Journal of Photochemistry
B: Biology, 5 (1990) 3 - 23
NEW TRENDS IN PHOTOBIOLOGY (Invited Review) BIOSYNTHESIS OF PHYCOBILINS. FORMATION OF THE CHROMOPHORE OF PHYTOCHROME, PHYCOCYANIN AND PHYCOERYTHRIN STANLEY B. BROWN, JENNIFER D. HOUGHTON and DAVID I. VERNON Department
University of Leeds, Leeds LS2 9JT (U.K.)
(Received October 9, 1989; accepted October 25,1989)
Keywords. Biosynthesis, phycobiliproteins, chromophore, 5-aminolaevulinic acid, photosynthetic bile pigments.
Summary Phycobiliproteins play important roles in photomorphogenesis and photosynthesis. The light-absorbing chromophores of the phycobiliproteins are linear tetrapyrroles (bilins) very similar in structure to the mammalian bile pigments. 5-Aminolaevulinate (5-ALA) is the first committed intermediate in phycobilin synthesis. The biosynthesis of 5-ALA, destined for phycobilins, occurs via the five-carbon pathway, now well established for tetrapyrrole synthesis in plants and distinct from the mammalian pathway. The phycobilins are formed by reduction of biliverdin which results from the synthesis and degradation of haem. This haem is an essential intermediate in the biosynthesis of phycobilins. Phycocyanobilin, the blue-green pigment found in certain algae and cyanobacteria, is formed from biliverdin via phytochromobilin, the chromophore of phytochrome. This leads to the likelihood that phytochromobilin is formed as an end product, or intermediate, in the synthesis of all phycobilins.
1. Phycobiliproteins: pigments
structure, function and relation to mammalian bile
In mammals and other animals it has been known for many years that bile pigments (also known as bilins) are formed by the metabolic breakdown of the haem of haemoglobin and other haemoproteins [l - 41. The chief mammalian bile pigments are bilverdin and bilirubin, which are formed from haem as shown in Fig. 1. In mammals, the bilins themselves play no functional role, other than in providing a pathway for the elimination of unwanted haem. loll-1344/90/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
d-CO +Fe H
Bilirubin Fig. 1. The degradation V, -CH=CH2).
to bilin in mammals
Bilins similar in structure to biliverdin occur also in plants and cyanobacteria. In contrast to the situation in animals, however, the plant and bacterial bilins carry out vital functions in photosynthesis and photomorphogenesis. For example phytochrome, which occurs in most if not all higher photosynthetic organisms and plays a major role in photomorphogenesis, is a phycobiliprotein which contains the bilin, phytochromobilin, as the active chromophore. Various types of algae including certain red algae and cyanobacteria contain the phycobiliproteins, phycocyanin and phycoerythrin. These proteins function as primary light-harvesting photosynthetic pigments and contain the bilins phycocyanobilin and phycoerythrobilin as chromophores. Figure 2 shows the structures of phytochromobilin, phycocyanobilin and phycoerythrobilin, and it can be seen that they are very closely related to each other and to the mammalian bile pigments. This close similarity suggests the possibility of a common biosynthetic pathway for the plant and bacterial bilins and the mammalian bile pigments. For comparative purposes, the chief steps in the mammalian biosynthesis of haem are shown in Fig. 3.
oJ$J$J$$o H Biliverdin
o;-fyJF(j&Jgo !+p&ygo H
Fig. 2. Structures -CH#.3H2COOH).
