Clostridium Tetanomorphum (Bilatriene/Uroporphyrin-Related/Enzymatic Formation/Purification/Characterization) PHILLIP J

Home , Corrin, Sirohydrochlorin, Tetrapyrrole

Proc. NatL Acad. Sci. USA Vol. 80, pp. 3943-3947, July 1983 Biochemistry

Bactobilin: Blue bile pigment isolated from Clostridium tetanomorphum (bilatriene/uroporphyrin-related/enzymatic formation/purification/characterization) PHILLIP J. BRUMM*t, JOSEF FRIED*t, AND HERBERT C. FRIEDMANN*§ Departments of *Biochemistry and tChemistry, The University of Chicago, Chicago, Illinois 60637 Contributed byJosef Fried, March 31, 1983

ABSTRACT A blue bile pigment, possessing four acetic and used were reagent grade or better. Silica gel for flash chro- four propionic acid side chains has been isolated from extracts of matography and a flash chromatography column, 3.4-cm di- the anaerobic microorganism Clostridium tetanomorphum and in ameter, were obtained from J. T. Baker. Whatman high-per- smaller amounts from Propionibacterium shermanii. The com- formance silica thin-layer chromatography plates with a pre- pound could be prepared in larger amounts by incubation of C. adsorbent spotting area (type LHP-K, 10 x 20 cm) and E. Merck tetanomorphum enzyme extracts with added 8-aminolevulinic acid. precoated cellulose thin-layer chromatography plates were ob- The ultraviolet-visible, infrared, and proton magnetic resonance tained from Anspec (Ann Arbor, MI). For fluorescence detec- spectra of the pigment indicate a chromophore of the biliverdin tion, a lamp emitting at 365 nm was used. C. tetanomorphum type. Field-desorption mass spectrometry of the purified methyl cells (ATCC 15920) were grown on a medium containing ester showed a strong molecular ion at m/e = 962. This corre- yeast sponds to the molecular weight expected for the octamethyl ester extract and monosodium glutamate (based on medium 163, of a bilatriene type of bile pigment structurally derived from uro- American Type Culture Collection) as described (18) and were porphyrin 1m or I. Of the five possible structures, two could be collected by centrifugation. Uroporphyrin and C-methylated eliminated by proton magnetic resonance spectroscopy. The name mono-, di-, and trimethylisobacteriochlorins (Factors I, II, and bactobilin is proposed for this previously unreported bile pigment. III) were isolated as their octamethyl esters by standard methods (19, 20). Sirohydrochlorin (Factor II) octamethyl ester was The open-chain tetrapyrrole compounds known as bile pig- given by A. I. Scott (College Station, TX); dimethyl- and ments are widely distributed. They are found free or bound to trimethylisobacteriochlorin dilactone octamethyl esters were protein in mammals, birds, amphibians, reptiles, fish, mol- given by V. Koppenhagen (Braunschweig-Stockheim, Federal luscs, and insects and in algae and higher plants (for reviews, Republic of Germany). see refs. 1-9). As side chains or 1&carbon ring substituents, all Enzyme Preparation. An acetone powder was prepared from bile pigments described thus far have four methyl groups, two the bacteria as follows. Immediately after centrifugation, 170- propionic acid groups, and two vinyl groups, one or both of which 180 g of packed cells (obtained from 56 liters of growth me- can be isomerized to ethylidene (4, 10, 11) or reduced to ethyl dium) were uniformly suspended in 170-180 ml of ice-cold 2% (6, 10, 11). These substituents are arranged in the sequence 2-mercaptoethanol with the help of a nonaerating stirrer (Kraft found in protoporphyrin IX; in fact, all these bile pigments are Apparatus, distributed by Glas-Col Apparatus, Terre Haute, formed from protoheme (8, 12-14), whose oxidative breakdown IN). The cold suspension was added slowly with stirring to 1.7- to biliverdin is catalyzed by the enzyme heme oxygenase (15- 1.8 liters of acetone at -100C; the stirring was continued for 17). Bile pigments thus far have not been detected in prokary- 5 min at about 0C. The material, collected by suction filtration, otes. The present paper reports the isolation and in vitro for- was resuspended uniformly in 1.7-1.8 liters of cold acetone with mation of a bile pigment from the bacterium Clostridium nonaerating stirring, collected again by filtration, and trans- tetanomorphum. This anaerobic organism makes uroporphyrin- ferred to a round-bottom flask. The