Catabolism of Tetrapyrroles As the Final Product of Heme Catabolism (Cf Scheme 1)
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CHIMIA 48 (199~) Nr. 9 (Scl'lcmhcr)
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Chimia 48 (/994) 352-36/ heme (1), at the a-methene bridge (C(5)) €> Neue Sclnveizerische Chemische Gesellschaft producing CO and an unstable Felli com- /SSN 0009-4293 plex. The latter loses the metal ion to yield the green pigment protobiliverdin IXa (usually abbreviated to biliverdin (2)), which is excreted by birds and amphibia, Catabolism of Tetrapyrroles as the final product of heme catabolism (cf Scheme 1). The iron is recovered in the protein called ferritin and can be reutilized Albert Gossauer* for the biosynthesis of new heme mole- cules. As biliverdin (2) has been recog- nized to be a precursor in the biosynthesis of phycobilins [9], a similar pathway is Abstract. The enzymatic degradation of naturally occurring tetrapyrrolic pigments probably followed for the biosynthesis of (heme, chlorophylls, and vitamin B 12) is shortly reviewed. this class oflight-harvesting chromophores
1. Introduction pounds known so far are synthesized, have Scheme I. Catabolism (!{ Heme ill Mammals been already elucidated, it may be antici- In contrast to the enormous amount of pated that the study of catabolic processes work accomplished by chemists in the will attract the interest of more chemists elucidation of biosynthetic pathways of and biochemists in the near future. secondary metabolites (terpenes, steroids, alkaloids, among others), only a few at- tempts have been made until now to un- 2. Heme Catabolism derstand the mechanisms oftheirdegrada- tion in living organisms. A possible rea- It has been known for over half a cen- son for this fact is the irrational association tury that heme, the oxygen-carrier mole- of degradation (catabolism: greek Kara= cule associated with the blood pigment down) with decay and, thus, with unattrac- hemoglobin, is converted in animal cells tive dirty colors and unpleasant odors. The to bile pigments, which are excreted with other reason is probably the meaning that the faeces and urine [I]. Owing to its degradation processes occur outside the significance in human medicine, the ca- cell rather by uncontrolled chemical reac- tabolism of the heme released upon the tions which are achieved by environmen- degradation of hemoglobin and related tal factors such as oxygen, water, temper- chromoproteins, like myoglobin and cyto- ature, etc., than by catabolic (i.e., enzyme- chromes, has been intensively studied catalyzed) processes. Thus, with excep- during the past decades. Particularly, in tion of proteins, fats, and sugars, which are recent years the application of modem subjected to a continuous degradation and spectroscopic, chromatographic, and iso- rebuilding in the living organism, and of topic techniques as well as the develop- nucleic acids, the well-documented catab- ment of methodologies which made the olism of which is treated in all text books most important bile pigments like biliver- on biochemistry, almost nothing is known din [2], bilirubin [3], phycocyanobilin (Proto)biliverdin IXa (2) concerning the fate of most of the compo- [4][5], phycoerythrobilin [6][7], and phy- nents ofliving organisms which are liber- tochromobilin [8] accessible by chemical ated from the organelles in the course of synthesis have enabled to elucidate a morphological differentiation of their cells. number of central aspects of the problem Actually, as the main pathways ofbiogen- but made others, of course, still more esis, by which virtually all natural com- puzzling. Ordinarily, heme degradation occurs in the phagocytic cells of the bone mar- row, spleen, and liver but can probably ·Correspondence: Prof. Dr. A. Gossauer Institut fUr Organise he Chemie occur in macrophages in other tissues as UniversiUit Freiburg i. Ue. well. The catabolic process is initiated by Ch. du Musee 9 the oxidative scission of the protoporphy- (Proto)bilirubin IXa (3) CH-1700 Fribourg rin IX macrocycle, the chromophore of CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 353
CHI~lIA 48 t 144-1.)Nr. l) (Sl'Ph..'lllht'r) which are associated with photosynthesis Scheme 2. Hypothetical Mechanismforthe Catabolism of Heme to Bilh'erdinlXa in vivo. Side chains in cyanobacteria and in two groups of and apoprotein have been omitted for clarity. eukaryotic algae, namely the red algae and the cryptomonads. H0PH Moreover, the occurrence of bile pig- heme -°0 ments in arthropoda (insects, crabs, oxygenas: n:S)0 shrimps, and barnacles), mollusca (squids, L'" ~:(I~;-~ clams, snails, etc.), as well as in egg shells, 4 / " fins, and scales has been recognized for a long time. Thus, e.g., biliverdin (2) is ° responsible for the green color of the pray- ing mantis (Mantis religiosa L.); one of its NADPH regioisomers, pterobilin (protobiliverdin ..• IX il, has been isolated from the wings of certain butterflies, another regioisomer (protobiliverdin IXO) from the ovaries of 5 the marine snail Turbo cornutus [10]. At heme Ioxygenase least for insects of two different taxonom- ° ic categories (Mantis religiosa L., which H,0",0, feeds on other insects, and Locusta migra- 00) H toria L., a herbivore), de novo biosynthe- sis of endogenous biliverdin has been dem- • onstrated [1 I]. As insects do not contain "F~llI) ~>4~~/" heme in their hemolymph, either a biosyn- thetic pathway peculiar to them or degra- dation of cytochromes may be taken into /O,(!)",H H (0 heme consideration to explain the presence of oxygenase biliverdin (2) in their tissues. In humans •• and mammals, enzymatic reduction of ~(~;RN" / ~ /Fe~l) ~ biliverdin, which is catalyzed by the NADPH-dependent enzyme biliverdin reductase, yields yellow bilirubin (strictly speaking protobilirubin TXa (3». Actual- H I ly, catabolism of heme is the only known (0 source of bilirubin (3), of which 300-500 ° mg are produced daily in adult human •• ,,1/ . H2O beings. Once formed, 3 is esterified with ~ ~ Fe(lIl) ~ ~ /"-... various sugars (mainly glucuronic acid) in 8 the liver, and these water-soluble esters (so-called bilirubin conjugates) are ex- creted via the bile duct into the gut where they undergo further reductive