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Home , Bacteriochlorophyll, Chlorophyllide, Chlorophyll a, Chlorophyll b, Phycoerythrobilin, Porphyrin

J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from Review article

Journal of Medical Genetics, 1980, 17, 1-14 Haems and : comparison of function and formation

G A F HENDRY AND 0 T G JONES From the Department ofBiochemistry, The Medical School, University ofBristol, Bristol BS8 ITD

In 1844 Verdeill reported that acid treatment of at the same time by McMunn3 of , or haem yielded apparently similar another group of haem . compounds; he even postulated that chlorophylls It was the demonstration by Nencki and co- would contain . Hoppe-Seyler2 confirmed the workers 45 that the degradation of both chlorophylls apparent similarity of acid derivatives of haems and and haems yielded monopyrroles that led them, in chlorophylls from their light absorption charac- true neo-Darwinian fashion, to postulate a common teristics, a point rather overshadowing the discovery origin for animals and .

0 0-'I CH2 II copyright. CH CH3 COOH CIH2 CH2 C-O CH2 http://jmg.bmj.com/ NH2 ( CH3' 'CH3 ® 5- Aminolaevulinic acid a CH2 2 1 12 2 CH2 )H COOH CD FIG 1 Structures ofprotohaem and Protoporphyrin IX and two of their precursors, acid and 5-aminolaevulinic on September 30, 2021 by guest. Protected protoporphyrin IX (with substituent numbering positions). CH2 CH CH.--,j

CH2 CH2 COOCH3

Protohoem (haem- b) CooC20H39 Chlorophyll a 1 J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

2 G A F Hendry and 0 T G Jones Following the work ofWillstatter6 and Fischer and particularly those of avian egg shells, have no Stern,7 the structure of most natural and many central complexed metal. There are only five metals unnatural was established. Although commonly found in natural porphyrins: copper in a Verdeil's prediction was wrong, he was correct in uroporphyrin III derivative in the flight feather of interpreting chlorophylls and haems as having the tropical Musophagidae family of birds; cobalt as essentially similar structures. the metal component of (cobalamins)9; Today we can show the similarity between haem iron in the metal complex in haems including and chlorophyll based on their common precursor, haemoglobin, , catalase, peroxidases, and protoporphyrin IX (fig 1). The carbon numbering cytochromes. The fourth metal, , is system used in fig 1 will be used in the subsequent characteristic of all chlorophylls and bacterio- text. chlorophylls. A fifth metal, zinc, may complex enzymically or non-enzymically to many porphyrins Natural occurrence of porphyrins and to the breakdown product (it is not known if complexing occurs before or after For over a century, scientists have been aware of the ring cleavage). Certainly the ease with which zinc is existence of the numerous types of porphyrin-based inserted non-enzymically into porphyrins in vitro compounds to be found in a wide range ofeukaryotic would make it surprising if zinc porphyrins did not and prokaryotic organisms. It was presumably only exist in vivo. It is indeed more surprising that nature a question of time before reports of extra-terrestrial has confined the metal complexes of porphyrins to porphyrins would be made.8 The earth-bound bio- four or five only; chemists have been able to logical porphyrins are diverse and range in colour insert well over 40 metals into porphyrins. from grey-blue (), (chloro- The biosynthetic pathway (fig 2) of porphyrins phyll), and red (protohaem) to yellow and brown from the monopyrrole begins with (avian egg porphyrins). the formation of the cyclic uropor- The natural occurrence of many porphyrins is phyrinogen IM and I. Isomers II and IV have shown in fig 2, together with their biosynthetic not been reported in biology; I is not ancopyright. relationships. It will be seen that many , intermediate in haem and chlorophyll synthesis, it

Porphobil inogen Cu III M bird integuments | Uroporphyrin I Uroporphytrinogen C Turacin http://jmg.bmj.com/ Co feathers

-*- CoproporptDhyrinogen III Fe- - --Vitamin B 1Certain I Avian eggs I hyi I * Sirohoem Plants, fungi, pbacnteria I Harder's gland ___H_rderopo_ in mammals .-Harderopo)rphyrinogen III Zn - egg IAvian eggs Protoporphyrinogen JX- verdin - sm on September 30, 2021 by guest. Protected II F~~~~~~~~~~~~~~~e Haemproteins |Echiuriod (e.g: cotalose) warm harmane Bonellin.s- ? Protohaem -_- Haem b Mg Mot_ rgnim cytochromes Most organisms Protoc hlorophyl l(ide) _,Haem a. c cytochromes Eukaryotic and some prokaryotic Dihydrochlorin a- Haem cl, s --w rProkoryotes | photosynthetic (chlorophylls Icertain fungiI organisms a.b.c.d Leghoemoglobin _| sybosesJPlant-bacteria Hoemoglobin Certain photosynthetic Tetrahydrochlorins [osivrtbrates, bacteria Myoglobin many invertebrates (bacteriochlorophylIs Chlorocruorin a,b.c.d.e ) FIG 2 Biosynthetic pathway oftetrapyrroles and their natural occurrence. Ki-dK k i J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

Haems and chlorophylls: comparison offunction andfotrmation rL occurs commonly as an excreted compound in the Anoplura (suckirn lice), and Ixodes (ticks), t1j group of disorders of haem synthesis known as the haemoglobin is derive ftrn the higher a-nalj o t . It also occurs in many mollusc shells and and the moiety I.d in nutrition. Full re integuments, in just those molluscs which do not of the occurrence of haiei6ilobins and myogtabih synthesise haemoglobin. This led Kennedy10 to in invertebrates are provided by Kennedy.13 The postulate that molluscs themselves may be porphy- following summary is very much a simplification of riacs! These molluscs must additionally make what is a complex story. Haemoglobins have been sufficient isomer III for the synthesis of reported in certain protozoan strains, in the larvae prosthetic groups. Uroporphyrin, coproporphyrin, ofthe insect Chironomus, in one sub-class ofmolluscs, and protoporphyrin are found in egg shells and in the bivalvia (Solen, Arca, and Pentunaulus where the some cases account for their characteristic colours. haemoglobin is present in corpuscles), and in one Intermediate between coproporphyrin and proto- gastropod genus Planorbis (as free haemoglobin in porphyrin is harderoporphyrin, which is a pigment the plasma). In the crustacea, haemoglobin appears found in the Harderian gland of the rodent eye, sporadically in all orders of the branchiopods. The though it is present in other tissues. most unusual case occurs in Daphnia, where the A equivalent to the porphyric mammal may haemoglobin is generally present in the eggs, but be found in some x-ray or UV treated seedling may be absent in the adult. Many crustaceans also mutants. Some such seedlings accumulate uropor- contain bile pigments, which by analogy with phyrin, coproporphyrin, and particularly proto- mammals are, probably, breakdown products of porphyrin IX. From such findings protoporphyrin protohaem. Among the nemerteans haemoglobin is IX has come to be placed in a central role in the present in some members but not all, published study of haem and chlorophyll . Proto- phyllogenies showing little correlation with presence porphyrin isomer IX, of the fifteen possible isomers, or absence of haemoglobin. Haemoglobin is more is believed to be a prccursor at the branch point of common in the annelid worms, generally as a freely of both haems and chlorophylls. dispersed pigment. However, in a few species,