of plant bilins and their relationship
to biliverdin (Me, -CH3;
The enzymes catalysing these steps and those involved in mammalian haem degradation are listed in Table 1. Most plants, algae and cyanobacteria synthesize chlorophylls, which are closely related in structure to haem and bilins. It is well established that, with the exception of the mode of formation of 5-aminolaevulinate (5-ALA), the pathway for biosynthesis of chlorophylls in plants is very similar to that of haem in animals, as far as protoporphyrin-IX (Fig. 3). The pathways then diverge, with the insertion either of iron for haem synthesis and subsequent bile pigment formation, or of magnesium for the chlorophyll branch. It is now known that plant bilins are also formed from 5-ALA in common with other tetrapyrroles [ 5 - 71. In recent years, detailed studies of the plant systems have revealed that the formation of 5-ALA occurs directly from the five-carbon skeleton of glutamate and is therefore different from the system operating in mammals. Because of the fundamental difference in animal and plant metabolisms there is presently intense interest in this early part of the pathway. The formation of &ALA in plants is also likely to be a key control point of the pathway and this regulation almost certainly involves light, though the mechanisms of control are not yet known. The latter part of the pathway of biosynthesis of plant bilins has also proved to be of great interest, because it is now known that bilins are
Porphobilinogen (3,4) I Uroporphyrinogen
(6) I Protoporphyrinogen
(7) I Protoporphyrin
ilaem Fig. 3. Steps in the mammalian biosynthesis of haem. Numbers in parentheses refer to the
enzymes listed in Table 1. TABLE 1 Enzymes in the mammalian biosynthetic pathway of haem Haem synthesising enzymes
Haem degrading enzymes
(1) 5-Amino laevulinate synthetase (EC. 22.214.171.124) (2) B-Amino laevulinate dehydratase (E.C. 126.96.36.199) (3) Porphobilinogen deaminase (E.C. 188.8.131.52) (4) Uroporphyrinogen cosynthetase (5) Uroporphyrinogen III decarboxylase (E.C. 184.108.40.206) (6) Coproporphyrinogen III oxidase (E.C. 220.127.116.11) (7) Protoporphyrinogen oxidase (E.C. 18.104.22.168) (8) Ferrochelatase (E.C. 22.214.171.124)
(9) Haem oxygenase (E.C. 126.96.36.199) (10) Cytochrome P-450 reductase (E.C. 188.8.131.52) ( 11) Biliverdin reductase (E.C. 184.108.40.206)
synthesised via formation and subsequent degradation of haem by the same mechanisms as occur in animal systems. The following brief review contains an account of recent work on the main features of phycobilin synthesis, concentrating especially on those areas of novel interest and likely growth points for the future.
2. Formation of 5-ALA As with mammalian haem synthesis, production of 5-ALA is the first committed step in chlorophyll synthesis, in phycobilin synthesis in both red and blue-green algae and in phytochrome synthesis in higher plants. It is now known that, in these organisms, 5-ALA is made from the intact fivecarbon skeleton of glutamate [8,9] rather than from the condensation of glycine and succinyl coenzyme A as occurs in animals. This appears to be true whether the 5-ALA is destined for chlorophyll, phycobilins or haem (all such organisms synthesize small amounts of haem for cytochromes). The five-carbon pathway of 5-ALA synthesis requires the activities of three soluble enzymes to bring about this conversion  as shown in Fig. 4: (1) a magnesium- and ATP-requiring ligase which activates glutamate by forming glutamyl tRNAGIU; (2) a NADPH-linked dehydrogenase responsible for the conversion of glutamyl tRNAGIU to glutamate-1-semialdehyde; (3) an aminotransferase which transforms the semialdehyde to form 5-ALA. Following a good deal of earlier suggestive evidence, the formation of 5-ALA exclusively from glutamate has been confirmed in maize [lo] and in the red alga Cyanidium caldarium [ll], and utilization of the five-carbon pathway has also been shown in a number of cyanobacteria including
Fig. 4. Formation of B-ALA from the intact carbon skeleton of glutamate. (1) Glutamyl tRNA ligase; (2) glutamate tRNA dehydrogenase; (3) glutamate-l-semialdehyde aminotransferase.