transfor- mations to urobilinoids by the intestinal Scheme 3. Possible Pathway of the Mode of Action of Heme Hydrogenase (side-view ofthe porphyrin flora, before their final excretion in the iron-complex chromophore represented by a bar) faeces. On the basis of the experimental evi- dence available at present, Scheme 2 is I 1111 proposed as an outline of the chemical Felli'0-0' Fe steps involved in the conversion of heme I I to biliverdin in vivo (cf [9]). The mode of Heme Heme action of microsomal heme oxygenase oxygenase may be analogous to the commonly ac- cepted mechanism of the autoxidation of ferrous porphyrins in vitro, as represented in Scheme 3 ((-1 [12]). Thus, the suggested estingly, heme oxygenase is inactive reducing agent (usually ascorbic acid). pathway explains the requirement for against protoporphyrin IX, suggesting that This reaction, named 'coupled oxidation' molecular oxygen and provides an expla- the central Fe-atom of heme is essential because it requires both oxygen and a nation for the need for NADPH and a for the activity of the enzyme. reductant, was thought in the past to be a functioning microsomal electron-transport It is known that pyridino-ferrous-por- suitable model for the enzymatic break- system. Moreover, it is consistent with the phyrin complexes (so-called pyridine he- down of heme, in which basic amino-acid finding that the microsomal heme oxyge- mochromes (4), L = pyridine) are readily residues of the apoprotein replace pyrid- nase is inhibited by CO as well as by azide oxidized and cleaved to give bile pigment ine as stabilizing ligands for ferrous iron and cyanide ions, which may interact with precursors by mild treatment with oxygen and NADPH acts as the reducing agent. the ferrous heme-protein complex. Inter- in the presence of an excess of a suitable However, after the origin of the a-atoms CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 354
CHIMIA 18 (1994) Nr. 9 \Scplcl11ocr) present in the two lactam groups of the atmosphere inappreciable amounts. There- biliary excretion, and5) transport and elim- biliverdin molecule has been soundly es- fore, it is particularly interesting, in this ination in the intestine. Bilirubin (3) re- tablished by a series of outstanding exper- connection that bile pigments (so-called leased into the plasma is firmly bound to iments using oxygen isotopes by Brown bactobilins) are formed from uroporphy- plasma albumin, which serves as its carri- and coworkers [13-15], the mechanism of rins I and III by cell extracts of Clostrid- er protein. The mechanism by which un- the enzymatic reaction appears to be more ium tetanomorphum under anaerobic con- conjugated bilirubin is removed from al- complex. According to these investiga- ditions [18][19]. bumin and transmitted into the liver cells tions, the cleavage of the protoporphyrin Tn summary, the mechanism of ring is unknown. Indeed, it is not even known macrocycle occurs by a 'two-molecule cleavage of heme is highly unusual. The with certainty, whether the hepatic uptake mechanism', i.e., the terminallactam 0- coupled oxidation reaction is unique in of3 is an active or a passive process. In the atoms in biliverdin (2) are derived from porphyrin chemistry in that it permits this liver, 3 undergoes ester formation with a two different O2 molecules. Thus, the for- particularly stable macrocycle to be easily variety of carbohydrate moieties and pos- mation of2 by a hydrolytic step involving opened under extremely mild conditions. sibly with other conjugating groups that the insertion of an a-atom from the medi- Furthermore, decarbonylation at room tem- render the pigment water-soluble. The so- um does not occur in the heme-oxygenase perature in the dark is a rare reaction in called conjugation of the bile pigment is system. This reaction pathway (7 ~ 8) has organic chemistry, specially in the con- catalyzed by membrane-bound enzymes been observed, however, in the coupled densed phase. Because of the lack of ap- localized primarily in the endoplasmic oxidation of hemoglobin in vitro, in which propriate chemical models for these un- reticulum of the liver cell, the carbohy- the formed biliverdin contains one a-atom usual reactions, the pathway suggested on drate moieties being transferred from their deri ved from H20 and one from molecular Scheme 2 remains speculative. None the respective uridinediphosphate nucleotides. O2 ((f. Scheme 2). Nevertheless, in a hy- less, it provides a valuable approach to the Conjugation of 3 appears to be essential drophobic environment, an intermediate better understanding of the mechanisms for biliary excretion, since normally only of the oxaporphin type 6 may be reduced of degradation of other tetrapyrrolic natu- minute quantities of unconjugated biIirubi n by NADPH to the corresponding ferrous ral compounds. occurs in mammalian tissue. The hepatic complex, which in turn would be able to As mentioned before, biliverdin (2) excretory mechanism for conjugated bi- react with O2 in the same manner as for- produced upon cleavage of heme in the lirubin is poorly understood, but it is known mulated for the original hemochrome com- mammalian liver is reduced to bilirubin that it is shared by diverse groups of en- plex, to yield ultimately a ferric complex 8 (3) by biliverdin reductase, before it is dogenous compounds that are secreted of biliverdin. On hydrolytic decomposi- conjugated and excreted in the bile. The into the bile. In normal human bile. bi- tion of the latter, biliverdin (2) and a Felli small amount of 2 frequently detectable in lirubin mono- or di-,B-glucuronoside con- ion are liberated. mammalian bile is probably formed by stitute the predominant bile-pigment frac- According to the reaction sequence oxidation of conjugated bilirubin in the tion. Conjugation of bilirubin (3) restricts outlined in Scheme 2, the transformation bile ducts, in the gallbladder, or during the back-diffusion of the excreted pigment of heme into biliverdin (2) requires three collection process. In contrast, avian, rep- across the intestinal mucosa, which is es-
