (SRCM) described by Naqvi et al. (1997) (Spectrochim Acta A Mol Biomol Spectrosc 53: 2229–2234) . In isolated chlorosomes, BChl a content was ca. 1% of the total BChl content and the stoichiometric ratio of BChl e to carotenoids was 6. In whole cells, however, BChl a content was 3–4%, owing to the presence of BChl a-containing elements, i.e. FMO protein and reaction centre. An average of 5 BChl e molecules per carotenoid was determined in whole cells.     

Probing the bacteriochlorophyll binding site by reconstitution of the light-harvesting complex of Rhodospirillum rubrum with bacteriochlorophyll a analogues

Structural features of bacteriochlorophyll (BChl) a that are required for binding to the light-harvesting proteins of Rhodospirillum rubrum were determined by testing for reconstitution of the B873 or B820 (structural subunit of B873) light-harvesting complexes with BChl a analogues. The results indicate that the binding site is very specific; of the analogues tested, only derivatives of BChl a with ethyl, phytyl, and geranylgeranyl esterifying alcohols and BChl b (phytyl) successfully reconstituted to form B820- and B873-type complexes. BChl analogues lacking magnesium, the C-3 acetyl group, or the C-13(2) carbomethoxy group did not reconstitute to form B820 or B873. Also unreactive were 13(2)-hydroxyBChl a and 3-acetylchlorophyll a. Competition experiments showed that several of these nonreconstituting analogues significantly slowed BChl a binding to form B820 and blocked BChl a-B873 formation, indicating that the analogues may competitively bind to the protein even though they do not form red-shifted complexes. With the R. rubrum polypeptides, BChl b formed complexes that were further red-shifted than those of BChl a; however, the energies of the red shifts, binding behavior, and circular dichroism (CD) spectra were similar. B873 complexes reconstituted with the geranylgeranyl BChl a derivative, which contains the native esterifying alcohol for R. rubrum, showed in-vivo-like CD features, but the phytyl and ethyl B873 complexes showed inverted CD features in the near infrared. The B820 complex with the ethyl derivative was about 30-fold less stable than the two longer esterifying alcohol derivatives, but all formed stable B873 complexes.     

A reconstituted light-harvesting complex from the green sulfur bacterium Chlorobium tepidum containing CsmA and bacteriochlorophyll a

Green sulfur bacteria possess two light-harvesting antenna systems, the chlorosome and the Fenna-Matthews-Olson (FMO) protein. In addition to self-aggregated bacteriochlorophyll (BChl) c, chlorosomes of Chlorobium tepidum contain a small amount of BChl a (ratio 100:1). The chlorosomal BChl a is associated with CsmA, a 6.2 kDa protein that accounts for more than 50% of the protein content of chlorosomes. This CsmA-BChl a complex is located in the chlorosome baseplate with the hydrophilic C-terminal part of CsmA in contact with the FMO protein. CsmA was purified from Chl. tepidum. Isolated chlorosomes were lyophilized and extracted with chloroform/methanol (1:1, v/v). The extract was further purified using gel filtration and reverse-phase HPLC and the purity of the preparation confirmed by SDS-PAGE. Mass spectrometric analysis showed an m/z of 6154.8, in agreement with the calculated mass of the csmA gene product after C-terminal processing. CD spectroscopy of the isolated protein showed that the main structural motif was an alpha-helix. We have reconstituted the isolated CsmA protein with BChl a in micelles of n-octyl beta-d-glucopyranoside. The resulting preparation reproduced the spectral characteristics of the CsmA-BChl a complex present in the chlorosome baseplate.     