Anabena variabilis , Agmenellum quadruplicatum  and Synechocystis 6803 [ 141. Characterisation of the three enzymic processes involved in the transformation of glutamate destined for both chlorophyll and for phycobiliproteins has been achieved in a number of species and is considered in detail below. 2.1. Glutamyl tRNA ligase The dependence of 5-ALA synthesis on an RNA fraction was first shown by the use of RNase and RNase inhibitors to respectively abolish and protect against abolition of 5-ALA formation from glutamate [15 - 171. The identity of the RNA involved in 5-ALA synthesis as a tRNAG1” came from studies using a purified RNA fraction and labelled glutamate to show specific incorporation of the charged amino acid into 5-ALA [15,18]. The RNA was found to hybridise to the chloroplast genome in barley  and has since been identified by sequence analysis as a tRNAGIU with a UUC anticodon [ 191. It has recently been confirmed by anticodon-based affinity chromatography that the active RNA components in C. caldarium and Synechocystis also carry the UUC anticodon [ 201. There has been some discussion as to whether the tRNA used to activate glutamate for 5-ALA synthesis is also involved in protein synthesis within a given organism. Early reports suggest that although tRNAs from other organisms were capable of heterologous reconstitution with plant extracts to form charged glutamate, many failed to reconstitute 5-ALA formation [16, 211. These results were interpreted to suggest that the RNA required for 5-ALA formation may be functionally distinct from that involved in protein synthesis [ 211. However, recently it has been shown that tRNAG1” isolated from Synechocystis 6803 by UUC anticodon affinity chromatography is equally effective in supporting 5-ALA formation and protein synthesis [ 201. It is in any case probable, in organisms whose chloroplast genes use a single codon for glutamate [22, 231, that one tRNAGIU is used for both protein and 5-ALA synthesis [ 181. It is interesting to note that in a number of species so far reported, fractionation of the 5-ALA synthesising components by affinity chromatography has resulted in a tRNA moiety being bound to a haem or chlorophyllin sepharose column [ 16, 18,241. The ligase enzyme itself has been purified from a number of higher plants [ 21,261 and algae [ 271 but purification has not yet been reported for a red or blue-green algal species. The barley ligase has been purified to homogeneity by immunoaffinity chromatography  and has a subunit molecular weight of 54 000 Da. Experiments on the purified enzyme from barley confirm the requirement for ATP in the aminoacylation reaction and that the enzyme is capable of accepting aI1 of the three tRNAGIUpresent in the chloroplast. As only one glutamate specific tRNA ligase was identified, it seems likely that a single enzyme provides glutamate for both chlorophyll and protein biosynthesis, and that it may have a role in coordination of the two biosynthetic pathways [ 261.
2.2. Glutamate tRNA dehydrogenase This enzyme, responsible for the conversion of glutamyl tRNA to glutamate-l-semialdehyde, is widely believed to catalyse the rate-determining step of the pathway . This step may be the point at which feedback inhibition operates [ 271 and Glutamate tRNA dehydrogenase is certainly the least characterised enzyme of the 5-ALA synthesis pathway. Dehydrogenase activity from both higher plants and algae [28 - 301 is adsorbed onto blue sepharose affinity columns, along with glutamyl tRNA ligase. Attempts to separate the two enzymes have resulted in a decrease of activity leading to the suggestion that they may exist as a complex in uiuo [ 81. The barley dehydrogenase has been purified sufficiently to demonstrate a concomitant oxidation of NADPH during the conversion of glutamyl tRNA to glutamate-l-semialdehyde [ 81. The property of the dehydrogenase as an NADPH-binding protein has been used to separate the ChZoreZZaenzyme on an ADP-agarose affinity column . A preliminary report of the separation of enzyme activity from Synechococcus has appeared  and confirms the similarity of the behaviour of the cyanobacterial enzymes on affinity chromatography with those of the barley systems. If this enzyme does represent a control point in porphyrin biosynthesis, it will be interesting to determine whether the controlling factors in higher plants, whose main porphyrin product is chlorophyll, are the same as those in the phycobilin-producing organisms.
2.3. Glutamate-1-semialdehyde aminotransferase The conversion of glutamate-l-semialdehyde to 5-ALA was confirmed as the next step in the pathway by chemical synthesis of the substrate  and identification of its structure by mass spectroscopy and ‘H and i3C nuclear magnetic resonance (NMR) spectroscopy [34 - 361. The enzyme responsible for this conversion has proved to be relatively stable and, as a result, glutamate-l-semialdehyde aminotransferase is perhaps the best characterised enzyme of the 5-ALA biosynthetic pathway in a wide range of organisms. In addition to these advantages, the enzyme is specifically acid), a known inhibited by gabaculine (3-amino, 2,3-dehydrobenzoic inhibitor of pyridoxal phosphate-linked enzymes [37 - 391 and this has proved a useful tool in the study of pigment synthesis in general. Gabaculine was first used to inhibit chlorophyll synthesis in barley chloroplasts  and leaves [35,41]. It is now known also to inhibit phytochrome synthesis in higher plants  and phycocyanin synthesis in a number of species [ 43 - 451. It is of particular interest that in the case of phytochrome synthesis, gabaculine has been shown to affect chromophore formation independently of apoprotein synthesis , indicating that synthesis of the apoprotein and chromophore are not coupled obligatorily, and that the apoprotein is stable within the cell. Similar studies have not yet been reported for the synthesis of other phycobiliproteins.