O2 molecules. The first intermediate tilian, and amphibian bile contains pre- sential for its efficient elimination in the formed in the heme-cleavage process is dominantly biliverdin (2), but most of this alimentary canal. As a matter of fact. 3 probably the oxyferriheme species (i.e., pigment appears to be unconjugated, since does not appear to be resorbed in signifi- an iron-oxophlorin chelate 5). The forma- biliverdin conjugates have not been iden- cant amounts from the human intestinal tion ofthis intermediate involves a mixed- tified with certainty. Most recently, bi- tract. In the distal portion of the intestine, function oxygenase type of reaction which lirubin-IO-sulfonic acid has been identi- the excreted pigment undergoes a series of results in the regioselective hydroxylation fied as the end product of heme catabolism stepwise reductions to urobilinoid chro- of one of the methene bridges of the tetra- in the gallbladder bile from adult bullfrogs mogens catalyzed by bacterial enzymes. pyrrole macrocycle. Thus, which isomer (Rana catesbiania) [20]. Although biliver- Removal of the conjugating carbohydrate of biliverdin (2) is produced, is deter- din (2) appears to be reduced to bilirubin functions probably also occurs in the in- mined at the very first stage of the degra- (3) in most fishes, it is interesting that testine, but it is not known whether this dation process. It is interesting that the some species have high concentrations of takes place before, during, or after reduc- edge of the heme ring that is preferentially 2 in the serum. It is not clear why 3 rather tion. The biological rationale for the fecal attacked in the coupled oxidation of heme than 2 is the end product of heme catabo- excretion of bile pigment in the form of proteins (the a bridge) is the most deeply lism in mammals. In contrast to biliverdin urobilinoids rather than as conjugated bi- buried within the hydrophobic core of the (2), bilirubin (3) is cytotoxic and not readi- lirubin remains obscure. It is possible that protei n lI 6]. The further transformation of ly excreted, necessitating the development formation of these chromogens is of no oxyheme to biliverdin is a complex one, of a complex series of conjugation and consequence to the host and is purely since the process involves incorporation transport functions for its removal and incidental to the presence of microorgan- oftwo additional a-atoms, loss of the Fe- elimination. The biological rationale for isms in the intestine. It is noteworthy, atom, and loss of the a-methene C-atom as the evolution of an apparently ubiquitous, however, that at least one of these intenne- co. presumably substrate-specific, and regio- diates, urobilinogen, is reabsorbed from On the other hand, the finding that selective biliverdin reductase remains ob- the gut to the portal circulation and is biliverdin (2) is a biogenetic precursor of scure. reexcreted by the liver (so-called entero- phycocyanin [9] (via a bilirhodin-like in- The transport of bilirubin 3 from its hepatic circulation). Part of the urobilino- termediate? [17]) gives rise to the question tissue sites of formation to its eventual gen escapes to the systemic circulation about the mechanism of the biosynthesis excretion in the intestinal tract can opera- and is excreted by the kidney. of this photoreceptor molecule in organ- tionally be characterized by five phases: Urobilinoidcompounds have also been isms (the cyanobacteria) which populated 1) transport in the plasma, 2) transfer into found in the phycobilisomes, the supramo- the earth before oxygen was present in the the hepatocyte, 3) hepatic conjugation, 4) Iecular assemblies attached to the outer CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 355
CHI MIA 48(1994) Nr. 9 (SCpIClIlhcr\ surface of the thy lakoid membrane which contains the chlorophyll-binding proteins and the reaction centre of the photosyn- thetic apparatus in cyanobacteria and some microalgae [2 I]. Their biosynthetic origin is so far unknown. The bacterial reduction of bilirubin to urobilinoids was studied extensively by Watson [22). The most representative de- rivatives are urobilinogen (properly: meso- urobi linogen (9)) and (meso )stercobilino- gen (10), which upon mild reoxidation by air yield urobilins (e.g. d-(meso)urobilin (11)) and stercobilins (e.g.l-(meso)sterco- bilin (12)), respectively (cf. Fig. 1). Al- urobilinogen (9) though intermediate pigments in the re- stercobilinogen (10) duction pathway have been found (e.g. an optically active d-monovinyl-urobilin, the structure and absolute configuration of which was established by synthesis [23]), the mechanism of the bacterial reduction of bilirubin (3) is not completely under- stood; apparently it depends on the strain of bacteria and conditions in the gut. The microbiological reduction of bi- lirubin (3) is stereose1ective. The occur- rence of an optically inactive mesourobi- lin ('i-urobilin') in faeces may be explained by racemization of the corresponding op- tically active compound. In fact, owing to d-mesourobilin-IXa (11) I-mesostercobilin (12) the acidity of their H-atoms attached to the stereogenic centres, urobilins are prone to isomerization both in basic and acidic Fig. I. Principal bile pigmentsformed by bacterial reduction of bilirubin in the intestine ofmallllllais medium. In the latter, a mixture of diaste- reoisomeric bilirhodines are formed, the structure and relative configuration of curs in solution in helical conformations, 3. Chlorophyll Catabolism which has been elucidated by synthesis which are dissymmetric. However, as the [24][25). energy barrier for the mutual transforma- Besides fucoxanthin, the characteris- Optical activity of the urobilinoids was tion of enatiomeric biliverdin conformers tic pigment of many marine algae and, first observed by Fischer et ai. [26] with is rather low (10 ± 0.5 kcal/mol at 200 K) without question the most abundant natu- laevorotatory stercobilin; a dextrorotatory [32], this bile pigment, in contrast to the ral carotenoid, chlorophyll is the natural urobilin was discovered later by Schwartz urobilinoids, is devoid of any optical ac- pigment that is present in the greatest and Watson in human fistula bile [27). tivity. Bound to proteins, however, amounts on earth (at a rough estimate Uro- and stercobilins are characterized by biliverdin derivatives may occur either as some 109 t of chlorophyll are destroyed their extremely