Takasada Ishii

The Effects of Epimerization at the 31‐position of Bacteriochlorophylls c on their Aggregation in Chlorosomes of Green Sulfur Bacteria. Control of the Ratio of 31 Epimers by Light Intensity

R‐ and S‐epimerization at the 31 position of bacteriochlorophyll (BChl) c and the formation of rod‐like aggregates in chlorosomes of green sulfur bacteria were markedly affected in Chlorobium (Cb.) tepidum and Cb.limicola by cultivation under various light intensities (photon fluence rate). The stronger the light, the higher the ratio of the S‐epimer to the R‐epimer for each homolog of BChl c in the bacteria. S[P,E] BChl cF and S[I,E] BChl cF were found to be the major S‐epimers in Cb. tepidum and Cb. limicola, respectively. R[P,E] BChl cF decreased markedly compared to R[E,E] BChl cF in Cb. tepidum, whereas no observable change in the ratio of R[P,E]/R[E,E] was detected for Cb. limicola. With increase in light intensity the Qy absorption maximum of the bacteria shifted to shorter wavelengths. In vitro spectroscopic studies of the aggregates showed a marked difference in the formation of aggregates from R‐ and S‐epimers of BChl c; the S‐epimers formed aggregates much more slowly than did the R‐epimers. These results suggest that the ratio of the epimers of BChl c might significantly affect the aggregation of BChl in the chlorosome. We propose different roles for the R‐ and S‐epimers in chlorosomes of Cb. limicola and Cb. tepidum.

The Role of Carotenoids in the Photoadaptation of the Brown-colored Sulfur Bacterium Chlorobium phaeobacteroides

The brown‐colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e. These carotenoids were identified with C18‐high‐performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two φ‐end groups such as isorenieratene and β‐isorenieratene and the second is carotenoid with one or two β‐end groups such as β‐zeacarotene, β‐carotene and 7,8‐dihydro‐β‐carotene. The latter 7,8‐dihydro‐β‐carotene was found to be a novel carotenoid in nature. OH‐γ‐Carotene glucoside laurate and OH‐chlorobactene glucoside laurate were also found as minor components. The distribution of BChl e homologs in Cb. phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the φ‐end group maintained the same ratio to BChl e, whereas that with the β‐end group increased with increasing light intensity. The cells cultured under low‐light intensity contained more φ‐end carotenoids than β‐end. In Cb. phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change. We suggested that Cb. phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.

How the formation process influences the structure of BChl c aggregates

The change of absorption spectra has been measured during the drying process of (31 R)bacteriochlorophyll (BChl) c from diethyl ether, dichloromethane (CH2Cl2) and carbon tetrachloride (CCl4) solutions. Absorption maxima of the Qy(0–0) transition of BChl c appear at 659 nm in diethyl ether, 680 nm in CH2Cl2 and 710 nm in CCl4. All these peaks are red-shifted to about 740 nm with formation of solid high aggregates when the solutions are completely dried. Fourier transform infrared spectra of the three solid aggregates are almost identical. However, magnetic circular dichroism and circular dichroism spectra are different and can be explained in terms of variations in stacking size of the aggregates and molecular arrangement of BChl c. Small-angle X-ray diffraction has been observed only for the aggregates treated with CH2Cl2, and the same sample gave rise to highly resolved cross polarization/magic angle spinning 13C nuclear magnetic resonance spectrum. The results suggest that molecular ordering of the solid-state BChl c aggregates is highly dependent on the formation process which is largely determined by the solvent used.

The Effects of pH and Ionic Strength on the Aggregation of Bacteriochlorophyll c in Aqueous Organic Media: The Possibility of Two Kinds of Aggregates

The aggregation behavior of two homologs of bacteriochlorophyll c (BChl c) in various media was investigated for the effects of pH and salt, and the corresponding structures were analyzed by Fourier transform (FT)‐IR spectroscopy. R‐[P, E] BChl cF (31‐R‐form of BChl c with a propyl group at the C‐8 position and an ethyl group at the C‐12 position) and R‐[E, E] BChl cF (31‐R‐form of BChl c with two ethyl groups at positions C‐8 and C‐12) were isolated from the green sulfur bacterium Chloro‐bium limicola. Aggregates of each homolog showed a pH‐dependent shift of the absorption maximum; at low pH, the peak moved to the red. This tendency was also revealed by circular dichroic spectra. A similar red shift of the peak was also induced by a high concentration of salt (NaCl) or buffer for both homologs. The FT‐IR spectrum indicates that at low pH, both homologs formed a rather amorphous aggregate. On the other hand, a regular structure of R‐[P, E] BChl cF was indicated in an acetone‐water mixture. This structure was stabilized by a triangular interaction among three pigment molecules through the Mg‐OH (3>) O = C (131) linkage. This structure was not found for R‐[E, E] BChl cF. These results indicate that the replacement of the side chain at the C‐8 position on the macrocycle induces a change in aggregation behavior. A possible heterogeneity of the in vivo rod structure of chlorosomes in green sulfur bacteria is discussed based on the above results.