erythrocyte corpuscles may be found (for example copyright. Naturally occurring iron porphyrins in Travisia and Trevella spp). In the polychaete Aphrodita, haemoglobin is present in the nerve cord. From fig 2 it will be seen that a minor pathway may Among the marine worm phyla, haemoglobin is to the iron-chelate sirohaem (see below) and a present in the echiurids and lophophorates, generally major pathway gives rise to a series of haem com- within corpuscles. In that 20th century discovery, the pounds, of which the cytochromes, in one form or deep sea dwelling pogonophora worms, haemoglobin another, are present in nearly all organisms. Haemo- has been reported, but in a freely dispersed form. http://jmg.bmj.com/ globin, a characteristic pigment of vertebrates, is It is towards the top of the invertebrate 'tree', the present in a wide range of organisms. One family of holothurian echinoderms or sea cucumbers, that vertebrates, however, the ice-fish (Chaenicthyidae) discoid cells containing haemoglobin are found, survive in the cold waters of the Antarctic apparently corresponding to the erythrocytes of mammals. without any haemoglobin; their requirement The monomer myoglobin is closely related in con- is met from oxygen dissolved in blood plasma. formation to one of the chains of the haemoglobin Similarly, the Leptocephalus eel larva has no haemo- tetramer. It is present throughout the vertebrates globin until it reaches the elver stage." A form of but appears only sporadically in the invertebrates. on September 30, 2021 by guest. Protected haemoglobin is found in plants under the name of Typically, myoglobin is present in the gastropod leghaemoglobin, It is, however, confined to those radula muscles and the body wall muscles of various plants which are able to fix nitrogen in co-operation polychaete fanworms and occasionally in other phyla. with the bacterium Rhizobium. Such plants include Haem s (also called Spirographis haem or chloro- the legume family. Leghaemoglobin appears to help cruorohaem) in combination with a protein serves to maintain a constant, but low, level of oxygen as the extra-corpuscular oxygen-carrying pigment in within nitrogen fixing cells. Haem synthesis appears four families of polychaete worms (Sabellidae, to take place in the bacteria, globin in the plant, the Serpullidae, Ampharetidae, and Flabelligeridae). completed molecule being located on the interface Almost on a belt-and-braces principle, members of of the two organisms.'2 the Serpula and Potamilla families contain both Plants do not, of course, have a complex gas haem s protein and haemoglobin. To show complete transport system; they obtain their oxygen from inconsistency, at least to reviewers, in the family solution in cell wall water films. Haemoglobin is also Spirorbis there are three groups, one containing found in the invertebrate phyla, though in some cases, haem s protein, one containing haemoglobin, and particularly in parasites such as Aplaniptera (fleas), the third containing neither. J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

4 G A F Hendry and 0 T G Jones Other pigments commonly used in oxygen carry- CH2 ing, other than blood plasma or water, include the II iron protein hemerythrin (despite its name not a CH CH3 haem protein) and the copper containing protein (also not a haem protein). The former has a restricted distribution among certain peanut CH3 worms, inarticulate brachiopods, and a few other phyla, always apparently in corpuscles. Hemo- cyanins, as oligomeric proteins, on the other hand are always free floating and are found in the non- haemoglobin containing molluscs, notably the Prosobranchia and Pulmonata, and in those suppo- sedly ancient mollusc ancestors, the chitons. These hemocyanin containing molluscs accumulate uro- CH3 porphyrins in their shells. are also found in scorpions, in the cephalopods (squids, cuttlefish, and octopods), and in the majority of crustaceans, including the malacostracan crabs, CH2 lobsters, and shrimps. Hemocyanins are also found CH in that other living fossil, the survivor ofthe trilobites, OH2 COOCH 0 the horse-shoe crab. It is quite clear, then, that COOH 3 alternative systems to haemoglobin for oxygen transport have existed for hundreds of millions of Protochlorophyl lide years, probably long before the appearance of the FIG 3 Structure ofprotochlorophyllide, the precursor of first land animals. chlorophylls and bacteriophylls.

Apart from the animal kingdom, all fungi and copyright. plants contain haem pigments, the cytochromes. One third or more of the total tetrapyrrole content of dark-grown seedlings consists of protohaem.'4 chlorophyll: the dihydroporphyrins or (for Plants, in common with most other organisms, also example chlorophylls a and b) present in all eukaryo- contain other haem proteins, notably catalase, tic photosynthetic organisms and some prokaryotes peroxidases, and tryptophan pyrrolase. (the blue-green ), and the tetrahydroporphyrins

(for example a and b) found in http://jmg.bmj.com/ Naturally occurring magnesium porphyrins the purple sulphur (Thiobacteriaceae) and non- sulphur bacteria (Athiobacteriaceae). In the green All chlorophylls are magnesium tetrapyrroles and it photosynthetic bacteria (Chlorobiaceae) bacterio- is likely that they are formed by successive modifica- chlorophyll a is a minor component; the major tions of magnesium protoporphyrin (see Jones15 for chlorophylls are bacteriochlorophylls c and d which a discussion of this). Indeed magnesium protopor- are chlorins. phyrin and its monomethyl have been found in mutant algae, plants, and bacteria in which normal Structures of naturally occurring porphyrins on September 30, 2021 by guest. Protected chlorophyll synthesis is inhibited, wholly or in part. A much more commonly found magnesium tetra- Table 1 lists, in an abbreviated form, the major and intermediate in cholorophyll synthesis is most of the known minor porphyrins (or porphyrin (see fig 3), in which the vinyl derivatives) found throughout the biological world. group at position 4 is reduced to the ethyl, and the The salient points from this table are that of the four fifth ring E, characteristic of the chlorophylls, has possible isomers, only isomer III been formed by a modification of the propionic is used in haem and chlorophyll synthesis; of the acid substituent at position 6. In higher plants (the fifteen possible isomers of protoporphyrin, only flowering plants or angiosperms), the porphyrin isomer IX has been reported from biological protochlorophyllide accumulates if seedlings are systems. There is a restricted number of side chain germinated in the dark. In most other plants (some modifications in naturally occurring porphyrins. algae being an exception) chlorophyll is formed even Once coproporphyrin III is formed, no further in the dark, with no detectable protochlorophyllide modification occurs at carbon position 1 in any accumulation. However, protochlorophyllide is an organism. Similarly, at carbon position 3, a methyl intermediate in the biosynthesis of two classes of group is present in all biological systems (except in J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