high optical activity (e.g. chiral helical-shaped molecules - e.g. in and re-synthesized each year on land and [a]D = + 5000 for natural d-mesourobilin, the wing pigment of the cabbage butterfly in the oceans). [a]D = - 4000 for i-stercobilin). The (Pieris brassicae L.) [33] - or as 'stretched' In some aspects heme (1) and chloro- optical activity of urobilinoids has been chromophores in the so-called phycobili- phyll are intimately related natural com- explained by the presence of an inherently proteins. It has been shown experimental- pounds, in another sense they are antithet- dissymmetric dipyrrin chromophore, the ly [34][35] that the enhanced extinction of ic. Thus, although, in contemporary or- helicity of which is conditioned by the the visible-absorption band of the latter is ganisms, protoporphyrin IX is a biosyn- absolute configuration of the asymmetric caused by the presence of 'stretched' con- thetic precursor of chlorophyll [38], it is C(4)- and C(l6)-atoms [28]. Only recent- formations of the chromophore, as origi- generally agreed that photosynthetic ly, however, the absolute configurations nally suggested by theoretical calculations. prokaryotes appeared on the earth before of two urobilinoid bile pigments could be Partially 'stretched' bile pigment chromo- aerobic live would be able to develop, i.e., assigned unequivocally by X-ray diffrac- phores result also from (2) ~ (E) isomer- the function of chlorophyll a, as an ubiq- tion analysis of their synthetic precursors ization of the exocyclic C=C bonds uitous photosynthetic pigment in nature, [29] [30], so that a relationship between [36][37). This process has been studied preceded the evolution of hemoglobin, as helicity of the chromophore and sign of intensively with bilirubin as a substrate the most common oxygen-carrier protein the Cotton effect in this class of com- owing to its relevance in the treatment of in eukaryotic cells. From this point of pounds can be now established experi- human hyperbilirubinemia by photothera- view, heme symbolizes the (most frequent- mentally [3l]. py. In the case of biliverdin and its deriv- ly destructive) heterotrophic metabolism, As a matter of fact, bi Iiverdin (2), which atives, however, this kind of isomers have whereas chlorophyll enables living organ- is intrinsically an achiral compound, oc- not yet been found in nature. isms to build up complex organic mole- CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 356
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cu]es from CO2 and H20. Thus, the psy- 0 HN---Q HN~OH chological response (aggressiveness and N oSCO," appeasement) to the colors red and green N ;:? N:X > I ' in higher developed living creatures may l~N N N:Xl~N >N ~ N be an atavism, which has its origin in the H H H chemical structure oftwo natura] pigments kinetin (13) zeatin (14) auxin (15) derived from the same biogenetic precur- sor. o It is all the more surprising that despite -~I~OH the fascination exerted by the yearly play Fig. 2. Some impor- H3C~~ of colors, which takes place in the plant tant plant honno- nes involved in se- HO kingdom at the beginning of the autumn in nescence of plant gibberellic acid = gibberellin A3 (16) (+)·abscisic acid (17) the nontropical countries, the natura] fate tissues of chlorophyll in the leaves turning yellow remained an enigma until four years ago [39). Despite subtle experiments, which were carried out at the end of the last century to get insight in this phenomenon, the only relevant finding from these ear]i- er studies, from the structural point of view, was the recognition that the yellow color of autumn leaves is due to caroti- noids (mainly lutein = xanthophyll) which are already present in the plant cells before
C02H their senescence, and are not related to the breakdown of chlorophyll [40). On the 20 (from C. protothecoides) 21 (from C. protothecoides) H H other hand, however, the regulation of senescence in plants has been object of a large number of investigations [4 t). The prominent role played by some endog- enous substances like ethylene, cytoki- HO nins (e.g. kinetin (13), zeatin (14), etc.), auxin (15), gibberellins (e.g. 16), abscisic acid (17), and other plant hormones has o o been recognized, although the regulation mechanisms are until now only rudimen- o 22 (from C. protothecoides) o 23 (from C. protothecoides) tary understood (Lf Fig. 2). Moreover, several enzymes involved in the degrada- tion of chlorophyll without affecting the integrity of the macrocycle, have been recognized in the past. Thus, chlorophyl- lase has long been known to catalyze the hydrolysis of phytol from chlorophyll (18) yielding chlorophyll ide [42). Removal of the complexed Mgll-ion by magnesium dechelatase leads to pheophytin. Through the action of both enzymes pheophorbide (19) is formed (cf Scheme 4). The corre- Fig. 3. Chlorophyll 24 (from C. prololhecoides) 25 (from C. kess/en) a cataholites from sponding pyropheophytin and pyropheo- Chlorella strains phorbide, which lack the methoxycarbo- nyl group on C(132), have been found in OH photosynthetic organisms [43). Both pig- ments have been characterized as chloro- phyll catabolites in the digestive tract and in the faeces of terrestrial herbivores. On OH the other hand, a few natural pigments which probably proceed from cleavage of the chlorophyll macrocycle have been found in organisms which do not produce endogenous chlorophyll, like krill (Eu- phausia pacifica) [44), dinoflagelates (Py- Fig. 4. Chlorophyll acatabolites isolat- rocystis lunula) [45), and insects (Anasa edfromphanerog- tristis Degger) [46]. Moreover, the pres- 29 (RP-14 from Hordeum vulgare cv. Gerbel) 30 (Bn-NCC-1 from Brassica napus L.) ams ence of a red-fluorescent antiviral glyco- CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 357
Scheme 4. Primary Products of Chlorophyll Degradation in vivo Scheme 5. Chlorophyll b Catabolites Isolated from Chlorella protothe- coides
chlorophyll a (18) pheophytin a Chlorophyll b (26) /Ph'IOI
pheophorbide a (19) 27 28
Scheme 6. Possible Mechanism of the Proteolytic Degradation ofChloro- Scheme 7. Suggested Mechanism of the Oxidative Ring Clem'age (!{ phyll Catabolitesfrol11 C. protothecoides Pyropheophorbide Metal Chelates in vitro
..
