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Review article

Journal of Medical Genetics, 1980, 17, 1-14

Haems and chlorophylls: comparison of functionand formationG A F HENDRY AND 0 T G JONES

From the Department ofBiochemistry, The Medical School, University of Bristol, Bristol BS8 ITD

In 1844 Verdeill reported that acid treatment ofchlorophyll or haem yielded apparently similar redcompounds; he even postulated that chlorophyllswould contain iron. Hoppe-Seyler2 confirmed theapparent similarity of acid derivatives of haems andchlorophylls from their light absorption charac-teristics, a point rather overshadowing the discovery

at the same time by McMunn3 of cytochromes,another group of haem proteins.

It was the demonstration by Nencki and co-workers 45 that the degradation of both chlorophyllsand haems yielded monopyrroles that led them, intrue neo-Darwinian fashion, to postulate a commonorigin for animals and plants.

0CH2IICH

COOH

CIH2CH2

C-OCH2

NH2

5- Aminolaevulinic acid

CH2

CH CH.

( CH3'

CD

0-'ICH3

'CH3 ®

a CH22 1 12

2 CH2)H COOH

Protoporphyrin IX

FIG 1 Structures ofprotohaem andchlorophyll a and two of their precursors,5-aminolaevulinic acid andprotoporphyrin IX (with substituentnumbering positions).

Protohoem (haem- b)

CH2CH2 COOCH3

CooC20H39Chlorophyll a

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G A F Hendry and 0 T G Jones

Following the work of Willstatter6 and Fischer andStern,7 the structure of most natural and manyunnatural porphyrins was established. AlthoughVerdeil's prediction was wrong, he was correct ininterpreting chlorophylls and haems as havingessentially similar structures.Today we can show the similarity between haem

and chlorophyll based on their common precursor,protoporphyrin IX (fig 1). The carbon numberingsystem used in fig 1 will be used in the subsequenttext.

Natural occurrence of porphyrins

For over a century, scientists have been aware of theexistence of the numerous types of porphyrin-basedcompounds to be found in a wide range of eukaryoticand prokaryotic organisms. It was presumably onlya question of time before reports of extra-terrestrialporphyrins would be made.8 The earth-bound bio-logical porphyrins are diverse and range in colourfrom grey-blue (bacteriochlorophyll), green (chloro-phyll), and red (protohaem) to yellow and brown(avian egg porphyrins).The natural occurrence of many porphyrins is

shown in fig 2, together with their biosyntheticrelationships. It will be seen that many tetrapyrroles,

particularly those of avian egg shells, have nocentral complexed metal. There are only five metalscommonly found in natural porphyrins: copper in auroporphyrin III derivative in the flight feather ofthe tropical Musophagidae family of birds; cobalt asthe metal component of vitamin B12 (cobalamins)9;iron in the metal complex in haems includinghaemoglobin, myoglobin, catalase, peroxidases, andcytochromes. The fourth metal, magnesium, ischaracteristic of all chlorophylls and bacterio-chlorophylls. A fifth metal, zinc, may complexenzymically or non-enzymically to many porphyrinsand to the porphyrin breakdown product biliverdin(it is not known if complexing occurs before or afterring cleavage). Certainly the ease with which zinc isinserted non-enzymically into porphyrins in vitrowould make it surprising if zinc porphyrins did notexist in vivo. It is indeed more surprising that naturehas confined the metal complexes of porphyrins tofour or five ions only; chemists have been able toinsert well over 40 metals into porphyrins.The biosynthetic pathway (fig 2) of porphyrins

from the monopyrrole porphobilinogen begins withthe formation of the cyclic tetrapyrrole uropor-phyrinogen isomers IM and I. Isomers II and IV havenot been reported in biology; isomer I is not anintermediate in haem and chlorophyll synthesis, it

Porphobil inogen

integuments | Uroporphyrin I Uroporphy

I Avian eggs I -*- Coproporpt

Harder's gland ___H_rderopo_in mammals .-Harderopo

Cu

trinogen III CM Turacin birdCo feathers

Dhyrinogen III Fe- - --Vitamin B 1Certain bacteria Ihyi

* Sirohoem Plants, fungi,pbacnteria I

)rphyrinogen III

Zn

IAvian eggs - Protoporphyrinogen JX- verdin - sm egg

II F~~~~~~~~~~~~~~~e|Echiuriodwarm harmane Bonellin.s- ?

