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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 b and g in the biosynthesis of (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- 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 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 , 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-, 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 products of the , 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 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 . Because of the extreme sensitivity of BChlg to stable free radical were formed by irradiation at 5 K with 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 /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 ESR from Bpheog, with BChlg are shown in Fig. 1. The differences in the visible no contribution from any stable free-radical species present spectra are sufficient to make it possible to detect either in the sample. compound in mixtures of the two. Molecular Weights ofBpheog and BChlg. The molecular ion RESULTS ofBpheog as measured by 252Cf-PDMS has an (average) mass Extraction and Purification of BChig. Frozen cells of H. of797.0 atomic mass units (Fig. 2A). The (average) molecular chlorum were extracted with methanol/water/pyridine weight calculated from the Brockmann and Lipinski (5) (95:4:1) or ethanol/diethylether/hexane (5:2:1) under a ni- structure is 865.2 atomic mass units. The difference corre- trogen atmosphere and in the dark. Four extractions were sponds to C5H8, which is the mass difference between needed for complete removal of the pigment from the cells. geranylgeraniol and farnesol. A fragment with a mass of592.8 The extract was then centrifuged to remove cell debris. The (M-farnesyl+H)+ strongly indicates that the esterifying al- absorption spectrum characteristic of BChlg is essentially cohol in Bpheog is indeed farnesol. The molecular weight of unchanged after storage of the crude extract for 4 hr in the a small sample of pure BChlg prepared by HPLC was found dark under nitrogen. On further standing, a peak at 665 nm to be 819.5; calculated (average) molecular weight with slowly grows in, which indicates the formation of small farnesyl as the esterifying moiety, 819.3; a molecular frag- amounts of chlorophyll a-like substances. ment with mass 615 can be assigned to (M-farnesyl+H)' (Fig. The crude extracts were evaporated to dryness at 280C and 2B). Farnesol, not geranylgeraniol, is the esterifying alcohol further dried under high vacuum for 1 hr. The residue was in BChlg. dissolved in a mixture of diethylether and ether Zero Field-Splitting Parameters of 3Bpheog. The light- (bp, 350C-60'C) (1:5). The solution was washed first with a modulated ESR spectrum of 3Bpheog (Fig. 3) showed a saturated solution of sodium chloride in water and then with typical triplet spectrum with strong Z-axis peaks and weaker pure water. After evaporation to dryness, the residue was X- and Y-axis peaks. The D value of the triplet is 0.0225 + dissolved in a small amount of the diethylether/petroleum 0.0010 cm-1 and the E value is 0.0015 ± 0.0010 cm-'. D is a ether mixture and absorbed on a sugar column. The pigments measure ofthe relative size ofthe conjugation system that an were separated by gradient elution with a petroleum electron is free to move through, and E is a measure of the ether/isopropanol mixture, whose concentration varied from axial symmetry ofthe conjugation system. Comparison ofthe an initial value of 0.2% to a final concentration of 1% zero field-splitting parameters of 3Bpheog with those of isopropanol. Elution was stopped when the pigments were 3Bpheoa shows that the D values are about the same for the well separated into two zones, one a slower-moving green two, but that the E value of Bpheog is about one-half. zone (A) and the faster-migrating blue gray zone (B). ESR of Bpheog+. The cation free radical was generated by The two zones were collected separately, and the pigments irradiation with red light in either the absence or presence of were eluted from the adsorbent with a mixture of ethyl tetranitromethane. The ESR signal for both the unstabilized alcohol/petroleum ether (1:5). Evaporation gave two prod- and tetranitromethane-stabilized samples is a featureless ucts. Fraction A showed no visible absorption maxima at gaussian line with a peak-to-peak linewidth of 11.6 gauss. wavelengths >665 nm and was clearly a chlorophyll a-like This signal is somewhat narrower than that of Bpheoa+, degradation product of BChlg formed primarily by photo- which is consistent with the absence of a proton at position isomerization. The blue gray fraction B absorbed light max- 4 in ring II of Bpheog; the single protons at positions 3, 4, 7, imally at 760 nm and was free of any contaminant absorbing and 8 have large hyperfline coupling constants, and the at 665 nm. absence of one ofthese protons would be expected to narrow Analysis of the blue gray zone by reversed-phase HPLC the signal. The light-induced free radical signal is not revers- shows one component migrating with a retention vol of 21.54 ible at S K. The g value of 2.0025 indicates that the unpaired ml in methanol/acetonitrile/tetrahydrofuran (10:7:3) and electron is confined entirely to the ir-system of the Bpheog' 29.40 ml in methanol/acetonitrile/tetrahydrofuran (90:7:3). cation. HPLC and UV/visible spectra show no significant Downloaded by guest on September 28, 2021 2572 Chemistry: Michalski et al. Proc. Natl. Acad. Sci. USA 84 (1987)

