PhotosynthesisResearch 46: 427-433, 1995. © 1995KluwerAcademicPublishers. PrintedintheNetherlands. Regular paper

Aggregation of 8,12-diethyl farnesyl bacteriochlorophyll c at low temperature A. Dudkowiak 1, C. Francke & J.

Amesz

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Department of Biophysics, Huygens Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands; 1Permanent address: Molecular Physics Laboratory, Institute of Physics, Poznah University of Technology, Piotrowo 3, 60-965 Pozna~, Poland; "Author for correspondence Received27 June 1995; acceptedin revisedform 26 September1995

Key words: aggregates, bacteriochlorophyll c, chlorosome, green sulfur bacteria

Abstract

The effect of temperature on the aggregation of 31R-8,12-diethyl farnesyl bacteriochlorophyll c in a mixture of npentane and methylcyclohexane (1/1, v/v) was studied by means of absorption, circular dichroism and fluorescence spectroscopy. At room temperature essentially only two aggregate species, absorbing at 702 nm (A-702) and 719 nm (A-719), were present. Upon cooling to 219 K, A-702 was quantitatively converted to A-719. Further lowering of the temperature led to the stepwise formation of larger aggregates by the conversion of A-719 to aggregate species absorbing at 743 nm (A-743) and 755 nm (A-755). All absorption changes were reversible. A-719 was highly fluorescent (maximum at 192 K: 744 nm), while A-743 and especially A-755 were weakly fluorescent. Below 130 K the mixture solidified, and no major changes in the absorption spectrum were observed upon further cooling. At 45 K, however, a relatively strong emission at 775 nm was observed. Below 200 K, the absorption, fluorescence and circular dichroism spectra resembled that of the chlorosome. These results open up the possibility to study higher aggregates of BChl c as models for the chlorosome by various methods at low temperature, thus avoiding interference by thermal processes.

Abbreviations: A-680, A-702, A-719, A-743 and A-755-BChl c aggregates absorbing at the wavelengths indicated; B C h l - bacteriochlorophyll; R[E,E] BChl cF - the 31 R isomer of 8,12-diethyl BChl c esterified with farnesol (F), analogously: M - methyl; P r - propyl and S - stearol (see Smith 1994); CD - circular dichroism

Introduction

Two types of bacteriochlorophyll (BChl) are found in the chlorosomes of green sulfur bacteria (Chlorobiaceae): BChl a and the BChls c, d and e (Olson 1980; Gerola and Olson 1986). When dissolved in solvents such as diethyl-ether and acetone, BChl c absorbs near 660 nm, whereas it has an absorption maximum near 750 nm in the chlorosome. Similar red shifts are observed for BChls d and e (Hoff and Amesz 1991; Otte et al. 1991, 1993). It is now generally agreed that these chlorophylls form large aggregated structures which are responsible for the spectral properties of the chlorosome (Smith et al. 1983a; Worchester et

al. 1986; Brune et al. 1987; Hildebrandt et al. 1991; Hirota et al. 1992; Holzwarth et al. 1992; Miller et al. 1993). The BChls c, d and e are intrinsically suited for intermolecular hydrogen bonding because of the presence of a hydroxyl group attached to the carbon atom at position 3 I. In contrast to other antenna systems, proteins are thought to play only a secondary role in BChl organisation. BChl a makes up about 1% of the chlorosomal pigments in green sulfur bacteria (Gerola and Olson 1986; van Noort et al. 1994) and is present in only one homologue form: BChl a esterified with phytol (van Noort et al. 1994). In contrast, many different homologues of the BChls c, d and e are present in the chlorosomes