Haems and chlorophylls: comparison offunction andformation 5 TABLE 1 Structures ofnaturally occurring porphyrins Porphyrin Peripheral carbon position Complexed metal 1 2 3 4 5 6 7 8 Uroporphyrin I Ac Pr Ac Pr Ac Pr Ac Pr Uroporphyrin III Ac Pr Ac Pr Ac Pr Pr Ac Sirohaemt Ac/Me Pr/H Ac/Me Pr/H Ac Pr Pr Ac Fe Coproporphyrin III Me Pr Me Pr Me Pr Pr Me - Harderoporphyrin III Me Vi Me Pr Me Pr Pr Me - Protoporphyrin IX Me Vi Me Vi Me Pr Pr Me - Bonellin 4-Me H Me H Me Pr Pr/H Me/Me - Protohaem (haem b) Me Vi Me Vi Me Pr Pr Me Fe Haem a Me RI Me Vi Me Pr Pr Fo Fe Haem c Me R2 Me R2 Me Pr Pr Me Fe Haem dt Me HO-Et Me Vi Me Pr Pr/H Me/H Fe Haem s Me Fo Me Vi Me Pr Pr Me Fe Lactoperoxidase haemt Me Vi Me Vi HO-Me Pr Pr HO-Me Fe Myeloperoxidase haemt Me Vi Me Vi Me Pr Pr Fo Fe Mg-protoporphyrin Me Vi Me Vi Me Pr Pr Me Mg Mg-proto methyl ester Me Vi Me Vi Me R3 Pr Me Mg Protochlorophyllide Me Vi Me Et Me CP Pr Me Mg a Me Vi Me Et Me CP Pr/H Me/H Mg Chlorophyll a Me Vi Me Et Me CP Phy/H Me/H Mg Me Vi Fo Et Me CP Phy/H Me/H Mg Chlorophyll cl Me Vi Me Et Me CP Acr Me Mg Chlorophyll c2 Me Vi Me Vi Me CP Acr Me Mg Me Fo Me Et Me CP Phy/H Me/H Mg Bacteriochlorophyll a Me R4 Me/H Et/H Me CP Phy/Ht Me/H Mg Bacteriochlorophyll b Me R4 Me/H R5 Me CP Phy/Ht Me/H Mg Bacteriochlorophyll c* Me HO-Et Me R6 Me or Et CP Far/H Me/H Mg Bacteriochlorophyll d Me HO-Et Me R6 Me or Et CP Far/H Me/H Mg Bacteriochlorophyll e* Me HO-Et Fo R6 Et CP Far/H Me/H Mg Abbreviations: Ac, -CH2-COOH; Pr, -CH2-CH2-COOH; Me, -CH3; Vi, -CH=CH2; Fo, -CHO; HO-Et, -CHOH-CH3; HO-Me'

-CH20H; Et, -CH2-CH3; CP, -cyclopentanone ring E; Phy, -Pr- ester; Far, -Pr- ester; Acr, -CH=CH-COOH; copyright. Rl, -CHOH-CH2-(CH2-CH=C.CH5-CH2)3-H; R2, -CH.S Cys. CH3; R3, -Pr-Me ester; R4, -C=O.CH3; R5, R6, -propyl, butyl, or ethyl. =CH-CH.; * 8-position-ethyl or methyl; t or Pr-geranylgeraniol ester; t proposed structure. chlorophyll b). Another conservative group is at The most varied substituents in biological systems position 5 which always retains the are found at positions 2 and 4. At position 2, the except in the somewhat uncommon green-sulphur vinyl groups of protoporphyrin IX are retained in http://jmg.bmj.com/ bacteria Chlorobacteriaceae, where the bacterio- protohaem, the chlorophylls a and b, in lacto- and chlorophylls c and d often include an ; myeloperoxidase. In the iron porphyrins the variation the haem of the lactoperoxidase also has an in the position 2 substituents is considerable, from oc-hydroxymethyl group at this position. Position 8 the simple reduction in bonellin (a worm sex commonly retains a methyl group except in haem a hormone) to the long chain 1-hydroxy-2-(E,E- and myeloperoxidase where a formyl substitution is farnesyl)-ethyl group substitution found in haem a present and in lactoperoxidase where again the (the prosthetic group of the cytochromes a). Among a-hydroxymethyl substitution takes place. the chlorophylls, position 2 changes occur in red on September 30, 2021 by guest. Protected So of the eight side group positions found in seaweeds (chlorophyll d) with formyl substitution for coproporphyrin Ill, positions 1, 3, 5, and 8 undergo the . In the prokaryote bacteriochloro- little or no further change. Even among the remain- phylls a and b, position 2 is modified to form a keto ing groups the changes are modest. The iron- group; in bacteriocholorophylls c, d, and e, it under- containing porphyrins retain the two propionic goes hydration to hydroxyethyl groups. acid side chains at positions 6 and 7. Among the Among the iron porphyrins the substituent at magnesium porphyrins, positions 6 and 7 undergo position 4 is fairly constant. The original vinyl modification; the group at 6, after group persists in haemoglobin, cytochromes a, b, esterification, forms the characteristic fifth ring E. and d, catalase, and most peroxidases. Only haem c, The propionate group at position 7 is esterified also, covalently bonded to its protein to form cytochrome but with the long chain fatty acid phytol (occasionally c, has thio-ether groups at positions 2 and 4 linking farnesol or geranyl-geraniol). Exceptions occur in it to the protein. Among the magnesium porphyrins, the case of the c chlorophylls from the brown sea- position 4 modifications are more common. The weeds where the propionate group at 7 is oxidised to original vinyl group at position 4 of protoporphyrin acrylate. IX is reduced to the ethyl state in chlorophylls a, b, J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