remaining acetone was re- ogen, the precursor of vitamin B-12, but not heme, protopor- moved in vacuo at room temperature with the aid of a rotary phyrinogen, or coproporphyrinogen. The isolated bile pigment evaporator. The powder could be stored over a desiccant at is of interest not only because of its detection in a prokaryote, -20'C for at least 3 months without significant activity loss. but also because its 13-carbon ring substituents, four acetic acid The average yield was 50 g (i.e., about 29% of the packed cell and four propionic acid groups, correspond to those of a uro- weight). For enzyme extraction, 30 g of powder was stirred for porphyrin and not of protoporphyrin. 10 min at room temperature without aeration in 300 ml of 100 mM Tris'HCI buffer, pH 8.0/0.2% 2-mercaptoethanol. The suspension was centrifuged for 20 min at 48,000 X g. MATERIALS AND METHODS Preparation of Bacterial Bile Pigment. To the clear super- Supplies. -Aminolevulinic acid was obtained from Sigma; natant solution, 60 mg of 8-aminolevulinic acid dissolved in 10 biliverdin IXa dimethyl ester and the fully esterified methyl ml of 100 mM Tris (pH 8.0) was added with gentle swirling. esters of uroporphyrin III, coproporphyrin III, protoporphyrin The mixture was incubated 18 hr in the dark at 370C. Precip- IX, and heptacarboxylporphyrin I, were from Porphyrin Prod- itated protein was removed by centrifugation for 20 min at 48,000 ucts (Logan, UT). The n-hexane used for flash chromatography X g. The uroporphyrins and related anionic substances in the was "95 + %" (Aldrich), whereas the n-hexane for thin-layer supernatant solution were esterified after retention on an anion chromatography was 99 mol % pure (Fisher). Other chemicals exchanger by a modification of the method of Bergmann et al.

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(19): about 5 g of DEAE-Sephadex A-25 that had been equil- Table 1. Ultraviolet-visible and infrared absorption maxima ibrated with 100 mM potassium phosphate buffer (pH 7.4) was in chloroform added in aqueous suspension with slight swirling. The ion ex- Ratio Ratio changer was collected by filtration and washed three times with UV-Vis 380/ IR 1,730/ 250 ml of water and three times with 250 ml of methanol. To Ama,) nm 650 Pmax, cm- 1,700 esterify the substances retained on this anhydrous DEAE- 200 Biliverdin IXa 1,732 (ester C=O) Sephadex, ml of methanol/sulfuric acid, 90:10 (vol/vol), dimethyl 376, 642-670 3.76 1,695 (amide C=O) 0.76 was added. The vessel was tightly capped. After 18 hr at 370C, ester the supernatant solution was collected by filtration, and the Bactobilin 1,735 (ester C=O) DEAE-Sephadex was washed with a small volume of chloro- octamethyl 369,644 3.56 1,705 (amide C=O) 1.27 form. The esters were extracted into chloroform by agitating ester the filtrate with about 200 ml each of water and of chloroform. The reddish chloroform layer was collected and washed twice Vis, visible. with 200 ml of 0.1 M ammonia. In this process yellow material was removed. Three washes with 200 ml of water followed. The RESULTS chloroform was removed in vacuo at room temperature, and the The present work resulted from studies of intermediates in vi- residue was dissolved in 10 ml of chloroform. The material was tamin B-12 biosynthesis. After incubation of enzyme extracts subjected to flash chromatography on 180 ml of silica gel that of C. tetanomorphum with 6-aminolevulinic acid at pH 8 in the had been equilibrated with a mixture of n-hexane/2-propanol/ presence of 2-mercaptoethanol and workup by conventional methanol, 5:2:1 (vol/vol) (refs. 19, 21, 22; and the unpublished methods (19), it was found that when the reaction products in method of A. I. Scott was incorrectly quoted in ref. 21 re- but the form of their methyl esters were submitted to thin-layer peated correctly in refs. 19 Pressure was and 22). applied with chromatography on silica plates in a widely used solvent (24), nitrogen or argon. was The column treated with 400 ml of this a certain amount of pigmented material remained near the or- solvent, followed by 200 ml of n-hexane/2-propanol, 1:1.5 (vol/ igin. Thin-layer chromatography of this material in chloroform vol). The eluates were combined, and the solvent was removed containing from 2% to 3% methanol showed small amounts of in vacuo. a striking hitherto