Gabaculine-treated leaves have been shown to accumulate glutamate-lsemialdehyde [ 341, but the exact mode of inhibition of the aminotransferase has been the subject of some discussion. Although gabaculine is known as an inhibitor of pyridoxal phosphate-linked transaminases [ 381, its inhibitory effect on the barley enzyme is only apparent in the presence of, or following pre-incubation with, an amino acceptor [ 361. This has led to the hypothesis that the enzyme exists in its native form as a pyridoxamine phosphate and only becomes sensitive to gabaculine on conversion to the pyridoxal form either during the normal catalytic cycle  or by incubation with alternative amino acceptors [ 361. The effect of pyridoxal phosphate on enzyme activity has been reported to be both inhibitory  and stimulatory  whilst pyridoxamine has been shown to activate the enzyme in barley , Synechococcus PC6301 and, more recently, in C. caldarium [30,32]. These results may not be entirely contradictory for two reasons. Firstly there may be species differences in cofactor requirements. Secondly if pyridoxamine is the true cofactor then pyridoxal phosphate could still have a stimulatory effect on activity of the apo-enzyme given the presence of an amino donor. The aminotransferases of barley and Synechococcus PC6301 have been purified, and their partial amino acid sequences reported [ 301. Interestingly, antibodies to the barley enzyme cross-react with the cyanobacterial protein. The N-terminal amino acid sequences of the two enzymes also show significiant homology. Given the reported differences in cofactor requirements and sensitivity to gabaculine of these two aminotransferases, further comparative studies may provide useful information on the catalytic mechanism of the enzyme. It is interesting to note that a gabaculine-resistant mutant of ChZamydomonas rheinhardtii has been reported , as has the ability of the red alga C. caldarium to adapt to the presence of gabaculine during heterotrophic growth [44,49]. The effect of gabaculine in these latter cells is to prolong the lag phase, which is followed by an exponential phase at near normal rates. Adapted cells are not only capable of producing normal growth curves when re-inoculated into fresh medium containing gabaculine, but will also synthesise phycocyanin and chlorophyll in the presence of gabaculine when exposed to the light . The mechanism of this adaptation has yet to be established, but it will be interesting to compare the gabaculine effects in this organism with the UV-induced mutation in Chlamydomonas. 2.4. Alternative five-carbon pathways Although it is now widely believed that the formation of 5-ALA from glutamate uses glutamate-1-semialdehyde as the substrate for the final step, with diaminovalerate as a possible enzyme-bound intermediate [30, 361, there is still a body of opinion which supports an alternative five-carbon pathway. The suggested intermediate in this pathway is 4,5-dioxovalerate (DOVA), with the enzyme DOVA aminotransaminase being responsible for its conversion to 5-ALA. This enzyme is found in plants, as well as in bacteria and animal tissues, although not always in high activity. The strongest evidence for DOVA transaminase involvement in 5-ALA biosynthesis is in green algae (
est evidence for DOVA transaminase involvement in 5-ALA biosynthesis is in green algae (e.g. ref. 50) and it has also been considered recently as a further component of the glutamate-1-semialdehyde route, replacing diaminovalerate as the final intermediate in the pathway [ 51] . Evidence that DOVA transaminase plays a major role in 5-ALA synthesis in higher plants or in the red and blue-green algae is so far not clear and for this reason the DOVA pathway will not be considered further here.
