Proc. Nati. Acad. Sci. USA Vol. 84, pp. 2570-2574, May 1987 Chemistry Bacteriopheophytin g: Properties and some speculations on a possible primary role for bacteriochlorophylls b and g in the biosynthesis of chlorophylls (photoisomerization/esterifying alcohol/electron spin resonance) T. J. MICHALSKI, J. E. HUNT, M. K. BOWMAN, U. SMITH, K. BARDEEN, H. GEST*, J. R. NORRIS, AND J. J. KATZ Chemistry Division, Argonne National Laboratory, Argonne, IL 60439 Contributed by J. J. Katz, January 14, 1987 ABSTRACT Bacteriopheophytin g and small amounts of characterized Bpheog by HPLC, and 252CF plasma desorp- bacteriochlorophyll g have been obtained in high purity from tion mass spectrometry (252Cf-PDMS). We have also studied the recently discovered photosynthetic bacterium Heliobacte- triplet Bpheog (3Bpheog) and the cation free radical BPheog' rium chlorum. Preparative methods and precautions in han- by ESR. Because of the high current interest in the structure dling these sensitive compounds are described. The compounds of bacterial photosynthetic reaction centers (6, 7), the reac- have been characterized by californium-252 plasma desorption tion center of H. chlorum has quickly attracted attention mass spectrometry, HPLC, visible absorption, and electron (8-10). The extreme sensitivity of BChlg to light and air was spin resonance spectroscopy. Our results agree with the struc- not adequately taken into consideration in the early reaction ture of bacteriochlorophyli g advanced by H. Brockmann and center work, and it will be necessary to do so in future A. Lipinski [(1983) Arch. Microbiol. 136, 17-191, with the reaction center research on this organism. exception that we find the esterifying alcohol to be farnesol and not geranylgeraniol as originally suggested. Zero field splitting MATERIALS AND METHODS parameters of triplet state bacteriopheophytin g and the ESR Cell Culture. H. chlorum (American Type Culture Collec- properties ofthe cation free radical ofbacteriochlorophyllg are tion 35205) was grown anaerobically at room temperature in reported. The photoisomerization of the subject compounds medium 112 ofthe ATTC, supplemented with 0.05% ascorbic has been studied. Bacteriopheophytin g undergoes photo- acid (1). Cultures were maintained at ambient temperatures isomerization in white light to pheophytin a with a half-time and in low light (1800 lx). Although growth is strongly of -42 min. We suggest that all of the chlorophylls are inhibited by small amounts of oxygen, we have found it biosynthesized from a common intermediate containing an practical to grow the organism in 4-liter cultures, even ethylidine group, ==CH-CH3, such as is present in bacte- without the use of an anaerobic hood. Cells were harvested riochlorophylls b and g. by centrifugation after 5-6 days of growth; between 25 and 30 g of cells was usually obtained from a 4-liter culture. All Gest and Favinger (1) have recently discovered a photosyn- harvests were monitored for possible contamination by thetic bacterium with such unusual features that it has been microscopic examination and Gram staining. placed in a new genus, Heliobacterium. This organism, Chromatography. HPLC separations were made on a Heliobacterium chlorum, is a brownish-green, rod-shaped Beckman two-pump gradient system, fitted with a Hewlett- organism, isolated from surface soil. H. chlorum is unable to Packard (model 8451A) diode array optical detector. Effluent grow aerobically in the dark, and photosynthetic growth is from HPLC columns (Altex 5-,pm ODS) can be monitored strongly inhibited even by minute traces of oxygen. The simultaneously at different wavelengths, which increases the photosynthetic apparatus ofthe organism differs significantly sensitivity of detection and the likelihood of detecting com- from other photosynthetic bacteria in that H. chlorum lacks ponents that have widely different absorbance maxima. The the well-developed intercytoplasmic photosynthetic mem- detector and data reduction are under computer control; we branes of purple photosynthetic bacteria (2). Instead, the used the Hewlett-Packard liquid chromatography program photosynthetic apparatus of H. chlorum is contained within LCSurvey in the software package 89082A. This instrument its cell wall (3). The organism also lacks chlorosomes (4), the is also a recording spectrophotometer, which can record organelle characteristically present in green photosynthetic absorption spectra in 0.1 s. This feature was used to follow bacteria. The visible absorption spectrum indicates the pres- the time dependence of the photoisomerization of Bpheog ence in H. chlorum of a bacteriochlorophyll hitherto un- spectrophotometrically. known in nature. On the basis of NMR studies on alteration Mass Spectrometry. Recent