Mg Protohaem -_-

Protoc hlorophyl l(ide)

Eukaryotic andsome prokaryotic Dihydrochlorinphotosynthetic (chlorophyllsorganisms a.b.c.d

Certain photosynthetic Tetrahydrochlorinsbacteria (bacteriochlorophylIs

a,b.c.d.e )

Haemproteins(e.g: cotalose)Haem b Mot_ rgnimcytochromes Most organisms

_,Haem a. ccytochromes

a- Haem cl, s --w rProkoryotes |Icertain fungiI

Leghoemoglobin _| Plant-bacteriasybosesJHoemoglobin [osivrtbrates,Myoglobin many invertebratesChlorocruorin

FIG 2 Biosynthetic pathway of tetrapyrroles and their natural occurrence.

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Haems and chlorophylls: comparison offunction andfot

occurs commonly as an excreted compound in thegroup of disorders of haem synthesis known as theporphyrias. It also occurs in many mollusc shells andinteguments, in just those molluscs which do notsynthesise haemoglobin. This led Kennedy10 topostulate that molluscs themselves may be porphy-riacs! These molluscs must additionally makesufficient isomer III for the synthesis of cytochromeprosthetic groups. Uroporphyrin, coproporphyrin,and protoporphyrin are found in egg shells and insome cases account for their characteristic colours.Intermediate between coproporphyrin and proto-porphyrin is harderoporphyrin, which is a pigmentfound in the Harderian gland of the rodent eye,though it is present in other tissues.A plant equivalent to the porphyric mammal may

be found in some x-ray or UV treated seedlingmutants. Some such seedlings accumulate uropor-phyrin, coproporphyrin, and particularly proto-porphyrin IX. From such findings protoporphyrinIX has come to be placed in a central role in thestudy of haem and chlorophyll metabolism. Proto-porphyrin isomer IX, of the fifteen possible isomers,is believed to be a prccursor at the branch point ofbiosynthesis of both haems and chlorophylls.

Naturally occurring iron porphyrins

From fig 2 it will be seen that a minor pathway maylead to the iron-chelate sirohaem (see below) and amajor pathway gives rise to a series of haem com-pounds, of which the cytochromes, in one form or

another, are present in nearly all organisms. Haemo-globin, a characteristic pigment of vertebrates, ispresent in a wide range of organisms. One family ofvertebrates, however, the ice-fish (Chaenicthyidae)survive in the cold waters of the Antarctic apparentlywithout any haemoglobin; their oxygen requirementis met from oxygen dissolved in blood plasma.Similarly, the Leptocephalus eel larva has no haemo-globin until it reaches the elver stage." A form ofhaemoglobin is found in plants under the name ofleghaemoglobin, It is, however, confined to thoseplants which are able to fix nitrogen in co-operationwith the bacterium Rhizobium. Such plants includethe legume family. Leghaemoglobin appears to helpto maintain a constant, but low, level of oxygenwithin nitrogen fixing cells. Haem synthesis appearsto take place in the bacteria, globin in the plant, thecompleted molecule being located on the interfaceof the two organisms.'2

Plants do not, of course, have a complex gas

transport system; they obtain their oxygen fromsolution in cell wall water films. Haemoglobin is alsofound in the invertebrate phyla, though in some cases,particularly in parasites such as Aplaniptera (fleas),