6 G A F Hendry and 0 T G Jones TABLE 2 Structures of naturally occurring compounds derivedfrom or related to porphyrins Product Original porphyrin carbon position 1 2 3 4 5 6 7 8 [Protoporphyrin IX] Me Vi Me Vi Me Pr Pr Me Biliverdin/ Me Vi Me Vi Me Pr Pr Me Me Et Me/H RI Me Pr Pr Me Me Vi Me/H RI Me Pr Pr Me (proposed) Me Vi Me/H R2 Me Pr R3 Me Phylloerythrin Me Et Me Et Me CP Pr Me Abbreviations as for table 1 except for R 1, = CH-CCH3; R2, = CH-CH3.protein; R3, -CH2-CH2-CO.protein.

c, and d and in bacteriochlorophyll a. In bacterio- amounts in autumn senescing leaves.23 One of the chlorophylls from the green sulphur bacteria, ethyl, great mysteries of porphyrin is the propyl, or even isobutyl groups may be present natural fate of chlorophylls in autumn leaves. Some depending on the species involved. Side chain varia- 1012 tonnes24 of chlorophyll disappear annually, tion among the porphyrin derivatives is for the much of it during autumn. greater part confined to positions 2 and 4 (table 2). Certain degradation products of porphyrins are Biosynthesis of porphyrins well known.16-18 Such products, usually bilins or bile pigments, are listed in table 2. The structural If animals and plants share a common evolutionary similarity between biliverdin, bilirubin, and meso- origin then it is likely that they would share a common bilirubin suggests that these compounds are derived biosynthetic pathway leading to tetrapyrroles, at from the breakdown of protohaem (from haemo- least in the initial stages of synthesis. If there were globin, myoglobin, cytochrome P-450, and cyto- major dissimilarities in biosynthesis, then it may be vitro. The a of biochemical convergent evolution has chrome b) and this has been confirmed in that form copyright. prokaryote blue-green algae and the red taken place. As will be seen, a study of porphyrin algae contain bile pigments (probably as accessory biosynthesis raises some unanswered questions. pigments in ), two of which have been Probably all animal haems, vitamin B12, and named phycocyanobilin and phycoerythrobilin.19 avian egg shell pigments, are derived ultimately from The absence of the fifth ring E and the presence of the condensation of and succinyl CoA to the propionate group at position 7 suggests that form the amino-acid 5-aminolaevulinic acid (ALA, were or fig 1) (see for a review). This is the first these compounds derived from protohaem Bogorad25 http://jmg.bmj.com/ protoporphyrin and not chlorophyll. The hormone committed step in tetrapyrrole biosynthesis. Two phytochrome is the only bile pigment known in such ALA molecules condense to form the mono- higher plants and it too would appear from its pyrrole porphobilinogen (PBG): structure20 to be a protohaem or a protoporphyrin Glycine ALA PBG derivative. There are many reports of bilins and + >-synthase--*ALA-- synthetase-*PBG related compounds whose structures have not been Succinyl CoA x 2 defined. Most are assumed to be oc-IX isomers,21 that is the tetrapyrrole ring has been split at the a. CO2 on September 30, 2021 by guest. Protected carbon atom, but at least one case of 'y-IX isomer formation is known from the pigment of butterflies.18 ALA was first shown to be an intermediate in Some of the bile pigments so far uncharacterised porphyrin synthesis by Shemin and Russell26; may be derived from haems a, c, and d and perhaps it is formed by ALA synthase (E.C.2.3.1.37) by coproporphyrin and protoporphyrin, although alter- avian and mammalian erythrocytes, bone-marrow native haem breakdown pathways have been cells, and liver, by yeast, bacteria, and insects (see suggested.22 The other interesting point to emerge review by Granick and Beale27). It was also shown in a from studies of naturally occurring porphyrin photosynthetic bacterium Rhodopseudomonas sphae- structures is the absence of any reports of bile roides,28 where its activity was found to be greatest pigments derived from chlorophylls. Indeed it is not at times of maximum bacteriochlorophyll synthesis.29 known how chlorophylls are degraded in vivo. The enzyme ALA synthase has not been unequivo- Chlorophyll degradation might involve the loss of cally demonstrated in higher plants. This much magnesium (to form phaeophytin) and the removal debated topic was recently reviewed by Porra and of the phytol side chain (to form phaeophorbide). Grimme30 and Beale.31 No enzyme has been described Such compounds have been found generally in trace from plants which could account for the rate of J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from Haems and chlorophylls: comparison offunction andformation 7 synthesis of chlorophyll in actively greening tissues.32 involving the co-operation of the enzyme porpho- Trace activities of ALA synthase have been reported bilinogen deaminase (also known as uroporphyrino- in nitrogen-starved green algae,33 in cold stored gen I synthase, E.C. 4.3.1.8.) and uroporphyrinogen potato peelings,34 and in soya bean callus under con- III co-synthase. A review describing these steps has ditions where chlorophyll synthesis was minimal or been provided by Battersby and McDonald.42 In repressed.35 An alternative biosynthetic pathway has this complex sequence of condensations there are a been shown to exist3' 36 which involves the conver- number of details to be cleared up.27 However, the sion of glutamate or glutamine to ALA with the basic mechanism involves the polymerisation of original 5-carbon skeleton intact. The precise four PBG units by the deaminase to form a transient mechanism for this conversion is uncertain.30 31 intermediate, which in the absence of co-synthase It has been known for some time37 that the ALA forms , but in the presence of synthesising system in plants, has, like that of co-synthase forms isomer III, this being the normal animals, a short half-life. The ALA synthase in product. The evidence suggests that essentially the animals is located in the mitochondrial matrix.25 same mechanism exists in human erythrocytes as in The location of the ALA-forming system in plants higher plants.27 The two are present only is not known though there is some evidence (for in the cytosol in animals but the precise location in example Kannangara and Gough32) that oc-oxogluta- plants has yet to be shown clearly, though one rate-derived ALA may be synthesised in the isolated location must be within the plastids. . It has been postulated that plants may Uroporphyrinogen III is a for at least have two ALA-forming systems.3' 38 One such three separate pathways: system, the classical ALA synthase system, may 4 x PBG operate in the mitochondria in the absence of light, while a quantitatively more important system Fe Uroporphyrinogen III Co utilising a glutamate derivative may be present in ) ( the chloroplast, functioning in chlorophyll synthesis. _ Some support for these suggestions has been pro- Sirohaem Coproporphyrinogen Cobalamin vided by the examples quoted above of plant ALA copyright. synthase activity from tissue synthesising little or Sirohaem is structurally closely related to uropor- no chlorophyll, operating perhaps, as in animals, phyrinogen (fig 4). Its relationship to uropor- with the mitochondrial enzyme. Interestingly, phyrinogen and its function in reducing environments Klein and Senger39 detected both a 'classical' has attracted considerable interest as a possible ALA-synthase enzyme and a second ALA-forming primitive cytochrome (see Granick and Beale27 and mechanism in the same cells of an algal mutant. On below). Sirohaem is the prosthetic group of sulphite balance, it appears that plants have evolved a reductases in several bacteria and fungi and nitrate http://jmg.bmj.com/ separate mechanism for the synthesis of their reductases in fungi and higher plants.4345 porphyrins, but whether this separate mechanism is The second compound closely related to uropor- in addition to or replaces the mammalian ALA phyrinogen III is cobalamin or vitamin B12. The synthase is not known with certainty. biosynthetic pathway and its unresolved problems The enzymatic condensation of two molecules of have been discussed46 with further information ALA to form porphobilinogen (PBG) is well charac- provided by Horig et al.47 The conversion of uro- terised. The soluble enzyme involved, PBG synthase III to the intermediate cobyrinic (E.C.4.2.1.24), is present in mammalian and avian acid involves cobalt insertion, and no less than seven on September 30, 2021 by guest. Protected cells, fungi, bacteria, algae, and higher plants.25 First steps and the elimination of the 6-meso observed in avian erythrocytes,40 it has subsequently carbon.46 The remaining steps involve seven amida- been found that the enzymes from different organisms tions of the acidic peripheral groups, bonding of have different co-factor requirements; potassium in deoxyadenosine to the cobalt atom, and incorpora- some photosynthetic bacteria, zinc in mammalian tion of dimethylbenzimidazole and ribose-5-phos- erythrocytes, and magnesium or manganese in phate. The final structure has been described as the higher plants. However, the molecular weight of the most complex non-polymeric biomolecule.27 Vita- enzyme (subunit mass about 35 000) appears to be min B12 is synthesised by a wide range of prokaryo- similar in all organisms. The enzyme is present in tes, including the reportedly haemless anaerobe extraplastidic fractions in plants,41 but there is Clostridium tetanomorphum, photosynthetic bacteria, uncertainty as to the location of the enzyme within blue-green algae, and the nitrogen fixing bacterium the plastids, whether soluble or bound to membranes. Rhizobium in higher plant root-nodules. Four molecules of porphobilinogen condense to The third and principal pathway for uroporphy- form the first porphyrinogen in a two-step reaction rinogen III utilisation is in the formation of the major J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from 8 G A F Hendry and 0 T G Jones planarians coproporphyrin (and uroporphyrin) deposits have been implicated in phototactic responses.'0 The conversion of coproporphyrinogen III to harderoporphyrinogen and then to protoporphy- rinogen IX involves the enzyme coproporphyrinogen oxidase [E.C.1.3.3.3.]. The first step in the conver- sion involves decarboxylation of the propionate group at position 2 to a vinyl residue giving enzyme- bound harderoporphyrinogen. Harderoporphyrin has been isolated from the Harderian gland of the rat, and is present in small amounts in bile and bone marrow,52 and in photosynthetic .55 An CH2 CH2 interesting point is that the oxidation of the vinyl group of harderoporphyrinogen to a formyl group, CH2 CH2 and decarboxylation of the propionate at position 4 COOH COOH to a vinyl group, would give rise to the porphyrinogen Uroporphyrinogen corresponding to compound haem s (or chloro- COOH cruorin). I{aem s acts in place of haemoglobin in several polychaete worm families (see above). Harderoporphyrinogen itself does not normally accumulate in tissue, but is a transient intermediate bound to the coproporphyrinogen oxidase enzyme, where it may undergo a rapid rotation in situ so that the second propionate group (position 4) is de- carboxylated to a vinyl group.54 The product, proto- porphyrinogen, may undergo spontaneous oxidationcopyright. to protoporphyrin IX, or a specific enzyme (proto- porphyrinogen dehydrogenase) may control this step.55 Such an enzyme has been found in several organisms including yeast and mammalian mito- CH2 CH2 chondria. Certainly the substrate for iron insertion, CH2 CH2 in the final step in protohaem (haem b) synthesis, is COOH protoporphyrin not protoporphyrinogen. http://jmg.bmj.com/ COOH Protoporphyrin IX is, in one sense, the key Sirohcern ( proposed) compound in porphyrin metabolism. It is the direct FIG 4 Structure of uroporphyrinogen III, a porphyrin or indirect precursor of the major haem compounds precursor, and ofsirohaem, the prosthetic group of a including haemoglobin, myoglobin, cytochromes a, possible primitive cytochrome. b, c, and peroxidases and catalase, as well as the chlorophylls, and of bacteriochlorophyll in photo- synthetic organisms (bacteriocholorophylls c and d including haems and chloro- on September 30, 2021 by guest. Protected metalloporphyrins, tissue phylls. In a series ofuroporphyrinogen modifications, may be exceptions, see Jbnes15). In healthy four carboxyl carbons are removed from the acetic protoporphyrin IX is present in trace amounts only. acid groups, one from each ring, by the enzyme Treatment by iron starvation, or -inducing uroporphyrinogen decarboxylase [E.C.4.1.1.37] to drugs, can lead to its accumulation in mammals, intermediate coproporphyrinogen M.48 birds, fungi, plants, and bacteria. In all cases, form the IX is The enzyme has been detected in a wide range of throughout biological systems, only isomer organisms including animals,49 photosynthetic bac- found. teria,50 and green algae.51 While detailed comparisons [E.C.4.99.1.1.] is a membrane- are incomplete it appears that the enzyme is present bound enzyme catalysing the insertion of iron (Fe2+) and in animals25 into protoporphyrin IX, yielding protohaem (haem in the cytosol in higher plants in with an additional enzyme, perhaps, in the chloro- b). Protohaem does not appear to accumulate plast. The product, coproporphyrinogen isomer m, cells. Granick and Beale27 believe it is usual to find does not normally accumulate. It may be deposited haem bound in the ratio 1 haem: 1 apoprotein, in avian egg shells, and in millipedes it may be implying a close regulation of apoprotein synthesis chelated with copper as an integument pigment. In with haem formation, and experimental evidence for J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