NEAR-INFRARED-FT-RAMAN STUDY OF AGGREGATION OF BACTERIOCHLOROPHYLL C IN WHOLE LIVING CHLOROBIUM LIMICOLA

Near‐infrared excited Fourier‐transform Raman spectra have been measured for whole living Chlorobium limicola f. thiosulfatophilum to explore in situ structure of bacteriochlorophyll c (BChl‐c). The spectra, whose Raman bands are preresonance enhanced via a Qy band of BChl‐c, did not contain contributions from the major components of bacteria such as proteins and lipids. Therefore, the spectra provide selective structural information about BChl‐c in the chlorosomes in a totally nondestructive manner. A marker band, appearing at 1605 cm‐1 for the coordination number of the Mg atom, shows that BChl‐c in the chlorosomes is five coordinate. The Raman spectrum of living bacteria closely resembles that of BChl‐c in water‐saturated carbon tetrachloride (w‐std CC14) where it is comprised of dimer, tetramer and polymer spectra. However, a band assigned to a C=O stretching mode of the 131‐keto group is identified only at 1641 cm‐1. A band arising from the free keto carbonyl group, which appears in the spectrum of BChl‐c in w‐std CC14, is not observed in the spectra of bacteria. These observations suggest that BChl‐c in the chlorosomes forms mostly coordinate polymeric species whose structure is very similar to that of BChl‐c in w‐std CC14.

Effects of Growth Condition Changes on the Photosynthetic Pigment Systems for Green Sulfur Bacteria. A Study on the Constituent of the Photosynthetic Pigment in Chlorosome.

Green sulfur bacteria such as Chlorobium limicola f. thiosulfatophilum 6230 (Cb. limicola) have characteristic light-harvesting vesicles called chlorosome, which is surrounded by the mono-layer envelope of galactolipid1). The chlorosomes contain bacteriochlorophyll (BChl) c, BChl d or BChl e molecules as a rod-like self-aggregates for light-harvesting. The BChls in chlorosomes of Cb. limicola consist of four major homologs possessing different alkyl groups at 8- and 12-position; [E,M]-, [E,E]-, [P,E]-, and [I,E]BChl (Fig.1) 2). The homologation process increases the hydrophobic interactions between the BChl molecules, giving rise to larger aggregates3). Nevertheless, it is not clarified why the BChls in the chlorosomes of green sulfur bacteria have so many different homologs.

Effects of Homologs for Aggregation of Bacteriochlorophyll c and Bacteriochlorophyll d in Chlorosomes of Green Sulfur Bacteria

Bacteriochlorophyll (BChl) c and BChl d in the chlorosomes play important role as photosynthetic antenna pigment systems of green sulfur bacteria1). The chlorosomes contain rod-like self-aggregates of BChl in the envelope of monolayer galactolipid (Fig. 1). Uehara et al.2) reported that BChl c homologs formed various artificial aggregates in an aqueous medium containing lipid, monogalactosyl diglyceride, and showed optical properties similar to those of BChl c in intact chlorosomes. The chlorosomal BChls for green sulfur bacteria consist of homologs possessing different alkyl groups at 8- and 12-position with 31-(R) and 31-(S) epimers. The major esterifying alcohol at 17-position is usually farnesol for Chlorobium (Cb.) (Fig. 2) 3). The BChls in chlorosomes of Cb. limicola f. thiosulfatophilum 6230 formed three major 31-(R) homologs [E,M]-, [E,E]- and [P,E]-BChl cF and corresponding homologs of BChl dF under normal and K+ limited growth condition respectively.4) In this study, we investigated the difference between BChl cF and BChl dF on the effects of homologs for artifical aggregation together with disaggregation behavior in aqueous dimethyl sulfoxide (DMSO) solution. Comparison with whole chlorosomes for Cb. limicola and Cb. tepidum was also carried out. It was found that [E,M]- and [E,E]-BChl dF formed extremely stable aggregates compared with corresponding BCh1 cF homologs.