Ki-dK k i

rmation rLAnoplura (suckirn lice), and Ixodes (ticks), t1jhaemoglobin is derive ftrn the higher a-nalj o tand the protein moiety I.d in nutrition. Full reof the occurrence of haiei6ilobins and myogtabihin invertebrates are provided by Kennedy.13 Thefollowing summary is very much a simplification ofwhat is a complex story. Haemoglobins have beenreported in certain protozoan strains, in the larvaeofthe insect Chironomus, in one sub-class ofmolluscs,the bivalvia (Solen, Arca, and Pentunaulus where thehaemoglobin is present in corpuscles), and in onegastropod genus Planorbis (as free haemoglobin inthe plasma). In the crustacea, haemoglobin appearssporadically in all orders of the branchiopods. Themost unusual case occurs in Daphnia, where thehaemoglobin is generally present in the eggs, butmay be absent in the adult. Many crustaceans alsocontain bile pigments, which by analogy withmammals are, probably, breakdown products ofprotohaem. Among the nemerteans haemoglobin ispresent in some members but not all, publishedphyllogenies showing little correlation with presenceor absence of haemoglobin. Haemoglobin is morecommon in the annelid worms, generally as a freelydispersed pigment. However, in a few species,erythrocyte corpuscles may be found (for examplein Travisia and Trevella spp). In the polychaeteAphrodita, haemoglobin is present in the nerve cord.Among the marine worm phyla, haemoglobin is

present in the echiurids and lophophorates, generallywithin corpuscles. In that 20th century discovery, thedeep sea dwelling pogonophora worms, haemoglobinhas been reported, but in a freely dispersed form.It is towards the top of the invertebrate 'tree', theholothurian echinoderms or sea cucumbers, thatdiscoid cells containing haemoglobin are found,corresponding to the erythrocytes of mammals.The monomer myoglobin is closely related in con-

formation to one of the chains of the haemoglobintetramer. It is present throughout the vertebratesbut appears only sporadically in the invertebrates.Typically, myoglobin is present in the gastropodradula muscles and the body wall muscles of variouspolychaete fanworms and occasionally in other phyla.Haem s (also called Spirographis haem or chloro-

cruorohaem) in combination with a protein servesas the extra-corpuscular oxygen-carrying pigment infour families of polychaete worms (Sabellidae,Serpullidae, Ampharetidae, and Flabelligeridae).Almost on a belt-and-braces principle, members ofthe Serpula and Potamilla families contain bothhaem s protein and haemoglobin. To show completeinconsistency, at least to reviewers, in the familySpirorbis there are three groups, one containinghaem s protein, one containing haemoglobin, andthe third containing neither.



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Other pigments commonly used in oxygen carry-ing, other than blood plasma or water, include theiron protein hemerythrin (despite its name not ahaem protein) and the copper containing proteinhemocyanin (also not a haem protein). The formerhas a restricted distribution among certain peanutworms, inarticulate brachiopods, and a few otherphyla, always apparently in corpuscles. Hemo-cyanins, as oligomeric proteins, on the other handare always free floating and are found in the non-haemoglobin containing molluscs, notably theProsobranchia and Pulmonata, and in those suppo-sedly ancient mollusc ancestors, the chitons. Thesehemocyanin containing molluscs accumulate uro-porphyrins in their shells. Hemocyanins are alsofound in scorpions, in the cephalopods (squids,cuttlefish, and octopods), and in the majority ofcrustaceans, including the malacostracan crabs,lobsters, and shrimps. Hemocyanins are also foundin that other living fossil, the survivor of the trilobites,the horse-shoe crab. It is quite clear, then, thatalternative systems to haemoglobin for oxygentransport have existed for hundreds of millions ofyears, probably long before the appearance of thefirst land animals.Apart from the animal kingdom, all fungi and

plants contain haem pigments, the cytochromes. Onethird or more of the total tetrapyrrole content ofdark-grown barley seedlings consists of protohaem.'4Plants, in common with most other organisms, alsocontain other haem proteins, notably catalase,peroxidases, and tryptophan pyrrolase.

Naturally occurring magnesium porphyrins

All chlorophylls are magnesium tetrapyrroles and itis likely that they are formed by successive modifica-tions of magnesium protoporphyrin (see Jones15 fora discussion of this). Indeed magnesium protopor-phyrin and its monomethyl ester have been found inmutant algae, plants, and bacteria in which normalchlorophyll synthesis is inhibited, wholly or in part.A much more commonly found magnesium tetra-pyrrole intermediate in cholorophyll synthesis isprotochlorophyllide (see fig 3), in which the vinylgroup at position 4 is reduced to the ethyl, and thefifth ring E, characteristic of the chlorophylls, hasbeen formed by a modification of the propionicacid substituent at position 6. In higher plants (theflowering plants or angiosperms), the porphyrinprotochlorophyllide accumulates if seedlings aregerminated in the dark. In most other plants (somealgae being an exception) chlorophyll is formed evenin the dark, with no detectable protochlorophyllideaccumulation. However, protochlorophyllide is anintermediate in the biosynthesis of two classes of