Haems and chlorophylls: comparison offinction andformation 9 haem in controlling globin synthesis is now available. Angiosperms), the conversion of protochlorophyllide In fresh lysates of rabbit reticulocytes, globin (a porphyrin) to chlorophyllide (a ) involves synthesis continues at a maximal linear rate for the reduction of the carbon-carbon bond at positions several minutes before ceasing abruptly in the absence 7 and 8 in an NADPH-light dependent reaction by of haem. Haem inhibits the activity of a protein the enzyme protochlorophyllide reductase.64 kinase which in turn is an inhibitor of the initiation The insertion of chlorophyllide a into the chloro- of protein synthesis. Exogenous haem inhibition of plast membrane requires the conversion of chloro- the kinase permits globin production to be main- phyllide to chlorophyll.65 The esterification of tained.56 57 chlorophyllide with a long chain fatty alcohol Apart from haem b synthesis the biosynthetic transforms the slightly polar precursor into a pathways for other haem compounds is less well lipid-membrane soluble end-product. The enzyme known. Colleran and Jones58 showed that the haem- chlorophyllase [E.C.3.1.1.14.] may catalyse this requiring slime mould Phycarum polycephalum used esterification. The consequence is a loss of polar protohaem, not protoporphyrin IX, in the formation and the early moments following chloro- of cytochrome c. Sinclair et a159 showed the conver- phyll formation are usually associated with changes sion of protohaem to haem a in a Staphylococcus in absorption spectra linked probably to successive mutant and similar results havc been obtained with rearrangements of the cholorophyll melecules within haem-requiring yeast mutant.60 It is presumed, membranes and their association with specific though without conclusive evidence, that all cyto- proteins and lipids. The membranes with their arrays chrome haems are synthesised from modifications of of chlorophylls and other pigmcnts are called protohaem rather than directly from protopor- and are the physical location of the phyrin IX. photochemical acts of photosynthesis. The pathway leading to magnesium protopor- The second chlorophyll of higher plants, chloro- phyrins is not clearly established. There is no phyll b, is likely to be formed from chlorophyll a. unequivocal proof for a Shlyk and his associates66 have reviewed much of activity in extracts of either plants or photosynthetic this evidence. bacteria. Evidence from mutants suggests that proto- Bacteriochlorophyll a is probably derived from copyright. porphyrin IX is the substrate for magnesium chlorophyllide a in a series ofreactions. The structure . The presumed product, Mg-protopor- of the other bacteriochlorophylls is established but phyrin, undergoes esterification of the propionate little is known of the biosynthetic pathways. group at position 6. The methyl donor is S-adenosyl Just as haemoglobins and indeed all other haem methionine in a reaction catalysed by a methyl- proteins are associations of haem and protein, transferase [E.C. 2.1.1.11.]. the chlorophylls are also associated with various The subsequent steps from Mg-protoporphyrin proteins within the lipid membrane. Some six or http://jmg.bmj.com/ monomethyl ester to protochlorophyllide have not seven chlorophyll-protein complexes have been yet been defined. The most likely sequence appears partially characterised, with molecular weights to be that suggested by Griffiths and Jones6' and ranging from 8000 to 70 000. One chlorophyll- involves the following reactions. protein complex contains about 20 chlorophyll a molecules (and one ); another complex (1) The esterified propionate group at position 6 which has been crystallised has been shown to con-