3. Formation of protoporphyrin IX from B-ALA By analogy with mammalian systems, the next phase in biosynthesis of phycobilins would be expected to lead to formation of protoporphyrin IX. The first evidence that a porphyrin precursor is involved was obtained by Troxler [ 521. Troxler showed that formation of [ i4C]phycocyanobilin from [ i4C] 5-ALA was accompanied by equimolar amounts of [ 14C]carbon monoxide. The only reasonable interpretation of this result was that the carbon monoxide arose by cleavage of a methene bridge of a cyclic tetrapyrrole (see Fig. 1 for mammalian comparison). Subsequent experiments with C. culdarium have directly demonstrated the intermediacy of uroporphyrinogen III, coproporphyrinogen III and protoporphyrin IX [ 531; in addition, a substantial amount of porphobilinogen was isolated. However, because these same intermediates would be expected to be formed en route to chlorophyll (I produced by the same organism, it is difficult to be certain that they are also precursors of phycocyanobilin. This problem is even more acute when considering the biosynthesis of phytochrome in higher plants, in view of the minute amounts of phytochrome fomed relative to chlorophyll. The best way to obtakproof of the involvement of these intermediates in phycobilin formation is by demonstration of incorporation of exogenous labelled intermediates into the phycobilins either in uiuo or in vitro. Few such studies have been carried out for this part of the pathway, an exception being the demonstration in our laboratory that [14C] protoporphyrin is incorporated both into chlorophyll a and into phycocyanin. In addition, administration of [ 14C]5-ALA to C. caldarium results in labelling of both chlorophyll a and phycocyanin at the same specific radioactivity suggesting formation of both pigments from a common metabolic pool. Detailed studies have been made on the formation of porphobilinogen in pea and Arum . Porphobilinogen deaminase has also been studied in Euglenu [ 551, spinach , and pea and Arum [ 541. Fewer investigations have been made on uroporphyrinogen cosynthetase because no simple assay system is available. Preparations of the enzyme have, however, been obtained from Euglenu [ 571 and spinach [ 561. Studies of the porphyrin biosynthetic pathway in higher plants between uroporphyrinogen III and protoporphyrin IX are very sparse, although it has been demonstrated that etiolated cucumber cotyledon homogenates incubated with [ 14C]5-ALA accumulated radio-
labelled coproporphyrin III and protoporphyrin IX as well as several other porphyrins [ 581. This part of the pathway has also proved of preparative value. Using C. caldarium both in vivo and in vitro we have developed methods to prepare radiolabelled porphyrins of high specific radioactivity. These include protoporphyrin IX, coproporphyrin III, uroporphyrin III and protoporphyrin IX monomethyl ester. We have used [ 14C]protoporphyrin IX to synthesise [ “C]haematoporphyrin derivative and a radiolabelled equivalent of Photoein II, the photosensitiser used in the photodynamic therapy of cancer [ 59,601.
4. Formation of bilins from protoporphyrin - the synthesis and degradation of haem The route by which protoporphyrin IX is converted into linear tetrapyrroles (bihns) in organisms synthesising phycobiliproteins was uncertain until 1981. By analogy with mammalian systems, a pathway involving biosynthesis and degradation of haem was clearly possible. However, unlike mammals, all organisms synthesising phycobilins also synthesise substantial amounts of chlorophyll, a pathway which involves the insertion of magnesium into protoporphyrin IX. The possibility that plant bilin synthesis might also occur via the magnesium pathway was therefore suggested and the feasibility of such a process was supported on the basis of chemical model systems . This question was finally resolved when it was shown that when [14C] haem was administered to whole cells of C. caldarium the resultant phycocyanobihn was radiolabelled . Samples of chlorophyll ~1isolated frgm C. caldarium in the same experiments contained no radioactivity. This proved that the incorporation of label into phycocyanobilin occurred by direct insertion of haem rather than by labelling of protoporphyrin IX caused by loss of iron, which would have resulted in chlorophyll labelling also. This direct proof that haem is a biosynthetic intermediate in phycobihn formation implied the existence of the enzymes ferrocheletase (E.C. 220.127.116.11) catalysing iron insertion into protoporphyrin IX, and haem oxygenase (E.C. 18.104.22.168) catalysing degradation of haem to biliverdin (Table 1). 4.1. Insertion of iron in to pro toporphyrin IX The first direct evidence for the presence of ferrocheletase in organisms synthesising phycobiliproteins arose from studies with the inhibitor Nmethylprotoporphyrin IX. Earlier work had shown that this material strongly inhibited mammalian ferrochelatase . In C. caldarium it was demonstrated that N-methylprotoporphyrin IX strongly inhibited phycocyanobilin synthesis, but not chlorophyll synthesis, in the dark, (following administration of &ALA ) providing further evidence for the intermediacy of haem and the role of ferrochelatase . These results were later confirmed
independently . Interestingly, in the light, both phycocyanobilin synthesis and chlorophyll synthesis were inhibited in parallel and it was concluded that the primary effect was to cause inhibition of phycocyanobilin synthesis and that the inhibition of chlorophyll a synthesis was a secondary effect due to an as yet undefined control mechanism. Direct measurement of ferrochelatase activity in C. caldarium  was demonstrated using cobalt (II) and deuteroporphyrin IX as substrates [ 681. This work showed that dark-grown cells (which do not produce phycobiliproteins) had relatively low levels of the enzyme but, on exposure to light, the enzyme activity increased approximately six-fold, in advance of phycocyanobilin synthesis. A partial purification of the ferrochelatase from C. culdarium has been achieved in our laboratory, but the enzyme has not yet been purified to homogeneity.