CH2II

CH CH3

CH3

CH3

CH2CHOH2 COOCH 0COOH 3

Protochlorophyl lide

FIG 3 Structure ofprotochlorophyllide, the precursor ofchlorophylls and bacteriophylls.

chlorophyll: the dihydroporphyrins or chlorins (forexample chlorophylls a and b) present in all eukaryo-tic photosynthetic organisms and some prokaryotes(the blue-green algae), and the tetrahydroporphyrins(for example bacteriochlorophylls a and b) found inthe purple sulphur (Thiobacteriaceae) and non-sulphur bacteria (Athiobacteriaceae). In the greenphotosynthetic bacteria (Chlorobiaceae) bacterio-chlorophyll a is a minor component; the majorchlorophylls are bacteriochlorophylls c and d whichare chlorins.

Structures of naturally occurring porphyrins

Table 1 lists, in an abbreviated form, the major andmost of the known minor porphyrins (or porphyrinderivatives) found throughout the biological world.The salient points from this table are that of the fourpossible uroporphyrinogen isomers, only isomer IIIis used in haem and chlorophyll synthesis; of thefifteen possible isomers of protoporphyrin, onlyisomer IX has been reported from biologicalsystems. There is a restricted number of side chainmodifications in naturally occurring porphyrins.Once coproporphyrin III is formed, no furthermodification occurs at carbon position 1 in anyorganism. Similarly, at carbon position 3, a methylgroup is present in all biological systems (except in

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Haems and chlorophylls: comparison offunction andformation

TABLE 1 Structures of naturally occurring porphyrins

5

Porphyrin Peripheral carbon position Complexedmetal

1 2 3 4 5 6 7 8

Uroporphyrin I Ac Pr Ac Pr Ac Pr Ac PrUroporphyrin III Ac Pr Ac Pr Ac Pr Pr AcSirohaemt Ac/Me Pr/H Ac/Me Pr/H Ac Pr Pr Ac FeCoproporphyrin 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 FeHaem a Me RI Me Vi Me Pr Pr Fo FeHaem c Me R2 Me R2 Me Pr Pr Me FeHaem dt Me HO-Et Me Vi Me Pr Pr/H Me/H FeHaem s Me Fo Me Vi Me Pr Pr Me FeLactoperoxidase haemt Me Vi Me Vi HO-Me Pr Pr HO-Me FeMyeloperoxidase haemt Me Vi Me Vi Me Pr Pr Fo FeMg-protoporphyrin Me Vi Me Vi Me Pr Pr Me MgMg-proto methyl ester Me Vi Me Vi Me R3 Pr Me MgProtochlorophyllide Me Vi Me Et Me CP Pr Me MgChlorophyllide a Me Vi Me Et Me CP Pr/H Me/H MgChlorophyll a Me Vi Me Et Me CP Phy/H Me/H MgChlorophyll b Me Vi Fo Et Me CP Phy/H Me/H MgChlorophyll cl Me Vi Me Et Me CP Acr Me MgChlorophyll c2 Me Vi Me Vi Me CP Acr Me MgChlorophyll d Me Fo Me Et Me CP Phy/H Me/H MgBacteriochlorophyll a Me R4 Me/H Et/H Me CP Phy/Ht Me/H MgBacteriochlorophyll b Me R4 Me/H R5 Me CP Phy/Ht Me/H MgBacteriochlorophyll c* Me HO-Et Me R6 Me or Et CP Far/H Me/H MgBacteriochlorophyll d Me HO-Et Me R6 Me or Et CP Far/H Me/H MgBacteriochlorophyll 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-phytol ester; Far, -Pr-farnesol ester; Acr, -CH=CH-COOH;Rl, -CHOH-CH2-(CH2-CH=C.CH5-CH2)3-H; R2, -CH.S Cys. CH3; R3, -Pr-Me ester; R4, -C=O.CH3; R5, =CH-CH.;R6, -propyl, butyl, or ethyl.* 8-position-ethyl or methyl; t or Pr-geranylgeraniol ester; t proposed structure.

