Photosynthesis Research 35: 159-169, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.

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Identification of the major chlorosomal bacteriochlorophyils of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer Stephan C.M. Otte I, Erik Jan van de Meent l, Peter A. van Veelen% Anne S. Pundsnes 1 & J a n Amesz ~'*

1Department of Biophysics, Huygens Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands; 2Centerfor Bio-Pharmaceutical Sciences, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands; *Author for correspondence Received 27 April 1992; accepted in revised form 18 August 1992

Key words: green sulfur bacteria, chlorosome, bacteriochlorophyll c, bacteriochlorophyll d, bacteriochlorophyll e, HPLC, absorption spectrum, mass spectroscopy, (Chlorobiaceae) Abstract The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and 252Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.

Abbreviations: BChl - bacteriochlorophyll; BPh - bacteriopheophytin (used as a general name for the Mg-free compound, irrespective of the esterifying alcohol); HPLC - high-performance liquid chromatography

Introduction Compared with other photosynthetic bacteria, such as purple bacteria, green sulfur bacteria have a relatively large antenna system. The most abundant bacteriochlorophylls in these organisms are the so-called chlorobium chlorophylls, which on the basis of their C-7 and C-20 substituents have been denoted BChls c, d and e.

All three types exist as a series of homologues and stereoisomers, depending on the nature of the substituents at C-8 and C-12, and on the stereochemical configuration at the C-31 carbon atom (Smith et al. 1983a,b, Smith and Simpson 1986). These pigments are contained in the chlorosomes, oblong bodies with dimensions in the order of 150 x 70 x 30 nm, which are attached to the cytoplasmic membrane. They contain rough-

160 ly 10 000 molecules of BChl c, d or e, and are energetically connected to 5-10 reaction centers (Amesz 1991). In a previous article (Otte et al. 1991), we studied energy transfer in intact cells of green sulfur bacteria containing BChl c, d or e. It was concluded that the chlorosomes are spectrally inhomogeneous and that they contain at least two pools of BChls c-e that form a lateral gradient within the chlorosome, thus promoting directional energy transfer within the chlorosome to the membrane. The strongest heterogeneity was observed in Chlorobium vibrioforme, which had been reported to contain BChl d (Gloe et al. 1975). In order to investigate if the pigment composition would provide a clue to the observed spectral inhomogeneities, we have determined the relative amounts of the various chlorobium chlorophylls in C. vibrioforme and C. phaeovibrioides by means of HPLC. The various fractions were characterized by means of their absorption and mass spectra. It was found that C. vibrioforme contains significant amounts of BChl c in addition to BChl d, which explains the spectral inhomogeneity of its chlorosomes. C. phaeovibrioides contained BChl e only. Besides farnesol, various other esterifying alcohols were found, some of which had not earlier been observed in green sulfur bacteria.

Pigments were extracted by a mild second sonication in a ca. 50-fold volume of acetone, for 2 min at 4 °C (Kobayashi et al. 1991). Repeating the extraction procedure on the residual of the sample showed an extraction efficiency of more than 99%. The extract was filtered through a teflon filter (Tosoh, H-13-5) to remove denaturated proteins, dried on a rotary evaporator, and subsequently dissolved in chloroform. 300 ~1 of the chloroform solution was injected into a preparative scale normal phase silica HPLC column (Senshupak 5251-N, 250 x 20mm i.d.) at 4°C. The pigments were eluted isocratically at a flow rate of 5 ml min -1. The eluens consisted of a degassed n-hexane/2-propanol/methanol mixture in a ratio of 100:1.8:1.0 (v/v) for C. vibrioforme, and of 100:2.0:1.0 for C. phaeovibrioides. Elution of pigments was detected with an Applied Biosystems Spectroflow 757 UV-detector. After rechromatography by analytical scale HPLC (Senshupak 1151-N, 150 x 4.6mm i.d., flow rate 1 ml min-l), the isolated fractions were used for further examination. Absorption spectra were recorded with a home-built single beam spectrophotometer (Rijgersberg et al. 1980), equipped with a personal computer for data reduction. The spectral resolution was 0.4 nm. 252Cf-plasma desorption mass spectroscopy was performed with a Bio-Ion 20 spectrometer (Applied Biosystems, Uppsala, Sweden). After being dissolved in acetone, pigments were deposited on a nitrocellulose target. The mass accuracy of the spectrometer was +0.1%.

Materials and methods

Chlorobium vibrioforme (strain 6030) and Chlorobium phaeovibrioides (strain 2631), which

Results

were a kind gift from Prof N. Pfennig (University of Konstanz), were grown in 11 cultures as described by Gloe et al. (1975), at a light intensity of 1000 lux from incandescent lamps. The cells were harvested by centrifugation for 10 min at 12 000 x g and resuspended in a 10 mM sodium p h o s p h a t e - 1 0 m M sodium ascorbate buffer, pH 7.4. The resuspended cells were sonicated for 45 min at 4 °C in dark. Unbroken cells and large cell fragments were removed by centrifugation for 20 min at 39 000 x g. The resulting supernatant was used as sonicated cells in this study.

Chlorobium vibrioforme Figure 1 shows the elution pattern in the time range of 100-200 min, obtained by preparative scale HPLC of an extract of sonicated cells of Chlorobium vibrioforme. The more hydrophobic pigments, carotenoids, BChl a and BChl 663 eluted at shorter times (Watanabe and Kobayashi 1990, Van de Meent 1992), and are not shown here. Eight different bands can be discerned in the chromatogram. Each band was rechromatographed on an analytical column for

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Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer.

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investig...
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