unreported nonfluorescent blue material. The solid residue was dissolved in 20 ml of chloroform and The first indication that this substance is a bile pigment and not to area applied the preadsorbent spotting of 10 Whatman LHP- a cyclic tetrapyrrole was provided by the fact that it gave a pos- K were silica plates, which then developed in dim light in chlo- itive Gmelin reaction (multiple, gradually developing colors upon roform/methanol, 100:3 (vol/vol). The solvent troughs were careful addition of concentrated nitric acid) (described by Tie- lined with saturation pads. The main component was rapidly demann and Gmelin in 1826; for recent references see refs. 2, moving uroporphyrin octamethyl ester. The blue bands (Rf 25, and 26). This conclusion was confirmed by the absorption 0.5) were scraped off, and the pigment was eluted with 50 ml spectrum of the compound in chloroform, which indicated a of methanol. After removal of the silica gel by suction filtration close relationship to biliverdin methyl ester. The data are sum- and of the methanol by evaporation in vacuo, the pigment was marized in Table 1. Both substances show a broad absorbance in on redissolved 2 ml of chloroform, rechromatographed 4 LHP- band above 640 nm and a much sharper and more intense band K plates as above, again scraped off the plates, and extracted in the near ultraviolet region. The close similarity between the into methanol. The solvent was removed as before. The pig- two sets of maxima, which is also true of the inflection seen in ment, which still contained traces of fluorescent impurities, the two absorption curves, indicates that the two chromophoric was redissolved in 1 ml of chloroform and immediately chro- systems are essentially identical. The slight shift in the sharper matographed on one LHP-K plate in chloroform/methanol, 100:1 band to a lower wavelength may be due to the absence of the (vol/vol) (22). As many as 8-10 repeat developments were re- two vinyl groups in the new substance. Neither of the absorp- quired to separate the blue nonfluorescent bile pigment from tion bands showed a Cotton effect, demonstrating the absence traces of reddish-fluorescent material. It is important to use a of chirality in the molecule. The position of the strong infrared fairly large volume (say 100 ml) of the solvent because, in low methanol concentrations, the bile pigment and fluorescent ma- Table 2. behavior of various esters terial are not separated. The blue band was scraped off and eluted Chromatographic methyl with methanol. The silica gel was removed by filtration, and the Rf values in different solvent solvent was evaporated as before. systems* About 2 mg of the purified ester was obtained after incu- Compound 1 2 3 4 bation with 8-aminolevulinic acid of enzyme extract from 145 Protoporphyrin IX 0.81 0.91 0.67 0.96 g of acetone powder, corresponding to about 500 g of packed Coproporphyrin III 0.69 0.78 0.59 0.78 bacterial cells (preparative conditions not optimized). The pig- Uroporphyrin III 0.31 0 0.33 0 ment could be stored in the dry state or in a concentrated meth- Heptacarboxylporphyrin I 0.44 - 0.40 - anol solution at -20°C. Storage in chloroform is not recom- Factor I 0.37 0.52 0.39 0.45 mended (23). In air and light, the pigment on the developed Factor II 0.35 0.57 0.39 0.59 thin-layer chromatography plates rapidly decomposed, but the Factor III 0.34 0.54 0.39 0.46 thin-layer chromatography patterns could be preserved at room Factor II dilactone 0.18 0.30 0.27 0.07 temperature for at least 6 months, provided the plates were stored Factor III dilactone 0.19 0.28 0.29 0.05 under argon in the dark. The compound was destroyed when Biliverdin IXa 0.29 0.78 0.30 0.59 chromatography was attempted on preparative silica plates. Bactobilin 0.05 0.50 0.06 0 Spectra. Infrared spectra were recorded on a Perkin Elmer * Solvents: 1 (on silica), benzene/ethyl acetate/methanol (85:13.5:1.5, Model 297 spectrophotometer; ultraviolet-visible spectra, on a vol/vol) (24); 2 (on silica), n-hexane/2-propanol/methanol (50:20:10, Cary 219; and proton magnetic resonance spectra, on a Uni- vol/vol) (19); 3 (on silica), chloroform/methanol (100:1, vol/vol) (22); versity of Chicago-built DS-1000 spectrometer at 500 MHz 4 (on cellulose), n-hexane/chloroform/1-propanol/2-propanoI (94: equipped with a Nicolet 1180 Data Acquisition system. 