366 A 392 754 516 I~~~~~M 682

365 405 76648

a) C.) I CO C Cs 0 0en 412 C

664 510 536 608 3!50 450 550 650 750

600 700 Mass, m/z 500 Wavelength, nm FIG. 2. 252Cf-PDMS mass spectra of Bpheog (A) and BChlg (B). The mass spectrum offully photoisomerized Bpheog is identical with FIG. 1. Visible absorption spectra in diethylether solution. (A) the spectrum in A. Bpheog; (B) BChlg; (C) fully photoisomerized Bpheog; (D) time- dependent photoisomerization of Bpheog in isopropanol/hexane provide strong confirmation of the validity of structure 1. It solution. should be recognized, however, that the structures assigned to the chlorophylls (other than chlorophyll a), especially the formation of alteration products of Bpheog in the course of positions of the side chains, are almost entirely based on these experiments. analogy to the structure of chlorophyll a. Although these Photoisomerization. BChlg and Bpheog, like their close molecular structures are probably correct in their general relatives BChlb and Bpheob, readily undergo photoisomer- aspects, some uncertainties remain, because the chromato- ization, the pheophytins much less so than the very light- graphic procedures and magnetic resonance spectroscopy, sensitive Mg-containing chlorophylls. On the basis of the on which the molecular structures of most ofthe chlorophylls Brockmann and Lipinski (5) structure, isomerization of are based, have not yet been demonstrated to provide Bpheog should produce a pheophytin identical in all other unequivocal assignments of the macrocycle side-chain posi- respects with pheophytin a, except that the isomerization tions. product is esterified with farnesol instead ofphytol. We have BChlg 1 and BChlb 2 both contain the unusual ethylidene verified by optical spectroscopy, HPLC, and 252Cf-PDMS group, which suggests at first sight a close relationship that this is in fact the case. Solutions of Bpheog in either between the two host organisms. However, the esterifying hexane or isopropanol/hexane (1:99) kept in the dark are alcohols of BChlb and -g differ to such an extent as to raise stable even in the presence of oxygen for periods of several questions about a close relationship between H. chlorum and days. In white light, rapid isomerization in these solvent Rhodopseudomonas viridis. In general, chlorophyll a of green systems occurs with a half-time of -42 min. In our experi- plants and , and BChla of purple photosynthetic ments with Bpheog, we have not observed any of the bacteria are esterified with C20 alcohols. Scheer and his collab- oxidation products detected by Steiner et al. (23). orators have shown that BChlb extracted from R. viridis is esterified with (14, 15), and BChlb from Ectothiorhodo- DISCUSSION spira halochloris is esterified with A2,10-phytadienol (16), both All of our optical and mass spectroscopic measurements are of which are C20 alcohols. BChlc (17), -d (18), and -e (19), the consistent with the structure of BChlg advanced by characteristic chlorophylls ofgreen photosynthetic bacteria, are Brockmann and Lipinski (5), with the important exception of esterified with farpesol, a C15 alcohol. The esterifying alcohol the nature of the esterifying alcohol. Structure 1 predicts that thus appears to be more species specific than the macrocycle isomerization should produce chlorophyll a, which of course structures ofthe chlorophylls. A possible reason for this may be would then be esterified with farnesol rather than the usual that the ethylidene group is a normal intermediate in the phytol. Our chromatographic, 252Cf-PDMS, optical, and biosynthesis ofall ofthe chlorophylls, but that the =CH-CH3 NMR measurements on the photoisomerization products of group is retained as such only by BChlg 1 and BChlb 2, and by Bpheog and BChlg show this to be the case. These findings (20, 21) and (22), the Downloaded by guest on September 28, 2021 Chemistry: Michalski et al. Proc. Natl. Acad. Sci. USA 84 (1987) 2573

FIG. 3. Light-induced ESR triplet spectrum: 1000-gauss scan centered at 3262 gauss; microwave power, 5 mW; frequency, 9.127 GHz; field modulation, 40 gauss; light modulation frequency, 400 Hz.