21 J H
•• ? 21 •
protein [47] in the digestive juice of silk- of chlorophyll a into the biliprotein may biliprotein isolated from the digestive juice worms (Bombyx mori L.) has been report- be inferred [49]. Feeding experiments car- of the caterpillars; the main radioactive ed by Japanese authors [48]. Later work ried out with 14C-Iabelled chlorophyll a, product being chlorophyllide a. Interest- proved that in vitro this biliprotein is only which was prepared from pheophorbide ingly, up to 9% transformation of chloro- formed in the presence of chlorophyll a by partial synthesis [50], revealed, how- phyll a into chlorophyll b in the intestine and chlorophyllase, so that a transformation ever, a very low incorporation of 14Cin the of the larvae could be demonstrated [511. CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 358
CHIMIA 48 (1994) Nr. 9 (Septemher)
The occurrence of a chlorophyll oxi- organs until it is finally accumulated in the of the isolated catabolites is the unexpect- dase which presumably transforms chlo- reserve proteins of storage tissues. Wheth- ed regioselectivity of the oxidative cleav- rophyll into the corresponding (R)- and er the four N-atoms of the chlorophyll age of the macrocycle at the C(4)=C(5) (S)-] 32-hydroxy derivatives (allomeriza- molecule are reutilized in the same way is bond instead of the C-C bonds adjacent to tion) has been discussed [52]. As the cor- still unknown [53]. the reduced pyrrolering, as generally prog- responding nonenzymatic reaction occurs In 1991, two Swiss research groups nosticated before. Meanwhile, a related readily in the dark under the influence of succeeded for the first time, independently chlorophyll a catabolite has been found in air in methanolic solution, it must be born of each other, in the isolation and charac- the dicotyledon Brassiea napus L. (rape) in mind that such compounds may be terization of tetrapyrrolic chlorophyll a [58] (ef Fig. 4), and a series of bilin formed during the procedure of isolation catabolites from dark-bleached excised derivatives, all of them resulting from from the organic preparations. primary leaves of barley (Hordeum vul- cleavage of the chlorophyll macrocycle at More recent investigations of the chlo- gare cv. Gerbel) [54] and from the culture the a-methene bridge, have been isolated rophyll catabolism in phanerogams sug- medium of the microalgae Chlorella pro- from the culture media of C. protothe- gested that the degradative processes oc- totheeoides [55]. Shortly afterwards, the eoides [59] and C. kessleri [60] (e.f Fig. 3). curring in senescent leaves serve the reut- structure of the pigment 21 (ef Fig. 3), Moreover, two degradation products, 27 ilization of nutrients in other parts of the isolated from the latter, was confirmed by and 28, of chlorophyll b (26) have been plant. This is particularly important re- X-ray diffraction analysis [56], as well as isolated from the culture medium of the garding the nitrogen which is continuous- by partial synthesis starting from pyro- former and characterized for the first time ly displaced from senescent into growing pheophorbidea [57]. The common feature [61] (cf Scheme 5).
Fig. 5. Some strains of the microalgae ChIarella can be stimulated experimentally to the formation of chlorophyll catabo/ites which are secreted into the culture medium. The structures of these catabolites have been elucidated at the Institute for Organic Chemistry of the University of Fribourg for the first time. Growing (left) and bleaching cells (right) of Chlorella kessleri.