4:2:0.5, vol/vol) (22). Downloaded by guest on September 27, 2021 Biochemistry: Brumm et al. Proc. Natl. Acad. Sci. USA 80 (1983) 3945 absorption peaks supports the conclusion that the two com- the others. This observation indicated the presence of eight pounds are structurally closely related. The fact that the iso- ester groups. A comparison of the chemical shifts of these groups lated bile pigment differed from biliverdin was confirmed by and those of the methine and methylene protons, with the cor- thin-layer chromatography in four different systems (Table 2). responding signals shown by biliverdin IXa dimethyl ester, is The compound moved as one band in all of the solvent systems given in Table 3. Three low-field signals at 86.784, 8 6.015, and tried. If one assumes a biliverdin-like bilatriene having the side 8 5.976 corresponded closely to the three meso-proton signals chains found in uroporphyrin III, one arrives at the four iso- in biliverdin dimethyl ester. meric structures, I-IV (Fig. 1), with a molecular weight of 962 for the octamethyl ester. Indeed, mass spectrometry of the DISCUSSION methyl ester of the compound gave rise to a strong molecular The new compound, whose chromophore structure and mo- ion, m/e = 962. lecular weight indicate the presence of oxygen attached to the The most detailed information regarding the structure of the two outer rings, qualifies as a bile pigment by the definition of bile pigment was obtained from the 500-MHz proton NMR Rudiger (2). We have considered two names for this substance: spectrum of the methyl ester in both C2HC13 and C2H2CI2. The bactoverdin, in analogy to biliverdin, and bactobilin, in analogy spectra in the two solvents were nearly identical. The spectrum to the bound phycobilins (ref. 1; see p. 88 of ref. 27) from cer- in C2HCl3 showed seven separate methylcarboxylate signals, tain algae and all higher plants and to pterobilin from Lepi- the one at chemical shift 8 3.653 having twice the intensity of doptera (28). The former name, although it indicates a 5,10,15- bilatriene, was discarded in order not to add to eccentricity and COOH COOHCOOH COON I I I confusion (7) because, like biliverdin, this substance is not green COOH CH2 COOH CH2 CH2 COOH COOH CH2 but blue. Hence, we propose the second name. I I I I I I I I The presence of but three methine singlets in the 500-MHz CH CHp CH2 CH2 CH2 CH2 CH+ CH2 spectrum provides strong evidence that the methyl ester of the purified substance constitutes a single product and not a mixture of isomers produced by cleavage at more than one of the methine bridges. The interpretation of the NMR spectra was 0N N N 0 (I) H H H greatly aided by the work of Bonnett and McDonagh (29), who prepared all four possible isomers of biliverdin and reported COON COOH COON COOH their properties. From the available data, it was not possible to I I I I COOH CH2 CH2 COON COON CH2 COOH CH2 deduce unambiguously which of the four possible isomers had I I I I I I I I been formed. However, structures III and IV (Fig. 1), corre- CH2 CH2 H CH2 CN2 CH2 CH2 CH2 sponding to cleavage at the y and 8 methine carbons, could be ruled out on the basis of the following considerations. Bonnett and McDonagh showed that the propionyl methylene protons 0 N N N N 0 (11) a to the pyrrole rings exhibited significantly different chemi- H H H cal shifts, depending on whether they were attached to an exo position (i.e., a to the amide carbonyl group) or to an endo COOH COON COON COON I I I I position (i.e., at all other positions of the pyrrole rings). CH2 COON COON CH2 COON CH2 COON CH2 The methylene signal in biliverdin IXy, which possesses both I I I I I I I I propionyl groups in exo positions, appeared at 8 2.51, where- CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 as the other biliverdins that possess endo-propionyl substituents showed this methylene signal at ca. 8 3.0. The signal for the methylene groups a to the carboxyl group always appeared at ca. 83.0. M NH NH HN In the octamethyl ester of the new metabolite, all eight propionyl methylene signals appeared as triplets. Three of these COON COON COON COON were centered at 8 8 8 I I I I 2.923, 2.847, and 2.818, while the fourth COON CH2 COON CH2 COON CH2 CH2 COON methylene group was represented by one of the triplets found I I I I I I I at higher field