undergoos dehydrogenation, hydration, and oxi- tain seven chlorophyll molecules on a single poly- on September 30, 2021 by guest. Protected dation to yield a r-oxo group. peptide chain.67 (2) This modified propionate group is attached to There is a considerable amount of restructuring the -y-meso bridge carbon to form the cyclo- of the photosynthetic apparatus in the green plant pentane ring E. The product, Mg-2,4-divinyl during the first few hours of exposure to light, phaeoporphyrin a5 monomethyl ester, has been whether as a seedling or as a plant initially grown in detected in photosynthetic bacteria62 and in the the dark. The restructuring involves formation of seed coat of marrow.63 discoid membranous structures known as thylakoids (3) Reduction of the vinyl group at position 4 to within which the chlorophylls are sited. The thyla- ethyl forms protochlorophyllide. koids, present usually as aggregates or grana, are surrounded by an aqueous stroma limited by a In most of the lower plants protochlorophyllide is double layered envelope, the whole forming the rarely detected. Thus, while higher plants (for chloroplast. The processes concerned with the bio- example grasses) remain yellow if deprived of light, physical and chemical changes linked to the forma- pine seedlings, mosses, and seaweeds grow green (or tion of a functional photosynthetic unit are complex. brown) with or without light. In higher plants (the The changes include the synthesis of sulpholipids and J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

10 G A F Hendry and O T G Jones galactolipids and additional amounts of a special 100 c-type cytochrome (known as cytochrome f), b cytochromes, , plastocyanin and plasto- quinones.

Function of chlorophyll-protein complexes and C haemoglobin 60 Neither protohaem nor the chlorophylls are found i- in cells in any appreciable concentration in the free form; they are invariably complexed to proteins.68 The structure of the protohaem complex, haemo- globin, is of course well known. In mammals it is a tetramer consisting normally of two pairs of unlike polypeptide globin chains (cx- and ,-chains), each of which contains a protohaem molecule constantly maintained in the ferrous state. Each haem is co- ordinated to a histidine nitrogen of a globin chain 0 20 40 60 80 100 which is folded so that the haem groups lie in clefts (mmHg) on the surface of the haemoglobin molecule approxi- PO mately equidistant from each other.69 Although one side of the iron atom of haem is co-ordinated to the FIG 5 Saturation ofhaemoglobin *-* and side is free and can myoglobin 0-* exposed to varying partial pressures imidazole of histidine, the other of oxygen. bind a molecule of oxygen forming oxyhaemoglobin. It is believed that on binding to oxygen the atomic radius of the haem iron is diminished sufficiently to chlorophylls) must be complexed with protein, allow it to move into the plane of the porphyrin although the structure of these proteins and thecopyright. ring; its location in deoxyhaemoglobin is displaced nature of their binding to the chlorophylls is far out of the plane by 0 06 nm. As the iron of the haem from clear. The function of the first group of chloro- moves, it pulls its co-ordinated histidine with it, phyll-proteins is to absorb light energy and to trans- causing a whole series ofchanges in tertiary structure fer the energy to the second smaller group of and breaking salt bridges and hydrogen bonds. The chlorophyll molecules which, when excited by this effect is to change the configuration of the molecule light energy, eject an electron which reduces a so that oxygen binds more readily to haemoglobin primary acceptor, leaving behind oxidised chloro- http://jmg.bmj.com/ once one haem group is oxygenated, so a plot of phyll. The transfer of the electron is organised across oxygen saturation of haemoglobin versus oxygen the photosynthetic membrane so that this primary tension is sigmoidal. In contrast, in myoglobin, charge separation is stabilised, and an array of which is a monomer, no such sub-unit co-operativity electron carriers converts this form of conserved is possible and the oxygen binding curve is hyper- energy into the free energy of the ATP/ADP couple bolic (fig 5). The details of these mechanisms are (by mechanisms described by Mitchell70) and the