of and , respective- From the information that has become available for the ly, the widely distributed auxiliary protein pigments in chromopeptides of the cyanobacteria, we can visualize the cyanobacteria. In all of these instances, the ethylidine group is biosynthesis of chlorophyll as occurring on the surface of a found in the same molecular position-i.e., position 4 ofring II. We have shown here that photoisomerization of Bpheog 2, -COCH 3 forms pheophytin a, and judging from optical spectra and mass measurements, BChlg is likewise photoisomerized to BChla 3, -CH 3, H chlorophyll a. Steiner et al. (23) have studied the photo- { 4, -CH 2CH 3, H isomerization and photooxidation of BChlb and found small A amounts of 2-desvinyl-2-acetyl-chlorophyll a together with various oxidation products, whose formation was attributed to residual oxygen in the system. 2-Desvinyl-2-acetyl-chlo- rophyll a is closely related to BChla. In principle, all of the H3C known chlorophylls can be derived from intermediates that BChlb Chla contain an ethylidene group by a combination of isomeriza- D tion and/or reduction reactions (24) (Fig. 4). 2, -COCH3 2, -CH=CH2 Nothing is known directly about the genesis of the 3, -CH3,H } {3, -CH3 ethylidene group in BChlb and -g. [Recently, Smith et al. (25) 4, =CH-CH3 H3C I 4, -CH 2 CH3 have shown that reduction of a macrocyclic double bond can result in inward migration of a vinyl double bond to form an 2 ethylidine group. Whether this reaction is relevant to the present discussion is uncertain.] Considerable insight into the origins of the =CH-CH3 side chain in the , however, has been achieved. Crespi and co-workers pro- posed that phycocyanobilin (26, 27) is linked to the Bc apoprotein by an ester link and by a thioether bond to the 2, -CH(OH)CH 3 -SH cysteine of a cysteine side chain at position 2 of ring II, BChlc 13, -CH3, H a linkage analogous to that found in cytochrome c. More l4, -alkyl, H recently, Rudiger et al. (28, 29) and Schoenleber et al. (30, 31) 1 0, 2H have collected a large amount of evidence that indicates that FIG. 4. Suggested scheme for formation of chlorophylls from a both phycocyanobilin and phycoerythrobilin are attached to common intermediate. The molecular structure in the center is that protein by thioether links to cysteine at the molecular of BChlg. The side chains present at positions 2, 3, and 4 are shown position that has the ethylidene side chain in the free for each chlorophyll. R1 is farnesyl for BChlg and BChlc and phytyl . Model experiments by Rudiger and co-workers for chlorophyll a (Chla), BChla, and BChlb. The reactions involved (32, 33) show that a thioether link, -CH-CH3, can form an in the transformations are as follows: A, hydration ofthe vinyl group to hydroxyethyl and oxidation to acetyl, and isomerization and S-R addition of 2H at positions 3 and 4; B, isomerization of the hydrogen atom at position 3 to position 4; C, hydration of the vinyl group at ethylidene group, =CH-CH3, by elimination. Leaving position 2 and isomerization in ring II; D, hydration and oxidation at groups other than the -S-R group formed by interaction position 2. Since BChlc is a pyrochlorophyll, decarboxylation at with nucleophilic groups in other amino acid side chains may position 10 must occur at some point, as well as alkylation at the 8 also be involved. methine position (R2). Downloaded by guest on September 28, 2021 25742574Chemistry:Chemistry:MichalskiMichaiskietetal.al.