428 (Caple et al. 1978; Smith et al. 1983b; Otte et al. 1993; Borrego and Garcia-Gil 1994). No clear picture exists, however, of the effect of homologue composition on pigment aggregation. Various studies have been reported on the aggregated forms of BChls c, d and e in organic solvents and in other systems, as models for the in vivo aggregates. Aggregation of BChl c in vitro was first observed by Bystrova et al. (1979), and numerous reports exist on the aggregation of mixtures of homologues or stereoisomers (Smith et al. 1983a; Worchester et al. 1986; Brune et al. 1987; Hirota et al. 1992; Nozawa et al. 1994). Studies on the aggregation of single homologues have been reported by Olson and coworkers (Olson and Pedersen 1990; Olson and Cox 1991; Uehara et al. 1991; Uehara and Olson 1992), Holzwarth and coworkers (Holzwarth et al. 1992; Chiefari et al. 1995) and Causgrove et al. (1993). Formation of large aggregates absorbing near 750 nm has been observed for dry films of R[Pr,E] BChl CF (Uehara et al. 1991), as well as for solutions of various mixtures of homologues. However, for R[E,E] BChl CF, the most abundant homologue in Prosthecochloris aestuarii (Smith et al. 1983b) and in many Chlorobium species and strains (Caple et al. 1978; Nozawa et al 1991; Otte et al. 1993), only smaller aggregates absorbing near 680 and 710 nm have been observed under these conditions. This report concerns a study on the aggregation properties of R[E,E] BChl cF in a mixture of npentane and methylcyclohexane at various temperatures. At room temperature only small aggregates were formed, but at lower temperatures the formation of larger oligomers was observed. The properties of these aggregates were studied by means of absorption, fluorescence and circular dichroism spectroscopy.

Materials and methods 31R-8,12-diethyl farnesyl bacteriochlorophyll c (R[E,E] BChl cF) was isolated from Prosthecochloris aestuarii. Cells were harvested by centrifugation and pigments were extracted by mild sonication in a ca. 50-fold volume of acetone, for 2 rain at 4 °C. The extract was filtered (TOSOH H-25-5, teflon 0.45 #m), dried and dissolved in a mixture of n-hexane, 2propanol and methanol (100/3/3, v/v/v). The pigments were separated on a normal phase silica HPLC column (Senshupak 5251-N, 250 X 20 mm i.d.), cooled to 4 °C in an ice-water bath, using the above-mentioned

mixture as eluent. Pigment elution was monitored by means of a Jasco MD-915 diode array detector. The pigments were identified by comparing the elution pattern with that reported by Smith et al. (1983b) and comparing the elution times with those obtained by Otte et al. (1993) and van Noort et al. (1994). R[E,E] BChl CF was dissolved in chloroform. The chloroform was evaporated and the pigment was dissolved in a mixture of n-pentane and methylcyclohexane (1/1, v/v), dried over molecular sieves. Steady-state absorption, fluorescence and circular dichroism (CD) spectroscopy were performed at room temperature as well as at low temperatures using a single beam spectrophotometer, equipped with two monochromators, described by Francke et al. (1994). Fluorescence was measured perpendicular to the excitation light. The sample, contained within a 2.5 mm glass cuvette, was placed in a helium flow-cryostat. Room temperature measurements were made approximately one hour after dissolving the bacteriochlorophyll in the solvent mixture. Measurements at lower temperatures were performed by cooling the sample stepwise, allowing ca. 45 min for temperature equilibration. The spectral resolution was 0.5 nm for absorption, 2.5 nm for fluorescence and 1.5 nm for the CD measurements.

Results

Absorption Figures 1 and 2 show the absorption spectra of 10/.tM of R[E,E] BChl CF in n-pentane-methylcyclohexane at various temperatures together with their second derivatives. The solvent mixture was chosen because it remained a clear solution down to about 130 K and has been found to promote the aggregation of chlorophyll a (Fong and Koester 1976). The room temperature absorption spectrum (Fig. 1, solid line) showed two major absorption bands at 673 nm and 707 nm. A similar spectrum (not shown) was obtained at a pigment concentration of 1 #M but now the amplitude ratio m673/m707 was larger (0.94 as compared to 0.59 in Fig. 1). The room temperature spectra did not show any changes for at least several days. It is of interest to compare the room temperature spectrum of Fig. 1A with spectra reported in the literature for BChl c homologues in solvents such as dichloromethane and carbon tretrachloride. The absence of an absorption band near 750 nm shows that

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Aggregation of 8,12-diethyl farnesyl bacteriochlorophyll c at low temperature.

The effect of temperature on the aggregation of 3(l)R-8,12-diethyl farnesyl bacteriochlorophyll c in a mixture of n-pentane and methylcyclohexane (1/1...
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