chlorophyll b). Another conservative group is atposition 5 which always retains the methyl groupexcept in the somewhat uncommon green-sulphurbacteria Chlorobacteriaceae, where the bacterio-chlorophylls c and d often include an ethyl group;the haem of the enzyme lactoperoxidase also has anoc-hydroxymethyl group at this position. Position 8commonly retains a methyl group except in haem aand myeloperoxidase where a formyl substitution ispresent and in lactoperoxidase where again thea-hydroxymethyl substitution takes place.So of the eight side group positions found in

coproporphyrin Ill, positions 1, 3, 5, and 8 undergolittle or no further change. Even among the remain-ing groups the changes are modest. The iron-containing porphyrins retain the two propionicacid side chains at positions 6 and 7. Among themagnesium porphyrins, positions 6 and 7 undergomodification; the propionic acid group at 6, afteresterification, forms the characteristic fifth ring E.The propionate group at position 7 is esterified also,but with the long chain fatty acid phytol (occasionallyfarnesol or geranyl-geraniol). Exceptions occur inthe case of the c chlorophylls from the brown sea-weeds where the propionate group at 7 is oxidised toacrylate.

The most varied substituents in biological systemsare found at positions 2 and 4. At position 2, thevinyl groups of protoporphyrin IX are retained inprotohaem, the chlorophylls a and b, in lacto- andmyeloperoxidase. In the iron porphyrins the variationin the position 2 substituents is considerable, fromthe simple reduction in bonellin (a worm sexhormone) to the long chain 1-hydroxy-2-(E,E-farnesyl)-ethyl group substitution found in haem a(the prosthetic group of the cytochromes a). Amongthe chlorophylls, position 2 changes occur in redseaweeds (chlorophyll d) with formyl substitution forthe vinyl group. In the prokaryote bacteriochloro-phylls a and b, position 2 is modified to form a ketogroup; in bacteriocholorophylls c, d, and e, it under-goes hydration to hydroxyethyl groups.Among the iron porphyrins the substituent at

position 4 is fairly constant. The original vinylgroup persists in haemoglobin, cytochromes a, b,and d, catalase, and most peroxidases. Only haem c,covalently bonded to its protein to form cytochromec, has thio-ether groups at positions 2 and 4 linkingit to the protein. Among the magnesium porphyrins,position 4 modifications are more common. Theoriginal vinyl group at position 4 of protoporphyrinIX is reduced to the ethyl state in chlorophylls a, b,



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TABLE 2 Structures of naturally occurring compounds derivedfrom or related to porphyrinsProduct Original porphyrin carbon position

1 2 3 4 5 6 7 8

[Protoporphyrin IX] Me Vi Me Vi Me Pr Pr MeBiliverdin/bilirubin Me Vi Me Vi Me Pr Pr MePhycocyanobilin Me Et Me/H RI Me Pr Pr MePhycoerythrobilin Me Vi Me/H RI Me Pr Pr MePhytochrome (proposed) Me Vi Me/H R2 Me Pr R3 MePhylloerythrin 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-chlorophylls from the green sulphur bacteria, ethyl,propyl, or even isobutyl groups may be presentdepending on the species involved. Side chain varia-tion among the porphyrin derivatives is for thegreater part confined to positions 2 and 4 (table 2).

Certain degradation products of porphyrins are

well known.16-18 Such products, usually bilins or bilepigments, are listed in table 2. The structuralsimilarity between biliverdin, bilirubin, and meso-

bilirubin suggests that these compounds are derivedfrom the breakdown of protohaem (from haemo-globin, myoglobin, cytochrome P-450, and cyto-chrome b) and this has been confirmed in vitro. Theprokaryote blue-green algae and the eukaryote redalgae contain bile pigments (probably as accessorypigments in photosynthesis), two of which have beennamed phycocyanobilin and phycoerythrobilin.19The absence of the fifth ring E and the presence ofthe propionate group at position 7 suggests thatthese compounds were derived from protohaem or