described in most modern textbooks of biochemistry potential of reduced NAD or NADP. on September 30, 2021 by guest. Protected and will not be covered here. Suffice it to say that the Chlorophyll thus functions either in light-harvesting iron in the centre of the tetrapyrrole ring plays a protein complexes or in the reaction centre protein crucial role in the function of haemoglobin and, complexes. indeed, in the function of all haem proteins. It is A representation of the photosynthetic electron concerned in binding the haem to the appropriate transport system of higher plants is given in fig 6. protein and in binding and releasing oxygen. In This shows the two reaction centres characteristic of the case of the cytochromes the iron is concerned in the chloroplast membrane, photosystems I and II accepting and donating electrons and itself undergoes (PSI and PSII). The reducing electron lost from the reversible changes from the ferrous to the ferric state. excited reaction centre of PSI passes on eventually Chlorophyll-protein complexes have two separate to NADP and is available for the reduction of CO2. but closely related functions, both concerned with In the oxidised reaction centre chlorophyll (positive the absorption of light energy and its use in driving a hole), the redox state of the central magnesium is variety of redox reactions which lead to the synthesis unchanged and the electron is lost from the of ATP and assimilation of CO2. In order to partici- t-electron system. It is re-reduced by an electron pate in these reactions chlorophylls (or bacterio- supplied by the excitation of the second reaction J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

Haems and chlorophylls: comparison offunction andformation I I

Light energy

FIG 6 Schematic representation of the role of tetrapyrroles in higher plant photosynthesis. 2H20 ' XCO2

* function not yet resolved

centre, PSII. The two reaction centres are connected detergent. Usually they are composed of three sub- copyright. by a chain of electron transport carriers (plasto- units and contain four bacteriochlorophylls, two quinones, cytochrome f, and the copper protein bacteriophaephytins (that is bacteriochlorophylls plastocyanin) which mediate this electron transfer. lacking magnesium), one iron atom, and two bound The oxidised PSII is itself re-reduced by electrons quinones. Two of the bacteriochlorophylls are in derived from the enzymic splitting of water: close association (the 'special pair' or dimer) and function as the primary photochemical electron ------+ 4H+ 4e-. '- + http://jmg.bmj.com/ 2H2 02 donor. One of the two bacteriophaeophytins is the Oxygen is evolved as a by-product and the protons primary ; that is, the primary, very serve to provide part of the proton motive force used rapid, charge separation occurs between these two. in ATP synthesis, as described by Mitchell.70 The electron is then further transferred to a bound The chlorophyll-protein complexes involved in quinone which is closely associated with the iron green plant photosynthesis have been partly purified. atom. There is reasonable evidence from EPR The complex corresponding to the PSI reaction spectroscopy that a similar ' special pair' of chloro- centre a weight of about 110 kD a molecules is present in the PSI reaction centre has molecular phyll on September 30, 2021 by guest. Protected composed of three sub-units of roughly equal size, of higher plants although there is no evidence for the with about 40 chlorophyll a molecules per reaction presence of a phaeophytin or a quinone in this centre. No chlorophyll b is present. The light- reaction centre. harvesting complex has a molecular weight of about Photosynthetic bacteria do not catalyse a water- 35 kD, contains three chlorophyll a molecules and splitting reaction of the type associated with PSI1 three chlorophyll b molecules per mole,7' and of green plants. When an electron is lost from the accounts for about 50% of the total chlorophyll reaction centre and donated eventually perhaps to present. Purification of the PSII reaction centre is NAD, it is replaced, not from a second light reaction, less advanced and it is not so well characterised. but by electrons donated from substrates present in Reaction centres from photosynthetic bacteria the environment. These may be organic materials, have been extensively purified and have given much such as succinate or glutamate, or inorganic sulphur information about the mechanism of photosynthesis. compounds, such as sulphide or thiosulphate. These They are smaller than green plant reaction centres donors are connected to the oxidised bacterial (molecular weight around 70 kD) but like them they reaction centre by a chain of electron transport are extracted from membranes with the use of components, including quinones and cytochromes.72 J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

12 G A F Hendry and 0 T G Jones The membranes of photosynthetic bacteria usually product of metabolic advantage to the cell. Subse- also contain light-harvesting pigment-protein com- quent modifications to the end product improved its plexes, two of which have been purified. They are properties and led to abandonment of the direct use named B850 and B870, where the postscript numbers of intermediates which now are unable to function indicate the wavelength maxima of their principal per se in primary metabolism. The evolutionary absorption bands. The B850 complex contains three process therefore represents a progressive elaboration bacteriochlorophylls per 20 kD sub-unit, and the of mechanisms for carrying out the original primitive B870 sub-unit two bacteriochlorophylls per 20 kD functions of porphyrins in a more efficient manner.75 sub-unit (see Cogdell and Thornber73 for a review). A problem arises in attempting to link today's end The ratio of B850 to B870 and to reaction centre products (haems and chlorophylls) with the functions varies with growth conditions. In general, the lower of porphyrins in the primitive environmental the light intensity during growth, the more light- conditions of early life. This problem has been harvesting complexes are synthesised. discussed by Granick75 and Mauzerall77 and several There is one case in which an unusual light- hypotheses have been made. harvesting pigment protein complex (isolated from Granick75 has shown that the monopyrrole PBG, the photosynthetic bacterium Prosthecochloris aestu- when heated in neutral or alkaline conditions, forms arii) has been crystallised and its structure deter- uroporphyrinogen isomers I or III (but not isomers mined by x-ray diffraction. The molecule contains II or IV). Such a chemical event, it is postulated, seven molecules of bacteriochlorophyll a, each on may have provided, in low concentrations, a com- average separated from an adjacent bacterio- pound used to advantage in the primitive cell. chlorophyll by 1 2 nm, and essentially completely Genes then evolved to synthesise the porphyrin surrounded by protein.67 These chlorophyll mole- more efficiently. The first tetrapyrrole in the bio- cules are held firmly in place with little or no freedom synthetic pathway is uroporphyrinogen. This com- of movement. The magnesium atoms of six of the pound can be oxidised to yield uroporphyrin which bacteriochlorophyll molecules interact, on one side absorbs light, is fluorescent, and can carry out only, with the protein and there is evidence from the photosynthetic reactions. It can form a metal copyright. electron density pattern that, in some cases, the side chelate to serve as a redox catalyst.65 The modifica- chain could be histidine, just as it is in haemo- tions to uroporphyrin(ogen) III that subsequently globin. Access of a sixth ligand to the magnesium is took place in all organisms would have reflected prevented by the presence of the phytyl side chain changing environmental conditions. One such traversing one face of the rings. change would have been the development of photo- In both green plants and bacteria the bulk of the synthesis and oxygen release into the atmosphere. chlorophyll pigments serves a light-harvesting Mauzerall65 postulates that the change from the function. They absorb light and channel it by free porphyrin to the metalloporphyrin initiated the http://jmg.bmj.com/ resonance energy transfer to the photochemical development ofefficient photosynthesis. Mg-porphy- traps which, in the PSI reaction centre and bacterial rins, in light-induced excited states, form strongly reaction centres, contain the 'special pair' of pigment reducing agents; coupling reducing reactions with molecules. The special properties of the chlorophylls electron donation from water-splitting (resulting in in vivo, the positions of their absorption maxima, electron, proton, and oxygen release) led ultimately and their photochemical activity are dependent on to modern photosynthesis. Increasing oxygen levels