80 100
65 81 80 60 58
60 53 40 41 40
20 20
o o 580 581 582 583 584 585 586 587 588 589 590 566 567 568 569 570 571 572 573 574 575 576
Fig. 6. /sotopical distribution in the range of the molecular ion peak of21, Fig. 7./sotopical distribution in the range of the molecular-ion peak (121, obtained by photooxidation of methyl pyropheophorbide-Cd/l in an 1801' obtained in vivo in an 1801'enriched atmosphere: • using 16011802 (55 enriched atmosphere:. using 160/8°2(62 :38); ~ using 160/802(31:69). : 45); ~ using 16011802 (II :89). Percent values have been determined Percent values have been determined by subtraction of the FAB-mass by subtraction ofthe FAB-mass spectrum of unlabelled 21 from that ofthe spectrum of unlabelled 21 from that of the labelled derivative. labelled derivative. CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 359
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In contrast to the pigments excreted into the culture medium by Chiorella cells, the chlorophyll catabolites 29 and 30 iso- lated by Matiie et at. from phanerogams are colorless compounds, which turn a rusty color only on exposure to air (ef Fig. 4). Thus, it becomes clear why the primary degradation products of chlorophyll have been overlooked for such a long time. On the other hand, the present results point out that chlorophyll catabolism may in- volve several reductive steps which are absent in the degradation of heme. As it is well documented that polypyrrolic com- pounds in which the pyrrole rings are joined by methylene bridges are readily cleaved by protolysis [62], a further deg- radation of compounds like 21 should be a rapid process (ef Scheme 6). Until now, however, the fate of the products formed 32 after cleavage of the chlorophyll macro- cycle is not known. Decisive for the results of the Fribourg group was not only the knowledge about Fig. 8. Hitherto characterized xanthocorrinoids obtained by 'coupled oxidation' in vitro of heptam- the earlier finding that, during the process ethyl dicyanocobyrinate and some of its derivatives. For the sake of clarity, only the part of the of bleaching of C. protothecoides cells, molecule, which is modified, is represented in the partial structure, remainder as in the preceding entire formula. which takes place when this green algae is grown in a medium rich in glucose but poor in nitrogen (ef Fig. 5), a red pigment CONH is excreted into the culture medium 2 [63][64], but also the discovery that pho- (, tooxidation of pyropheophorbide metal ! chelates in vitro yields similar products as those isolated from the culture medium of C. protothecoides [65], the regioselectiv- ity of the ring cleavage being dependent on the complexed metal ion [66]. Howev- er, an investigation of the photooxidative ring cleavage of the CdII-chelate of py- ropheophorbide a in vitro and of the enzy- matic transformation of chlorophyll a into 34 18 21, both in the presence of 02, revealed that both transformations proceed by dif- ferentmechanisms. As a matteroffact, the Fig. 9. Xanthocorrinoids obtained by 'coupled oxidation' of cyanocobalamin (vitamin BJ 2) in vitro pattern observed in the range of the molec- ular-ion peaks of the photooxidation prod- uct, after reductive demetallation, agrees On the basis of the above results, a 4. Vitamin B12 Catabolism with the incorporation of two a-atoms tentative pathway for the enzymatic deg- proceeding from the same molecule (cf radation of chlorophyll can be suggested, Despite the fundamental structural dif- Fig. 6). Most likely, therefore, the ob- in which the 1,2-diol formed by hydroly- ferences between the chromophores of tained formylbilinone derivative is formed sis of a primary pheophorbide-4,5-epox- heme and the corrinoids (to which adeno- via a dioxetane, resulting from the cy- ide is cleaved by a retroaldol-like reaction sylcobalamin - the vitamin B 12 coenzyme cloaddition of a singlet oxygen molecule (ef Scheme 8). As the presence of a MgII_ - belongs), the latter are usually associat- to the C( 4)=C( 5) bond of the substrate [67] dechelatase in senescent chloroplasts has ed to the tetrapyrrolic pigments owing to (el Scheme 7). On the other hand, after been recently demonstrated by the Zurich their manifest global appearance which bleaching of C. protothecoides cells in the group [69], it may be anticipated that loss reflects their common biosynthetic origin. 18 presence of 02, it could be proved by of the Mg-atom precedes the oxidative As the vitamin B 12 requirement of man mass spectrometric analysis of the red cleavage of the macrocycle in the enzy- is very low (ca. 2-51lg daily), the prooffor bilin derivative 21 excreted into the cul- matic degradation process. Moreover, as a metabolism of this compound in the ture medium that only the a-atom of the microalgae are considered to be the phy- human body is difficult. On an adminis- formyl group proceeds from molecular logenetic ancestors of green plants, it may tered oral dose ofO.5-2Ilg of pure vitamin
O2, whereas the a-atom of the lactam be expected that this pathway holds true B12' 60-80% is absorbed. As the oral dos- carbonyl originates from H20 [68] (cf for protista and multicellular organisms as age increases, the percentage absorbed Fig. 7). well. decreases; at a dose of 5 mg 30% or less is CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 360
l'HIMIA.8 (1994) Nr. 9 {S.p'
Pheophorbide a Cunninghamella blakesleeana, Beauve- Epoxydase ria bassiana, and Corynebacterium sp.) 19 .. [85], the possibi lity that they are formed as artefacts cannot be ruled out at present. Thus it must be concluded that a convinc- ing evidence for an enzymatic degrada- tion of cobalamin derivati ves is still miss- mg.