between 8 2.62 and 8 2.49, together with the CN2 CN2 CN2 CN2 CNH CN2 CH2 CN2 signals for the four methylene protons a to the carboxyl groups. This indicates the presence of one exo- and three endo-propionyl groups. Of 0 N N N 0 (IV) the four possible structures, I-IV (Fig. 1), whose side chains N N ~~~N N correspond to those of uroporphyrin III, only structures I and II (corresponding to cleavage at the a and ,B methine carbons) possess one exo- and three endo-propionyl groups, as indicated by the presence of three lower-field and one higher-field methylene signals. Structure Ill would require two lower-field methylene signals, and structure IV, none. The NMR evidence therefore rules out these latter two structures, and bactobilin hence may represent either structure I or II. Structure I corresponds to that of the common biliverdin, H H H designated as IXa. Although the vast majority of bile pigments are derived by cleavage at the a methine carbon of heme, there FIG. 1. Alternative structures for bilatrienes derived by loss of the are biological precedents for bile pigments cleaved at other methine bridges in uroporphyrin III (structures I-IV) and uropor- methine carbons. Thus, the pigment pterobilin from Lepidop- phyrin I (structure V). tera (28, 30-32) has been shown to be biliverdin IXy (33, 34); Downloaded by guest on September 27, 2021 3946 Biochemistry: Brumm et al. Proc. Natl. Acad. Sci. USA 80 (1983) Table 3. 500-MHz proton magnetic resonance signals in C2HC13 Chemical shifts, 8 CO2CH3 bridge-CH* CH2CH2CO2CH3 Biliverdin IXa dimethyl ester 3.674 6.799, 6.070, 2.933 (m,t 2H), 2.563 (t,t J = 7.5 Hz, 2H) 6.015 Bactobilin octamethyl ester 3.719, 3.712, 3.693 6.784, 6.015, 2.923 (t, J = 7.5 Hz, 2H), 2.847 (t, J = 7 Hz, 2H) 3.687, 3.668, 3.659 5.976 2.818 (t, J = 7 Hz, 2H), 2.651 (t, J = 7 Hz, 2H) 3.653 2.62-2.49 (m, 8H) * The lowest-field signal arises from the central methine proton, while the two higher-field signals are due to the remaining two methine protons. Protons giving rise to these signals are underlined. t m, Multiplet. t t, Triplet. biliverdin IXf3 has been reported in hepatic catalase (35); traces not react with metal-free protoporphyrin (44). Iron copropor- of bilirubin IXM and IX, have been reported in pig bile (36); phyrin III is a poor substrate (43). At this point it is not known and the 8, 1B, and y isomers have been reported in man, pig, whether a metal complex of uroporphyrin III, such as uro- dog, and rat bile (37). heme, is changed enzymatically to bactobilin. Preliminary ex- The above discussion assumes that the sequence of side chains periments have shown that under the conditions used, the in bactobilin corresponds to that found in uroporphyrin III. present enzyme does not convert uroporphyrin III or I to bac- However, under the conditions of the present experiments (in- tobilin, or protoheme IX to biliverdin. Although the initial en- cubation of cell-free extracts with added 5-aminolevulinic acid), zyme incubation was carried out under anaerobic conditions, it the alternating sequence of side chains, corresponding to those is possible that bactobilin is formed from an anaerobically pro- of uroporphyrin I, cannot be excluded. Analysis of the isolated duced enzyme product during workup in the presence of ox- uroporphyrin by high-performance liquid chromatography ygen. Bactobilin octamethyl ester is obtained also when boron showed it to be a mixture of about equal parts of uroporphyrins trifluoride methanol is used instead of methanol sulfuric acid I and III. Analogous conclusions have been reached with uro- for esterification. The nature of the compound leading to bac- porphyrin prepared from suspensions of Propionibacterium tobilin remains unknown, and the presence of bactobilin in vivo shermanii fortified with 5-aminolevulinic acid (38). If the side has not been demonstrated. chains are indeed arranged as in uroporphyrin I, then only one There is one well-established process associated with the an- product (structure V, Fig. 1) would result, no matter which one aerobic ring opening and carbon loss of a cyclic tetrapyrrole. of the four bridge carbons is eliminated. Structure V cannot be This process occurs during the so-called ring contraction, when excluded from the NMR data. On the other hand, the fact that a porphyrin type of ring structure is changed to