their association with specific proteins. In this, in the environment of the metalloporphyrin would on September 30, 2021 by guest. Protected chlorophylls and protohaem can be seen to have lead to the quenching of the photo-excited state of great similarity. Stripped of their proteins they lose chlorophyll. However, in a lipid environment the their desirable properties. Their spectra change photo-pigment would be protected sufficiently to dramatically and their redox properties are quite allow electron ejection and so highly hydrophobic different. metalloporphyrins would be favoured. Haem por- phyrins, as cytochromes, are able to act as electron Evolution acceptors and donors and may therefore have evolved together with photosynthesis. Attempts have been made in the past to unify the Iron porphyrins are relatively photo-inactive but evolution of haems, haemoglobin, cytochromes, and may readily complex oxygen. The metabolic advan- chlorophylls.65 7 The basis of these attempts has tage gained from exploiting this property would been largely speculative though with some support- provide for the development of haemoglobin (or a ing evidence.65 The postulates assume that in the myoglobin precursor) in oxygen transport, storage, course of evolution, each of the intermediates in and release. When linked to cytochrome oxidase porphyrin biosynthesis was, at one time, an end these would provide the essential components of J Med Genet: first published as 10.1136/jmg.17.1.1 on 1 February 1980. Downloaded from

Haems and chlorophylls: comparison offunction andformation 13 respiration in higher organisms. Such a respiratory Rudd JT. Vertebrates without erythrocytes and blood function presumably arose after the development of pigments. Nature 1954;173:848-50. 12 Cutting JA, Schulman HM. The control of synthesis a mechanism for the continuous production of in soybean root nodules. Biochim Biophys Acta 1972 ;261: oxygen (photosynthesis) to complete the mechanism 321-7. of modern respiration. 13 Kennedy GY. Porphyrins: structure, distribution and Porphyrins function in a number of different ways metabolism. In: Florkin M, Mason HS, eds. Comparative biochemistry. vol IV-B. London: Academic Press, 1962: in biological systems, including oxygen carrying and 557-614. storage, light energy transfer, and light induced 14 Hendry GAF, Stobart AK. Haem and chlorophyll electron ejection. Even more variation occurs in formation in etiolated and greening leaves of barley. porphyrin structure, with many side group modifica- Phytochem 1977;16:1545-8. tions and a limited number of 15 Jones OTG. Biosynthesis of porphyrins, and central metal chlorophylls. In: Clayton RK, Sistrom WR, eds. The substitutions. Despite these variations in structure photosynthetic bacteria. New York: Plenum, 1978: and function, the evidence suggests that porphyrin 751-78. biosynthesis is essentially similar in all biological 16 Bogorad L. and complementary chromatic adaptation. Annu Rev PI Physiol 1975 ;26:369- systems. While there is a possible difference in the 401. synthesis of the porphyrin precursor 5-amino 17 Rudiger W. Ueber die Abwehrfarbstoffe von Aplysia- laevulinic acid in plants from animals and bacteria, Arten. II. Die Struktur von Aplysiaviolin. Hoppe Seylers the subsequent steps in the formation of proto- Z Physiol Chem 1967;348:1554. porphyrin IX are similar in all organisms. The 18 Rudiger W, Klose W, Vuillaume M, Barbier M. On the structure of pterobilin, the blue pigment of Pieris similarity is sufficiently great to support the conten- brassicae. Experientia 1968 ;24:1000. tion that porphyrins have arisen in the course of 19 Chapman DJ, Cole WJ, Siegelman HW. evolution from a common origin. The variation in of and R-. Biochem J function between the two great families, the haems 1967;105:903-5. the 20 Grombein S, Rudiger W, Zimmerman H. The structure and chlorophylls, is perhaps the result of the of the phytochrome in both photoreversible adaptability of the porphyrin structure to changing forms. Hoppe Seylers Z Physiol Chem 1975 ;356:1709-14.

biochemical requirements. 21 O'Carra P, Colleran E. Separation and identification of copyright. isomeric deuterobiliverdins andmesobiliverdins. J Chroma- GAFH acknowledges financial support from the togr 1975;108:212-5. Medical Research Council and OTGJ from the 22 Maines MH. Heme metabolism: factors affecting the in vivo oxidation of heme. In: Gordon AS, Silber R, Science Research Council. LoBue J, eds. The year in hematology. New York: Plenum, 1977:1-45. References 23 Sanger JE. Quantitative investigations of leaf pigments from their inception in buds through autumn coloration 1Verdeil F, 1844. Quoted by Aranoff S. The chlorophylls- to decomposition in falling leaves. Ecology 1971 ;52:1075- http://jmg.bmj.com/ an introductory survey. In: Vernon LP, Seely GR, eds. 89. The chlorophylls. London: Academic Press, 1966:1-20. 24 Smith KM. 1st Tetrapyrrole Discussion Group meeting, 2 Hoppe-Seyler F, 1880. Quoted by Aranoff S. The chloro- Cardiff, 1976. phylls-an introductory survey. In: Vernon LP, Seely GR, 25 Bogorad L. Chlorophyll biosynthesis. In: Goodwin TW, eds. The chlorophylls. London: Academic Press, 1966: ed. Chemistry and biochemistry ofplant pigments. vol 1, 1-20. 2nd ed. London: Academic Press, 1976:64-148. 3McMunn CA. On myohaematin, an intrinsic muscle- 26 Shemin D, Russell CS. I-aminolaevulinic acid, its role in pigment of vertebrates and invertebrates, on histo- the biosynthesis of porphyrins and purines. J Am Chem

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