4.2. Degradation of haem to biliverdin In mammals the breakdown of haem to biliverdin (as shown in Fig. 1) is catalysed by the microsomal enzyme haem oxygenase . The mammalian enzyme requires O2 and NADPH and a second microsomal enzyme, NADPH-cytochrome P-450 reductase, and it is thought that the first step involves the formation of the mesohydroxyhaem species  (see Fig. 1). A particular feature of this reaction is its regioselectivity i.e. whilst there are four possible nonequivalent positions at which hydroxylation and subsequent macrocyclic ring cleavage can occur, degradation occurs only at the cr-meso position to yield biliverdin 1X-o and hence bilirubin 1X-o (see Fig. 1). This regioselectivity is not an intrinsic property of the haem molecule itself, but is directed by haem oxygenase. It is therefore highly significant that all of the phycobilins are also a-isomers and this fact, in itself, is good evidence for the existence of a plant haem oxygenase. More direct evidence for the enzyme came from studies in which [ 14C]mesohaem (the product obtained when the vinyl groups of haem are reduced to ethyl) was aministered to whole cells of C. caldarium. In these experiments, no incorporation of label into phycocyanobilin was observed (showing that mesohaem itself is not an intermediate on the pathway) but large quantities of mesobiliverdin were excreted into the medium [ 691. This is the material which is formed when mesohaem is degraded in the same way as haem itself. Since it is known that mesohaem is a good substrate for mammalian haem oxygenase [ 11, this may be taken as evidence for the presence of a corresponding algal enzyme. Using ‘so labelling techniques, it was shown by Brown and coworkers [70,71] that mammalian haem degradation occurred via a “two-molecule mechanism” whereby the oxygen atoms incorporated into biliverdin (Fig. 1) were derived from two different oxygen molecules. This is in direct contrast with other dioxygenase reactions where the two inserted oxygen atoms are derived from the same oxygen molecule. The “two-molecule mechanism” along with the release of CO is unique in biochemistry. It was highly signif-
icant therefore, when phycocyanobilin isolated from algal cells exposed to ‘s*i802 was shown to be also formed by the “two-molecule mechanism” [ 721 providing further strong evidence in favour of an algal haem oxygenase. The direct measurement of algal haem oxygenase activity in vitro has proved difficult. This was finally achieved using the fact that mesohaem is a substrate for the enzyme, but the product mesobiliverdin is not further metabolised [69, 731, and has aIlowed characterisation of the enzyme activity in algae which shows a number of parallels with its mammalian counterpart. Algal haem oxygenase requires O2 and NADPH and produces only the physiological IX Q isomer. In addition it is strongly inhibited by tin protoporphyrin IX, a known competitive inhibitor of animal haem oxygenases. It differs, however, in a number of important respects, the most striking of which is that, unlike animal haem oxygenases which are membrane bound, the algal enzyme appears to be entirely soluble [ 731. This soluble activity has been recently fractionated into three protein components, all of which are required to reconstitute enzyme activity. They consist of a low molecular weight (22 000 Da) ferredoxin-like component, a ferredoxin-linked cytochrome c reductase (37 000 Da) and a fraction of 38 000 Da corresponding to haem oxygenase itself [ 741. A further interesting example of the similarity between mammalian and algal haem oxygenase was our demonstration of cross reactivity of polyclonal rabbit antibody, raised against the mammalian enzyme, to the algal enzyme. Western blotting clearly showed an increased level of cross-reactivity during induction of pigment synthesis in C. caldarium [321. A preliminary report has appeared on the use of carbon monoxide release to measure haem oxygenase activity in higher plants where its activity might be expected to be required for phytochrome synthesis [ 751. This technique is potentially much more