Seeing green bacteria in a new light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and filamentous anoxygenic phototrophic bacteria

Based upon their photosynthetic nature and the presence of a unique light-harvesting antenna structure, the chlorosome, the photosynthetic green bacteria are defined as a distinctive group in the Bacteria. However, members of the two taxa that comprise this group, the green sulfur bacteria (Chlorobi) and the filamentous anoxygenic phototrophic bacteria (Chloroflexales), are otherwise quite different, both physiologically and phylogenetically. This review summarizes how genome sequence information facilitated studies of the biosynthesis and function of the photosynthetic apparatus and the oxidation of inorganic sulfur compounds in two model organisms that represent these taxa, Chlorobium tepidum and Chloroflexus aurantiacus. The genes involved in bacteriochlorophyll (BChl) c and carotenoid biosynthesis in these two organisms were identified by sequence homology with known BChl a and carotenoid biosynthesis enzymes, gene cluster analysis in Cfx. aurantiacus, and gene inactivation studies in Chl. tepidum. Based on these results, BChl a and BChl c biosynthesis is similar in the two organisms, whereas carotenoid biosynthesis differs significantly. In agreement with its facultative anaerobic nature, Cfx. aurantiacus in some cases apparently produces structurally different enzymes for heme and BChl biosynthesis, in which one enzyme functions under anoxic conditions and the other performs the same reaction under oxic conditions. The Chl. tepidum mutants produced with modified BChl c and carotenoid species also allow the functions of these pigments to be studied in vivo.     

High-pressure and stark hole-burning studies of chlorosome antennas from Chlorobium tepidum

Results from high-pressure and Stark hole-burning experiments on isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum are presented, as well as Stark hole-burning data for bacteriochlorophyll c (BChl c) monomers in a poly(vinyl butyral) copolymer film. Large linear pressure shift rates of -0.44 and -0.54 cm(-1)/MPa were observed for the chlorosome BChl c Q(y)-band at 100 K and the lowest Q(y)-exciton level at 12 K, respectively. It is argued that approximately half of the latter shift rate is due to electron exchange coupling between BChl c molecules. The similarity between the above shift rates and those observed for the B875 and B850 BChl a rings of the light-harvesting complexes of purple bacteria is emphasized. For BChl c monomer, fDeltamu++ = 0.35 D, where Deltamu+ is the dipole moment change for the Q(y) transition and f is the local field correction factor. The data establish that Deltamu+ is dominated by the matrix-induced contribution. The change in polarizability (Deltaalpha) for the Q(y) transition of the BChl c monomer is estimated at 19 A(3), which is essentially identical to that of the Chl a monomer. Interestingly, no Stark effects were observed for the lowest exciton level of the chlorosomes (maximum Stark field of 10(5) V/cm). Possible explanations for this are given, and these include consideration of structural models for the chlorosome BChl c aggregates.     

Characterization of a plant-like protochlorophyllide a divinyl reductase in green sulfur bacteria

The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this ethyl group is subsequently methylated once or twice by the radical S-adenosylmethionine enzyme BchQ. The C. tepidum genome contains homologs of two genes, bchJ (CT2014) and CT1063, that are highly homologous to genes, bchJ and AT5G18660, and that have been reported to encode C-8 vinyl reductases in Rhodobacter capsulatus and Arabidopsis thaliana, respectively. To determine which gene product actually encodes a C-8 vinyl reductase activity, the bchJ and CT1063 genes were insertionally inactivated in C. tepidum. All three Chls synthesized by the CT1063 mutant of C. tepidum have a C-8 vinyl group. Using NADPH but not NADH as reductant, recombinant BciA reduces the C-8 vinyl group of 3,8-divinyl-protochlorophyllide in vitro. These data demonstrate that CT1063, renamed bciA, encodes a C-8 divinyl reductase in C. tepidum. The bchJ mutant produces detectable amounts of Chl a(PD), BChl a(P), and BChl c(F), all of which have reduced C-8 substituents, but the mutant cells secrete large amounts of 3,8-divinyl-protochlorophyllide a into the growth medium and have a greatly reduced BChl c(F) content. The results suggest that BchJ may play an important role in substrate channeling and/or regulation of Chl biosynthesis but show that it is not a vinyl reductase. Because only some Chl-synthesizing organisms possess homologs of bciA, at least two types of C-8 vinyl reductases must occur.     

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.     

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.     

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.