the corrin type bactobilin could be obtained from C. tetanomorphum or, in of ring structure. A two-carbon compound, namely acetic acid, smaller amounts, from P. shermnanii that had been grown with- is expelled from a repeatedly C-methylated intermediate (45, out S-aminolevulinic acid makes it unlikely that this substance 46), but the mechanism, the enzymology, and the exact sub- is related to uroporphyrin I. Uroporphyrin III apparently free strate of this reaction are unknown. It is possible that some from uroporphyrin I could be isolated from another Clostri- analogies exist between the first step in bactobilin and in corrin dium grown without added 5-aminolevulinic acid (39). If the ring formation. compound would be formed by a random nonenzymatic cleav- The physiological significance of bactobilin remains un- age of cyclic tetrapyrroles, it would be possible to observe more known. It may be more than a coincidence that one organism than one bile pigment in P. shermanii because, under those cir- contains apparently two separate systems for the anaerobic cumstances, coproporphyrinogen or coproporphyrin III and the cleavage of cyclic tetrapyrroles, but further work is needed to corresponding hepta- and hexacarboxylic tetrapyrroles could be shed light on this puzzle. expected to be cleaved as well. The mechanism of formation of bactobilin is still unknown. We thank Dr. Alan R. Battersby, Cambridge University, for the field Biliverdin IXa formation from protoheme IX, as catalyzed by desorption mass spectrum and stimulating discussions during his ten- the enzyme heme oxygenase, requires the participation of mo- ure as Kharasch Visiting Professor in the Department of Chemistry; lecular oxygen, NADPH-cytochrome c reductase, and NADPH Dr. Jerry C. Bommer, Porphyrin Products (Logan, UT), for the high- (8, 16, 17, 40): a reactive oxygen radical first forms an a-hy- performance liquid chromatography; and Mr. Barry Williams for ob- is added to this com- taining the various spectra. This work was supported by National In- droxyheme (41, 42), and further oxygen stitutes of Health Grants AM-09134 to H.C.F. and K06 AM-21846 to plex with resulting ring cleavage, carbon monoxide expulsion, J. F. and by The Burroughs Wellcome Fund. Funds provided by the and iron release (see, for example, refs. 8 and 17). In control National Science Foundation (GP 33116), the National Institutes of experiments on the formation of bactobilin, however, the com- Health (Cancer Center Grant CA 14999), and the Louis Block Fund to pound was still formed when the reaction was initiated under purchase the NMR equipment used in this work are gratefully ac- strictly anaerobic conditions (2-mercaptoethanol present as usual, knowledged. but tubes filled to the top), so that the red fluorescence indic- ative of exposure of the liquid to air was completely absent. 1. Lemberg, R. (1930) Liebig's Ann. 477, 195-245. The clostridial extracts, again, do not contain cytochrome c. They 2. Riidiger, W. (1971) in Fortschritte der Chemie Organischer Na- are active after dialysis without added NADPH (unpublished turstoffe, eds. Herz, W., Grisebach, H. & Kirby, G. W. (Spring- data). It appears likely, hence, that the present enzyme differs er, New York), pp. 60-139. A difference between the for- 3. Needham, A. E. (1974) The Significance of Zoochromes (Spring- from heme oxygenase. further er, New York), p. 88. mation of bactobilin and biliverdin lies in the intriguing like- 4. Bennett, A. & Siegelman, H. W. (1979) in The Porphyrins, ed. lihood that bactobilin, unlike biliverdin, is derived from a metal- Dolphin, D. (Academic, New York), Vol. 6A, pp. 493-520. free tetrapyrrole. The substrate specificity of heme oxygenase 5. Colleran, E. & Heirwegh, K. P. M. (1979) Comp. Biochem. Phys- has been thoroughly investigated (17, 43, 44). This enzyme does iol B 64, 133-139. Downloaded by guest on September 27, 2021 Biochemistry:BmProc.Brumm et al. Natl. Acad. Sci. USA 80 (1983) 3947 6. Lightner, D. A. (1979) in The Porphyrins, ed. Dolphin, D. (Ac- 25. Gossauer, A. & Plieninger, H. (1979) in The Porphyrins, ed. Dol- ademic, New York), Vol. 6A, pp. 521-584. phin, D. (Academic, New York), Vol. 6A, pp. 639-640. 7. McDonagh, A. F. (1979) in The Porphyrins, ed. Dolphin, D. (Ac- 26. Petryka, Z. J. & Howe, R. 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