~~~Proc.Nati. Acad. Sci. USA 84 (1987)

protein to which the macrocycle is tethered by a thioether (or Schiffer, M. (1986) FEBS Lett. 205, 82-86. 8. R. S. H. & R. C. similar linkage) at position 4 of ning II to one of the amino acid Fuller, C., Sprague, G., Gest, Blankenship, from (1985) FEES Lett. 182, 345-349. side chains. Cleavage of the chlorophyll intermediate 9. Nuijus, A. M., van Dorsen, R. J., Duysens, L. M. N. & the protein by an elimination reaction would leave an Amez, J. (1985) Proc. Nati. Acad. Sci. USA 82, 6865-6868. ethylidene group on the chlorophyll macrocycle at position 4 10. Brok, M., Vasmal, H., Horikx, J. T. G. & Hoff, A. J. (1985) of ring II and a vinyl group at position 2 of ring I. The ethyl FEES Lett. 194, 322-326., group at position 4 of chlorophyll a and BChl'a could be 11. Fenselau, C., Yergey, J. & Heller, D. (1983) Int. J. Mass formed from the ethylidine group following a 1,3 proton shift Spectrom. Ion Physics 53, 5-36. of the proton at position 3; Walsh (34) describes a number of 12. Hunt, J. E., Schaber, P. M., Michalski, T. J., Dougherty, well-characterized -catalyzed rearrangements of this R. C. & Katz, J. J. (1983) Int. J. Mass Spectrom. Ion'Physics 53, 45-58. kind. The acetyl or hydroxyethyl functions present in the 13. J. R. Katz, J. J. & bacterial can be derived from' a Hunt, E., Macfarlane, D., Dougherty, chlorophylls readily vinyl R. C. (1980) Proc. Nati. Acad. Sci. USA 77, 1745-1748. group. All of the necessary chemical reactions have well- 14. Scheer, H., Svec, W. A., Cope, B. T., Studier, M. H., Scott, established precedents, and there do not appear to be any R. G. & Katz, J. J. (1974) J. Am. Chem. Soc. 96, 3714-3716. major mechanistic obstacles in a unified scheme for the 15. Steiner, R., Wieschoff, H. & Scheer, H. (1982) J. Chromatog- generation of all of the known chlorophyll from a common raphy 242, 127-134. intermediate as proposed here. 16. Steiner, R., Schdfer, W. Blos, I., Wieschoff, H. & Scheer, H. In green photosynthetic bacteria, it has long been known (1981) Z. Naturforsch. 36c, 417-420. that the major chlorophylls, BChlc, -d, and -e, are accom- 17. Caple, M. B., Chow, H.-C. & Strouse, C. E. (1978) J. Biol. Chem. 253, 6730-6737. panied by small amounts of a long-wavelength chlorophyll 18. Gloe, A. & Pfennig, N. (1974) Arch. Microbiol. 