In the present work. a number of the author's own contributions to the subject treated are re- ported. All colleagues and coworkers, whose names are mentioned in the references, deserve my sincere thanks for their valuable help. encour- aging enthusiasm, and fruitful experimental abil- ities. Likewise, I wish to thank the Deutsche Forsclulllgsgellleinsclwf/ and the SlI'iss Na/ional Science Foundation for their generous financial support of our research projects, before and after October 1st, 1982. respectively. CHEMIE IN FREIBURG/CHIMIE A. FRIBOURG 361
C'HIMIA 48 (IW4l Nr. q (September)
Received: July 20, 1994 [32] H. Lehner, W. Riemer, K. Schaffner, Lie- Mooser, A. Gossauer, Photochem. Photo- bigs Ann. Chel1l. 1979, 1798. bioi. 1993, 58, 116. [II For a review see: R. Schmid, A.F. McDon- [33] R. Huber,M. Schneider, I. Mayr,R. Muller, [60] N. Engel, J. ItulTaspe, A. Salathe, A. Gos- agh in The Porphyrins', Ed. D. Dolphin, R. Deutzmann, F. Suter, H. Zuber, H. Falk, sauer, unpublished results. Academic Press, Inc., New York, 1979, H. Kaiser,./. Mol. BioI. 1987,198,499. [61] J. [turraspe, N. Engel. A. Gossauer. Phyto- Vol. VI, p. 257. [34] P. Nesvadba, A. Gossauer, ./. Am. Chem. chemistry 1994, 35,1387. [2\ H. Fischer, H. Plieninger, Hoppe-Seyler's Soc. 1987, 109,6545. [62] A. Gossauer, 'Die Chemic der Pyrrole', Z. Physiol. Chem. ]942,274,231. [35] For a recent review see: K. Abou-Hadeed, Springer-Verlag, Berlin, 1974, p. 113fl'. [31 H. Plieninger, F. EI-Barkawi, K. Ehl, R. P. Nesvadba, A. Gossauer, Chimia 1988, [63] Y. Oshio, E. Hase, Plant Cell Physiol. Kohler, A.F. McDonagh, Liebigs Ann. 42,282. 1969, 10.41; ibid. 1969, 10.51. Chem. 1972, 758, 195. [36] H. Falk, K, Grubmayr, E. Haslinger, T. [64] The author is greatly indebted to Prof. 141 A.Gossauer, W. Hirsch, LiebigsAnn. Chem. Schlederer, K. Thirring, Monatsh. Chem. Horst Senger(Fachbereich Biologiel Botan- ]974, 1496. 1978, 109, 1451. ik der UniversitlitMarburg, Marburg/Lahn, 151 A. Gossauer, R.-P. Hinze, J. Org. Chem. [37] A. Gossauer, M. Blacha-Puller, R. Zeis- Germany) for having drawn our attention 1978.43,283. berg, V. Wray, Liebigs Ann. Chem. 198], to Hase's work as well as for having pro- 161 A. Gossauer, E. Klahr, Chem. Ber. ]979, 342. vided us with cells of Chlorella protothe- 112,2243. [38] F.J. Leeperin 'Chlorophylls', Ed. H. Scheer, caides for our initial experiments. [71 A. Gossauer, J.-P. Weller, ./. Am. Chem. CRC Press, Inc., Boca Raton, Florida, 1991, [65] J. Iturraspe, A, Gossauer, P/lOtochem. Pho- Soc. 1978, 100,5928. p. 407-431; S. I. Beale, J.D. Weinstein, in tobial. 1991,54,43. [81 J.-P. Weller,A. Gossauer, Chel1l.Ber. ]980, 'Biosynthesis of Tetrapyrroles', Ed. P. M. [66] J.lturraspe, A. Gossauer. Hel\'. Chim. Acta 113.1603. Jordan, Elsevier Sci. Publ. B. V., Amster- 1991, 74, 1713. 191 S.l. Beale, Chem. Re\'. 1993,93,785. dam, 1991, p. 179ff. [67] J. Iturraspe, A. Gossauer, in preparation. IIOJ E. Benedikt, A. Gossauer, H.-P. Kost, W. [39] G.A.F. Hendry, J. D. Houghton, S.B. [68] N. Engel, e. Curty, A. Gossauer, in prepa- Miki, K. Yamaguchi,Eur.1. Biochem.1988, Brown, New Phytol. 1987, 107,255. ration. /75.643. [40] E. Stahl, 'Zur Biologie des Chlorophylls', [69] M. Langmeier, S. Ginsburg, P. Matile, IIII M. Passama- Vuillaume, M. Barbier, C. R. Verlag Gustav Fischer, Jena, 1909, p. Physiol. Plant. 1993,89.347. A cad. Sci. Paris 1966, 263D, 924. I32ff. [70] K. Helgeland, 1. Jonsen. S. Laland. Rio- [121 J. P. Collman, Acc. Chem. Res. 1977, 10, [41] L.D. Nooden, A.e. Leopold, 'Senescence chem. J. 1961,81,260, and ref. cit. therein. 265. and Aging in Plants', Academic Press, Inc., [71] S.Y.S. lng, J.J. Pfiffner, Arch. Biochem [131 S.B. Brown, R.F.G.J. King, Biochel1l. 1. New York, 1988. Biophys.1968.128, 281, and previous com- 1978,170,1297. [42] R. Willsttitter, A. Stoll, 'Untersuchungen munications cited therein. r 141 R.F.G.J. King, S.B. Brown, Biochel1l. J. uberChlorophyll', J. Springer Verlag, Ber- [72] R.R. Engel, S. ModicI', .I.M. Matsen, Z.J. 1978, 174, 103. lin, 1913. Petryka, Biochim. Biophys. Acta 1973,313, 1151 J.e. Docherty, B.A. Schacter, G.D. Fir- 1431 R. Ziegler, A. Blaheta, N. Guha, B. 150. neisz, S.B. Brown, J. Bioi. Clwn. 1984, Schonegge, ./. Plant Physiol. 