developments in heavy ion products of the chlorophyll, Brockmann and Lipinski (5) desorption mass spectrometry (11), particularly 252Cf-PDMS, assigned structure 1 (see Fig. 4) to the chlorophyll, designat- have made it a relatively simple matter to obtain molecular ed bacteriochlorophyll g (BChlg). [Structure 1 is shown with weights of chlorophylls and chlorophyll derivatives (12, 13). farnesol as the esterifying alcohol in place of the geranylge- This is a time-of-flight technique in which molecular ions of raniol originally suggested by Brockmann and Lipinski (5).] the sample are produced by impact of fission fragment ions BChlg is exceptionally sensitive to oxygen and becomes resulting from the spontaneous fission of 252Cf. The measured increasingly sensitive to light in the process of purification. molecular weights of molecular ions (or ions formed by We have now prepared pure bacteriopheophytin g (Bpheog) corre- on the multimilligram scale, and BChlg itself has been fragmentation) are average molecular weights-i.e., isolated in high purity on a very small scale. We have Abbreviations: Bpheo, bacteriopheophytin; BChl, bacteriochloro- phyll; 252Cf-PDMS, californium-252 plasma desorption mass spec- The publication costs of this article were defrayed in part by page charge trometry. payment. This article must therefore be hereby marked "advertisement" *Permanent address: Photosynthetic Bacteria Group, Department of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Biology, Indiana University, Bloomington, IN 47405. Downloaded by guest on September 28, 2021 2570 Chemistry: Michalski et al. Proc. Natl. Acad. Sci. USA 84 (1987) 2571 sponding to the following atomic weights: C, 12.011; H, The molecular weight of the product as determined by mass 1.0079; N, 14.0067; 0, 15.9994; Mg, 24.3096. spectroscopy is consistent with the structure assigned by ESR. ESR spectra were recorded on a Varian E-9 Spec- Brockmann and Lipinski (5) to BChlg. Exposure of the trometer with a TE102 rectangular cavity. The sample tem- 760-nm absorbing pigment to light and air causes complete perature was regulated with an Air Products Heli-Tran transformation within 3 min to a species absorbing maximally low-temperature ESR accessory. The Bpheog cation free at 660 nm, which, based on the observations of the radical was prepared from a frozen solution of Bpheog in photoisomerization ofBpheog described below, is very likely methanol/methylene chloride (4:1). Small amounts of a chlorophyll a. Because of the extreme sensitivity of BChlg to stable free radical were formed by irradiation at 5 K with red light and air, we have not as yet been successful in producing light of a wavelength of >750 nm. Much larger amounts of multimilligram amounts of BChlg, but microgram amounts free radical were produced when a small amount (5%) of can be readily prepared by the procedures we describe here. tetranitromethane (extreme caution: hazardous chemical) Preparation of Bpheog. Frozen cells of H. chlorum were was incorporated into the solution before freezing and extracted with ethanol/diethylether/hexane (5:2:1). The ex- illumination. Tetranitromethane forms charge transfer com- tract was centrifuged to remove debris, and the supernatant plexes with molecules containing delocalized ir-electron solution was acidified with 2 M hydrochloric acid. After systems. Illumination above the charge transfer band pro- washing with water, the solvent was removed by evapora- duces a radical cation and a tetranitromethane radical anion, tion, and the residue was redissolved in a small amount of which promptly dissociates, leaving the radical cation stabi- diethylether/hexane (1:9) and chromatographed on a sugar lized in the frozen solution. column using 1% isopropanol in hexane as the eluting agent. The Bpheog triplet state was produced by illumination at A dark green fraction absorbing maximally at 752 nm was 5 K of a solution of Bpheog in methanol/methylene chloride collected and further purified by HPLC on an Altex silica (10 (4:1). The light from a Varian Eimac 300W Xe arc lamp was ,4m) column (10 x 250 mm) with the same eluting agent. All filtered to produce light with a wavelength of >750 nm. The operations were conducted insofar as possible in the dark lamp intensity was modulated sinusoidally. The unfiltered with rigorous exclusion of oxygen. Bpheog is considerably output of the ESR spectrometer 100-kHz receiver was fed to more stable than BChlg. Minimizing exposure to light and air an Ithaco Dynatrac 391A lock-in amplifier and demodulated is all that is necessary to avoid alteration and loss of material. with respect to the modulation frequency of the lamp. The Optical Spectra. Visible absorption spectra of Bpheog and output was the photoexcited triplet