Transmission electron microscopic study on supramolecular nanostructures of bacteriochlorophyll self-aggregates in chlorosomes of green photosynthetic bacteria

doi:10.1263/jbb.102.118

Journal of Bioscience and Bioengineering

Volume 102, Issue 2, August 2006, Pages 118-123

https://doi.org/10.1263/jbb.102.118 Get rights and content

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.

Section snippets

Materials and methods

Two substrains of Chl. vibrioforme f. sp. thiosulfatophilum NCIB 8327 containing BChl-c and BChl-d, which were isolated by colony preparation, were grown as reported previously (28). Chl. tepidum ATCC 49652 and Cfl. aurantiacus Ok-70-fl were grown at 45°C and 55°C, respectively (29, 30). Cultured cells were harvested by centrifugation and stored in the dark at −20°C before TEM observation.

Freeze-fracture replicas were prepared at approximately −120°C in a JEOL freeze-specimen preparation

Nanostructures in chlorosomes of Chl. vibrioforme

Chl. vibrioforme NCIB 8327 C-substrain and D-substrain were cultured under continuous illumination by fluorescent lamps, and the cells that reached the stationary phase were used for freeze-fracture TEM observation. The two substrains would have the same chlorosomal components except BChls-c or d (9, 28). In this way, the effects of C20 methyl group on BChl macrocycles on nanostructures of BChl self-aggregates in chlorosomes will be unraveled.

Figure 2 depicts a typical TEM image of

Discussion

In this work, rod-shaped nanostructures parallel to the long axis of chlorosomes of four green photosynthetic bacteria could be visualized in freeze-fracture TEM. These nanostructures in chlorosomes of Chl. vibrioforme and Chl. tepidum have approximately 10-nm diameters, whereas those of Cfl. aurantiacus have approximately 5-nm diameters. These observations are compatible with the previous reports on chlorosomes of Chl. limicola and Cfl. aurantiacus by Staehelin and his colleagues (20, 21, 22).

Acknowledgments

We thank Ms. Yumi Kondo, JEOL, for experimental assistance. This work was partially supported by Grants-in-Aid for Scientific Research (nos. 15033271/17029065) on Priority Areas (417) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government, for Scientific Research (B) (no. 15350107) from the Japan Society for the Promotion of Science (JSPS), and by the “Academic Frontier” Project for Private Universities: matching fund subsidy from MEXT,