1988, 132, [73] J.M. Pratt, 'Inorganic Chemistry of Vita- 259, 13066. 327. min B 12', Academic Press Inc., London, 116] M.F. Perutz, Proc. R. Soc. London [Ser.} B [44] H. Nakamura, B. Musicki, Y. Kishi, O. 1972, p. 290ff. ]963,173,113. Shimomura, J. Am. Chem. Soc. 1988, /10, [74] A. Gossauer, B. Grlining, L. Ernst, W. [17 J A. Gossauer, F. Nydegger, E. Benedikt, H.- 2683. Becker. W.S. Sheldrick, Angel\'. Chem. P. Kost, Helv. Chil1l. Acta 1987, 72,518. [45] H. Nakamura, Y. Kishi, O. Shimomura, D. ]977.89.486; ibid. lilt. Ed. ]977,16.481. 1181 P.J. Brumm,J. Fried, H.e. Friedmann, Proc. Morse, J. W. Hastings, J. Am. Chem. Soc. [75] B. Gruning, A. Gossauer, Tetrahedroll Lett. Natl. Acad. Sci. USA 1983,80,3943. 1989, 111,7607. 1979,3497. [191 e.H.A. Chen, H.e. Friedmann, FEMS [46] R.L. Metcalf, Entomol. Soc. Am. 1945,38, [76] B. Gruning, A. Gossauer. in 'Vitamin BI~" Microbiol. Lett. 1993, 110,77. 397. Eds. B. Zagalak and W. Friedrich,Walter [20j .I.-S. Ma, A.F. McDonagh, L.A. Palma, (471 M. Doi, Y. Shioi, T. Sasa, Camp. Biochem. de Gruyter, Berlin, 1979, p. 141ff. unpublished results. Physiol .. B 1986, 83, 569. [77] B. Gruning, G. Holze, T.A. Jenny, P. Nes- 1211 P. O'Carra, R.F. Murphy, S.D. Killilea, [48] .1.1.Kusuda, J.-I. Mukai, Camp. Biochem. vadba, A. Gossauer, L. Ernst, W.S. Shel- Biochem. J. 1980, 187,303. Physiol., B ]971, 39, 317. drick, Hell'. Chim. Acta 1985, 68, 1754. 1221 For a review see: C.J. Watson, Z. Klin. [49] K. Hayashiya, Ent. Exp. Appl. 1978, 24, [78] B. Grlining, G. Holze. A. Gossauer, L. Chem. Klin. Biochel1l. 1971, 9,295. 228. Ernst, Hell'. Chim. Acta 1985, 68. 1771. 1231 A. Gossauer,J.-P. Weller, Chel1l.Ber.1978, [50] R. Fischer, N. Engel, A. Henseler, A. Gos- [79] G. Holze, A. Gossauer, L. Ernst, Hell'. 111,486. sauer, He/v. Chim. Acta] 994, 77, 1046. Chim. Acta 1986, 69, 1567. 1241 A. Gossaucr, D. Miehe, Liebigs Ann. Chem. [51] R. Fischer, A. Gossauer, unpublished re- [801 G. Holze, T.A. Jenny, P. Nesvadba, A. 1974,352. sults. Gossauer, L. Ernst, W. Keller. C. Kratky. 125\ A.Gossauer,G. Kuhne, LiebigsAnn. Chem. [52\ S. Schoch, W. Rudiger, B. Luthy, P. Mat- Hell'. Chim. Acta 1991, 74, 1287. 1977, 664. ile, J. Plant Physiol. 1984, 115,85. [81] B. Krautler, HelP. Chim. Acta 1982, 65, 1261 H. Fischer, H. Halbach, A. Stern, ./ustus [53] P. Matile, Chimia1987, 41, 376. 1941. Liebigs Ann. Cherrl. 1935,519254. [54] B. Krautler, B. Jaun, K. Bortlik, M. Schel- [82] B. Krautler, R. Stepanek, Photochem. Pho- 1271 S. Schwartz, e.J. Watson, Proc. Soc. Exp. lenberg, P. Matile, Angew. Chem. 1991, tobiol. 1991, 54, 585. Bioi. Med. 1942, 49, 641. /03,1354; ibid. 111I.Ed. 199],30,1315. [83] L. Debussche, D. Thibaut, M. Danzer, F. 1281 A. Moscowitz, W.e. Krueger, I.T. Kay, G. [55] N. Engel, T.A. Jenny, V. Mooser, A. Gos- Debu. D. Frechet, F. Herman, F. Blanche, Skewes, S. Bruckenstein, Pmc. Nat. Acad. sauer, FEBS Lett. 1991,293,131. M. Vuilhorgne,./. ClWII. Soc" Chem. COIll- Sci. U.S.A., 1964, 52,1190. [56] N. Engel, A, Gossauer, K. Gruber, C. mun. ]993, 1100. 1291 C. Pasquier, A. Gossauer, W. Keller, e. Kratky, Helv. Chim. Acta 1993, 76, 2236. [84] A.I. Scott, e.A. Roessner. N.J. Stolowich, Kratky, He/v. Chim. Acta ]987, 70,2098. [57] J. Itun-aspe, A. Gossauer, Tetrahedron J.B. Spencer, e. Min, S.-I. Ozaki, FEBS 1301 L. Floch, F. Nydegger, A. Gossauer, e. ]992,48,6807. Lett. 1993,331,105. Kratky, Hel\'. Chim. Acta ]994, 77,445. [58] W. Muhlecker, B. Krautler, S. Ginsburg, P. [85] D. Arnold. Ph.D. thesis, University of Fri- [311 A. Gossauer, F. Nydegger, unpublished Matile, Helv. Chim. Acta 1993, 76, 2976. bourg, 1988. results. [59] J. [turraspe, N. Engel, P. Matzinger. V.