sensitive than those measuring accumulation of the porphyrin products, and it has been used successfully on human tissue homogenates and whole tissue biopsy preparations where the amount of material available is very small [ 751. The suitability of this assay for use in measuring plant haem oxygenase activity has yet to be fully established and will depend on the demonstration of specific inhibition of CO release by tin protoporphyrin IX. Following the cloning and expression of the cDNA for rat haem oxygenase [ 761 and subsequent characterisation of the cDNA for the human enzyme  it has become possible to study the regulation of haem oxygenase at the molecular level. It is of particular interest to note that in human skin fibroblasts the enzyme has been shown to be induced by UV-A radiation [78, 791. There may be parallels between this induction and that of the plant haem oxygenases. Although it is not expected that the plant enzymes should have the same controlling factors as the mammalian haem oxygenases, whose function within the cell is degradative rather than biosynthetic, it may well be that the molecular biological approach will prove as useful in the study of the plant enzymes as it has already in mammalian
studies, where information on gene structure and organisation is now being obtained [ 801. 4.3. Inhibition of chlorophyll a and phycocyanin synthesis by cobalt ions As already described, the activities of ferrochelatase and the algal haem oxygenase increase during the formation of photosynthetic pigments in C. caldarium. Recent studies in our laboratory have shown that cobalt II ions, which are known to induce mammalian haem oxygenase, inhibit the synthesis of both chlorophyll a and phycocyanin in this alga. The effect however is more pronounced on chlorophyll a synthesis than on phycocyanin synthesis. A concentration of 1 PM cobalt chloride significantly inhibits chlorophyll synthesis whereas phycocyanin formation is only slightly affected. This effect can be duplicated by incubating the algae with cobalt protoporphyrin IX during illumination. Cobalt II is known to be an effective substrate for ferrochelatase . It is believed that, in uiuo, cobalt II acts as a competitive substrate for the enzyme, producing cobalt protoporphyrin. This, in turn, may inhibit the algal haem oxygenase in a manner similar to inhibition of the mammalian enzyme by tin protoporphyrin. The reasons for the greater effect of the cobalt (II) ions and cobalt protoporphyrin IX on chlorophyll a synthesis are unknown, but the differences may be a reflection of a stronger inhibition of magnesium chelation, or a compartmentalisation effect. It is also possible that the enzymes in the biosynthetic pathway between magnesium protoporphyrin and chlorophyll a are strongly inhibited by cobalt protoporphyrin IX. Preliminary studies have suggested that chlorophyll a synthesis in higher plants is also inhibited by cobalt protoporphyrin, but the effect is not as dramatic as with C. caldarium. 5. Phycobilin formation from biliverdin The immediate product of the reaction of haem oxygenase is biliverdin IX-a. The latter stages in the production of phycobilins from biliverdin have been studied mainly in one organism, C. caldarium. The direct evidence for these intermediates therefore relates mainly to the biosynthesis of phycocyanobilin, though it is likely that other phycobilins are formed by the same initial step. When cells of these algae are incubated with 5-ALA they excrete a large number of porphyrins and bile pigments . One of these pigments has been identified as biliverdin IX-a  confirming that this is an intermediate in the formation of phycocyanobilin. The direct incorporation of biliverdin into phycocyanobilin has now been established by in uiuo feeding experiments. Cells of C. caldarium were incubated with [ “C]biliverdin 1X-a and radioactivity was recovered in phycocyanin and its chromophore after isolation and purification of the protein [ 82,831. Studies in our laboratory have been able to show that 9 - 12% of the synthesized pigment was derived from added biliverdin.