96, 93-101. that has been reported to be BChla. Other than photosyn- 19. Brockma~ni, H., Gloe, A., Risch, N. & Trowitzsch, W. (1976) thetic green bacteria, no organism's that contain a dihydro- Liebigs Ann. 1976, 566-577. are known to contain a tetrahydroporphyrin. The 20. Crespi, H. L., Boucher, L. J., Norman, G. D., Katz, J. J. & reverse, is also the case: no photosynthetic organism whose Dougherty, R. C. (1967) J. Am. Chem. Soc. 89, 3642-3643. principal chlorophyll is BChla is known to contain significant 21. Cole, W. J., Chapman, D. J. & Siegelman, H. W. (1967) J. amounts of dihydroporphyrins. We think it is possible that Am. Chem. Soc. 89, 3643-3644. the long-wavelength chlorophylls in green photosynthetic' 22. Chapman, D. J., Cole, W. J. & Siegelman, H. W. (1976) J. bacteria may be surviving dihydroporphyrin precursors con- Am. Chem. Soc. 89, 5976-5977. 23. Steiner, R., Cmiel, E. & Scheer, H. (1983) Z. Naturforsch. taining ethylidene groups, the greater parts of which have 38c, 748-752. isomerized to form I3Chlc, -d, or -e. It is evident that 24. Castelfranco, P. A. & Beale, S. I. (1981) in The Biochemistry photoisomerization of the --=CH-CH3 grou~p in chlorophylls of Plants: , eds. Hatch, M. D. & Boardman, can take place very rapidly in vitro. Nothing is known about N. K. (Academic, New York), Vol. 8, pp. 375-421. the rate of such processes in vivo, or about the mechanisms 25. Smith, K. M., Simpson, D. J. & Snow', K. M. (1986) J. Am. that prevent isomerization in BChlb'and -g in the intact Chem. Soc. 108, 6834-6835. organisms. If such chlorophylls are in fact intermediates in 26. Crespi, H. L., Smith, U. & Katz, J. J. (1968) Biochemistry 7, the biosynthesis of chlorophyll, it will probably be necessary 2232-2242. to look for them in total darkness and in the absence of 27. Crespi, H. L. & Smith, U. (1970) Phytochemistry 9, 205-212. 28. Thuimler, F. & Rudiger, W. (1983) Z. Naturforsch. 38c, oxygen. 359-368. 29. Muckle, G., Otto, J. & Ruidiger, W. (1978) Hoppe-Seylers Z. 1. Gest, H. & Favinger, J. L. (1983) Arch. Microbiol. 136, 11-16. Physiol.'Chem. 359, 345-355. 2. Oelze, J. (1983) in The Phototropic Bacteria, ed. Ormerod, 30. Schoenleber, R. W., Leung, S.-L., Lundell, D. J., Glazer, J. G. (Univ. California Press, Berkeley, CA), pp. 8-34. A. N. & Rapoport, H. (1983) J. Am. Chem. Soc. 105, 4072- 3. Miller, K. R., Jacob, J. S., Smith, U., Kolaczkowski, S. & 4076. Bowman, M. K. (1986) Arch. Microbiol. 146, 111-115. 31. Schoenleber, R. W., Lundell, D. J., Glazer, A. N. & 4. Stehlin, L. A., Golecki, J. R. & Drews, G. (1980) Biochem. Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484. Biophys. Acta 589, 30-45. 32. E. H.-P. & W. (1975) Liebigs Ann. 5. Brockmann, H. & Lipinski, A. (1983) Arch. Microbiol. 136, Kost-Reyes, Kbst, Rudiger, 17-19. 1975, 1594-1600. 6. Michel, H. & Deisenhofer, J. (1986) in Encyclopedia of Plant 33. Schoch, S., Klein, G., Linsenmeier, U. & Ruidiger, W. (1976) Physiology: Photosynthesis III, eds. Staehlin, A. C. & Liebigs Ann. 1976, 549-558. Arntzen, C. J. (Springer, Berlin), Vol. 19, pp. 371-381. 34. Walsh, C. (1979) Enzymatic Reaction Mechanisms (Freeman, 7. Chang, C. H., Tiede, D., Tang, J., Smith, U., Norris, J. & San Francisco), pp. 599-609. Downloaded by guest on September 28, 2021