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After spin-coating and evaporating the solution of assembled Zn-2 on a highly oriented pyrolytic graphite (HOPG) substrate, the Zn-2 aggregates were found as nanorods with a height of approximately 5 nm (Figs. 6c/d). The height of the synthetic aggregates was comparable to those of the BChl-c rods in Chloroflexus aurantiacus determined by EM measurements [42,44,90] and their tubular model previously reported by Holzwarth and Schaffner [47]. To construct rod-shaped aggregates, the effect of the esterifying groups at the 17-propionate residue was also considered by our group using the ZnChls possessing a different oligomethylene chains [R = (CH2)nH, n = 2–18, see the right bottom drawing of Scheme 1] as a model of BChl-d [91].
Photosynthetic light-harvesting antennas possess a variety of supramolecular structures with the same function (collecting sunlight energy), dependent on their living habitats and environments of phototrophs. Notably, the main antenna in green photosynthetic bacteria called “chlorosome” is structurally unique. Its core is constructed solely from self-aggregates of chlorophyll molecules without the support of any protein scaffolds. The supramolecular structures of the chlorophyll aggregates were already estimated, but have not been determined completely due to the natural diversity of chlorosomes. The static structures of chlorosomes are somewhat difficult to be characterized, and also the determination of their dynamic construction processes in vivo, such as their biogenesis and growth, is more challenging. Consequently, the measurement and observation of a simplified chlorosome model prepared by in vitro self-assembly of native or non-native (semisynthetic) chlorophylls are important. The present review focuses on evaluation of the static and dynamic nanostructures of chlorosomal aggregates. The construction, observation, and analysis of such models could be translated into the supramolecular aggregates of native chlorosomes. Additionally, synthetic aggregates display remarkable properties, which would be valuable for the development of solar cells and artificial photosynthesis. Such systems could potentially also be applied as photofunctional materials.
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In vitro self-assemblies of bacteriochlorophylls-c from Chlorobaculum tepidum and their supramolecular nanostructures
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This 5-nm rod was identical to the in vivo and in vitro rod self-assemblies of R/S[E,M]BChls-c from Cfl. aurantiacus [22,77], which possessed several other long alkyl chains (cetyl, geranylgeranyl, oleyl, phytyl and stearyl) at the 17-propionate residue [82]. The 5-nm rods of any R[E,M]BChls-c were visible in AFM images, regardless of the variation of their esterifying hydrocarbon chains.
Chlorosomes are major light-harvesting antenna apparatuses in green photosynthetic bacteria. Chlorosomes contain a large amount of bacteriochlorophyll(BChl)-c, d, e and f molecules and the supramolecular nanostructures of their self-assemblies are suggested to be rods with 5-nm or 10-nm diameters and/or lamellas with approximately 2-nm spacing. BChls-c were extracted, isolated and purified from cultured cells of a green sulfur photosynthetic bacterium, Chlorobaculum (Cba.) tepidum. Self-assemblies of the natural composite BChls-c as well as each pure homolog/epimer were prepared in a hydrophobic hexane-based solution and the resulting self-assembled solids were investigated by spectroscopic and microscopic techniques. Visible and near-infrared absorption and circular dichroism spectra of the in vitro self-assemblies of the natural composite BChls-c on a quartz substrate were closely similar to those in cells of Cba. tepidum in an aqueous buffer solution. Self-assemblies of (3¹R)-epimerically pure BChl-c molecules showed a wider Qy absorption band with increase in the steric bulkiness of hydrocarbon substituents at the 8- and 12-positions, which were effective for capturing wider wavelength light. (3¹S)-BChls-c gave similar self-assembly bands, but more monomeric and/or dimeric species than (3¹R)-BChls-c. Atomic force microscopic images of in vitro self-assemblies of natural composite BChls-c showed a rod with a 5-nm height and bundles of fibrils with 1–2-nm heights. Spectroscopic and microscopic analysis suggested that the 8²-methylation enhanced π–π stacking of self-assemblies by hydrophobic interaction, but the 12¹-methylation slightly weakened the intermolecular interactions due to its steric bulkiness.
Introduction of perfluoroalkyl chain into the esterifying moiety of bacteriochlorophyll c in the green sulfur photosynthetic bacterium Chlorobaculum tepidum by pigment biosynthesis
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The BChl self-aggregates are formed by coordination of the 31-hydroxy group in one BChl to the central magnesium of another BChl as well as formation of the hydrogen bond between the 31-hydroxy group and the 13-keto group in a third BChl.9–11 The mesoscopic structures of the BChl aggregates in chlorosomes have been reported to be rod-shaped,12–14 lamellar,15,16 and rolled-up structures.17 Such unique supramolecular architectures of BChl self-aggregates in chlorosomes have attracted considerable attentions from the basic research area of photobiological chemistry to the developments of artificial photosynthesis.