Similar experiments in vitro have demonstrated the transformation of biliverdin to phycocyanobilin in extracts of C. caldarium . Beale and Cornejo have shown that two products are formed from biliverdin and these have been proposed as the Z- and E-ethylidine isomers of phycocyanobilin, the Z isomer being a possible precursor of the E isomer which in turn binds to the apoprotein. The transformation of biliverdin to phycocyanobilin requires two reduction steps. These may occur by one of three possible pathways, each of which proceeds via different intermediates (Fig. 5). Using i4C radiolabelled 3-vinyl-lðyl biliverdin and 3-ethyl-l&vinyl biliverdin in feeding experiments similar to those for biliverdin IX-o!, we have been able to eliminate these compounds as intermediates in the formation of phycocyanobilin  and thereby have shown that Pathways II and III (Fig. 5) do not operate. By elimination we therefore concluded that Pathway I must be in operation. This study therefore suggests that phytochromobilin, the chromophore of the plant regulatory protein phytochrome, is the true intermediate between biliverdin and phycocyanobilin. Unfortunately, because 14C-labelled phytochromobilin is not available, no direct proof of this pathway can be obtained. Recently, it has been shown that incubation of explants of phytochrome depleted oat seedlings with [ “C]biliverdin gives rise to rapid incorporation of isotope into phytochrome. The newly transformed phytochrome had characteristic spectral properties identical to those of the native phytochrome [ 861. In addition, exogenous biliverdin was able to overcome inhibition of phytochrome chromophore biosynthesis by gabaculine in Auena seedlings . These experiments support the hypothesis that biliverdin is the precursor of phycobilin in higher plants as well as in ‘algae.
6. Incorporation of phycocyanobilin
The chromophore of phycocyanin is attached to the apoprotein via a thioether bond to cysteine [88 - 901. There has been some debate as to whether phycocyanobilin is synthesised completely independently of apoprotein before attachment or whether an earlier intermediate may be attached to apoprotein, with the remaining biosynthesis of chromophore occurring subsequently. The fact that phycocyanobilin can be excreted by C. culdarium in the absence of apoprotein on incubation with 5-ALA  favours the former alternative. However, the ethylidene group is chemically more stable than the vinyl isomer and it is possible that the excreted pigment may arise as the vinyl derivative and rapidly isomerize in the incubation medium. In principle therefore, the formation of the thioether linkage could arise by a vinyl-cysteine reaction, rather than an ethylidene-cysteine reaction. However, recent work in our laboratory has shown that phycocyanobilin that has been cleaved from phycocyanin can be re-incorporated into
native phycocyanin by C. caldarium actively synthesising pigment. The balance of evidence therefore suggests that the pathway involves the independent formation of phycocyanobilin, followed by incorporation into protein. Another study demonstrated that exogenous phycocyanobilin could be accepted by phytochrome apoprotein to form a “pseudo-phytochrome”
1861. 7. Future prospects The recent studies on the biosynthesis of phycobiliproteins described here have led to the elucidation of almost all of the metabolic steps involved and have revealed several features of novelty and interest. The biosynthesis of &ALA destined for phycobilins appears to be identical to that operating in all plants for chlorophyll synthesis as distinct to that occurring in animals and most bacteria. On the other hand, the formation of phycobilins from protoporphyrin-IX occurs via formation and breakdown of haem in precisely the same way as occurs in animals. Indeed, since the pathways for biosynthesis of phycobilins in, for example, cyanobacteria, are very much older in evolutionary terms than the mammalian pathways, it is reasonable to assume that mammalian haem metabolism evolved from the existing pathway in photosynthetic organisms. The most recent work showing that phytochromobilin is an intermediate in the biosynthesis of phycocyanobilin is particularly significant because it implies that all three major plant bilins may be formed by a single unified pathway as shown in Fig. 2. This phytochromobilin is formed directly from biliverdin by reduction of ring A. A further reduction at the ring A side chain can produce phycocyanobihn as shown. However, an alternative reduction in a methene bridge position can produce phycoerythrobilin. Although there is no evidence as yet for this latter possibility, such a unified mechanism is very attractive. In spite of recent progress in elucidating the metabolism of phycobilins, very little is known about the control of the pathways. In particular, the mechanisms by which light exerts its influence remain rather obscure and it may be expected confidently that studies in this area will produce interesting findings. The whole area of control of pigment synthesis, including the role of light, seems ripe for study by molecular biology techniques. Recent progress in studies of control of mammalian haem oxygenase by these approaches have been revealing and there seems no reason why a similar strategy could not now be adopted for study of photosynthetic systems.
Acknowledgment We wish to thank the Yorkshire Cancer Research Campaign and the Science and Engineering Research Council for financial support.
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