The green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum was grown in liquid cultures containing perfluoro-1-decanol, 1H,1H,2H,2H-heptadecafluoro-1-decanol [CF₃(CF₂)₇(CH₂)₂OH] or 1H,1H-nonadecafluoro-1-decanol [CF₃(CF₂)₈CH₂OH], to introduce rigid and fluorophilic chains into the esterifying moiety of light-harvesting bacteriochlorophyll (BChl) c. Exogenous 1H,1H,2H,2H-heptadecafluoro-1-decanol was successfully attached to the 17²-carboxy group of bacteriochlorophyllide (BChlide) c in vivo: the relative ratio of the unnatural BChl c esterified with this perfluoroalcohol over the total BChl c was 10.3%. Heat treatment of the liquid medium containing 1H,1H,2H,2H-heptadecafluoro-1-decanol with β-cyclodextrin before inoculation increased the relative ratio of the BChl c derivative esterified with this alcohol in the total BChl c in Cba. tepidum. In a while, 1H,1H-nonadecafluoro-1-decanol was not attached to BChlide c in Cba. tepidum, which was grown by its supplementation. These results suggest that the rigidity close to the hydroxy group of the esterifying alcohol is not suitable for the recognition by the BChl c synthase called BchK in Cba. tepidum. The unnatural BChl c esterified with 1H,1H,2H,2H-heptadecafluoro-1-decanol participated in BChl c self-aggregates in chlorosomes.
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The only exception is found in the light-harvesting antenna complexes of green photosynthetic bacteria called chlorosomes [6–9], whose main photofunctional parts are the self-assemblies of 100,000–200,000 BChls c, d, e, and f molecules [10,11]. These BChl self-assemblies form organized mesoscopic structures in chlorosomes by interactions among BChl pigments [12–17]. No protein participates in the BChl assemblies in chlorosomes.
Green sulfur photosynthetic bacteria Chlorobaculum (Cba.) tepidum and Cba. limnaeum were cultivated with the supplementation of 9-decyn-1-ol, 9-decen-1-ol, and decan-1-ol by two methods to biosynthesize unnatural chlorosomal bacteriochlorophyll (BChl) pigments possessing an (un)saturated bond at the terminus of the esterifying chain. The supplementation of the acetone solutions of the exogenous alcohols every 24 h to Cba. tepidum (denoted as Method A) successfully produced unnatural BChls c esterified with the alcohols in vivo. The ratios of unnatural BChls c esterified with 9-decyn-1-ol, 9-decen-1-ol, and decan-1-ol over the total BChls c in Cba. tepidum were not so different from each other, indicating that terminal unsaturated moieties in the exogenous alcohols barely affected the last reaction in the BChl c biosynthesis catalyzed by the BChl c synthase. Cba. tepidum grown in a liquid culture that initially contained 9-decyn-1-ol and 9-decen-1-ol (denoted as Method B) also accumulated BChls c esterified with these alcohols; the relative ratios of the unnatural BChls c were analogous to those obtained by Method A at the corresponding final concentrations. In contrast, Cba. limnaeum did not synthesize BChl e esterified with the three exogenous alcohols. The incorporation of unsaturated bonds into the esterifying moieties of BChl c by a biosynthetic reaction will be useful as structural probes for the investigation of pigment interactions and the biogenesis of chlorosomes as well as the scaffolds for bioorthogonal modifications.
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The green sulfur photosynthetic bacterium Chlorobaculum tepidum newly produced BChl c derivatives possessing a chlorine or bromine atom at the terminus of the esterifying chain in the 17-propionate residue by cultivation with exogenous ω-halo-1-alkanols. The relative ratios of BChl c derivatives esterified with 8-chloro-1-octanol and 10-chloro-1-decanol were estimated to be 26.5% and 33.3% by cultivation with these ω-chloro-1-alkanols at the final concentrations of 300 and 150 μM, respectively. In contrast, smaller amounts of unnatural BChls c esterified with ω-bromo-1-alkanols were biosynthesized than those esterified with ω-chloro-1-alkanols: the ratios of BChl c derivatives esterified with 8-bromo-1-octanol and 10-bromo-1-decanol were 11.3% and 12.2% at the concentrations of 300 and 150 μM, respectively. These indicate that ω-chloro-1-alkanols can be incorporated into bacteriochlorophyllide c more than ω-bromo-1-alkanols in the BChl c biosynthetic pathway. The homolog compositions of the novel BChl c derivatives possessing a halogen atom were analogous to those of coexisting natural BChl c esterified with farnesol. These results demonstrate unique properties of BChl c synthase, BchK, which can utilize unnatural substrates containing halogen in the BChl c biosynthesis of Cba. tepidum.

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¹: Present address: Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka, Osaka 577-8502, Japan.

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Transmission electron microscopic study on supramolecular nanostructures of bacteriochlorophyll self-aggregates in chlorosomes of green photosynthetic bacteria

Section snippets

Materials and methods

Nanostructures in chlorosomes of Chl. vibrioforme

Discussion

Acknowledgments

Bioorg. Med. Chem.

Coord. Chem. Rev.

J. Photochem. Photobiol. C: Photochem. Rev.

J. Photochem. Photobiol. B: Biol.

Biophys. J.

Biophys. J.

Biophys. J.

Photosynthetic membranes and their light-harvesting antennas

Antenna complexes from green photosynthetic bacteria

Chlorophyll organization and function in green photosynthetic bacteria

Photochem. Photobiol.

Chlorins programmed for self-assembly

Topics Curr. Chem.

A comparative study of the optical characteristics of intact cells of photosynthetic green sulfur bacteria containing bacteriochlorophyll c, d, or e

Photosynth. Res.

Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria

Nature

Crystal structure of the RC-LH1 core complex from Rhodopseudomonas palustris

Science

Three-dimensional structure of cyanobacterial photosystem I at 2.5 angstrom resolution

Nature

Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II

Nature