protoporphyrin and not chlorophyll. The hormonephytochrome is the only bile pigment known inhigher plants and it too would appear from itsstructure20 to be a protohaem or a protoporphyrinderivative. There are many reports of bilins andrelated compounds whose structures have not beendefined. Most are assumed to be oc-IX isomers,21that is the tetrapyrrole ring has been split at the a.carbon atom, but at least one case of 'y-IX isomerformation is known from the pigment of butterflies.18Some of the bile pigments so far uncharacterisedmay be derived from haems a, c, and d and perhapscoproporphyrin and protoporphyrin, although alter-native haem breakdown pathways have beensuggested.22 The other interesting point to emergefrom studies of naturally occurring porphyrinstructures is the absence of any reports of bilepigments derived from chlorophylls. Indeed it is notknown how chlorophylls are degraded in vivo.Chlorophyll degradation might involve the loss ofmagnesium (to form phaeophytin) and the removalof the phytol side chain (to form phaeophorbide).Such compounds have been found generally in trace

amounts in autumn senescing leaves.23 One of thegreat mysteries of porphyrin biochemistry is thenatural fate of chlorophylls in autumn leaves. Some1012 tonnes24 of chlorophyll disappear annually,much of it during autumn.

Biosynthesis of porphyrins

If animals and plants share a common evolutionaryorigin then it is likely that they would share acommonbiosynthetic pathway leading to tetrapyrroles, atleast in the initial stages of synthesis. If there weremajor dissimilarities in biosynthesis, then it may bethat a form of biochemical convergent evolution hastaken place. As will be seen, a study of porphyrinbiosynthesis raises some unanswered questions.

Probably all animal haems, vitamin B12, andavian egg shell pigments, are derived ultimately fromthe condensation of glycine and succinyl CoA toform the amino-acid 5-aminolaevulinic acid (ALA,fig 1) (see Bogorad25 for a review). This is the firstcommitted step in tetrapyrrole biosynthesis. Twosuch ALA molecules condense to form the mono-pyrrole porphobilinogen (PBG):Glycine ALA PBG+ >-synthase--*ALA-- synthetase-*PBG

Succinyl CoA x 2

CO2

ALA was first shown to be an intermediate inporphyrin synthesis by Shemin and Russell26;it is formed by ALA synthase (E.C.2.3.1.37) byavian and mammalian erythrocytes, bone-marrowcells, and liver, by yeast, bacteria, and insects (seereview by Granick and Beale27). It was also shown in aphotosynthetic bacterium Rhodopseudomonas sphae-roides,28 where its activity was found to be greatestat times ofmaximum bacteriochlorophyll synthesis.29The enzymeALA synthase has not been unequivo-

cally demonstrated in higher plants. This muchdebated topic was recently reviewed by Porra andGrimme30 and Beale.31 No enzyme has been describedfrom plants which could account for the rate of

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synthesis of chlorophyll in actively greening tissues.32Trace activities ofALA synthase have been reportedin nitrogen-starved green algae,33 in cold storedpotato peelings,34 and in soya bean callus under con-ditions where chlorophyll synthesis was minimal orrepressed.35 An alternative biosynthetic pathway hasbeen shown to exist3' 36 which involves the conver-sion of glutamate or glutamine to ALA with theoriginal 5-carbon skeleton intact. The precisemechanism for this conversion is uncertain.30 31

It has been known for some time37 that the ALAsynthesising system in plants, has, like that ofanimals, a short half-life. The ALA synthase inanimals is located in the mitochondrial matrix.25The location of the ALA-forming system in plantsis not known though there is some evidence (forexample Kannangara and Gough32) that oc-oxogluta-rate-derived ALA may be synthesised in the isolatedchloroplast. It has been postulated that plants mayhave two ALA-forming systems.3' 38 One suchsystem, the classical ALA synthase system, mayoperate in the mitochondria in the absence of light,while a quantitatively more important systemutilising a glutamate derivative may be present inthe chloroplast, functioning in chlorophyll synthesis.Some support for these suggestions has been pro-vided by the examples quoted above of plant ALAsynthase activity from tissue synthesising little orno chlorophyll, operating perhaps, as in animals,with the mitochondrial enzyme. Interestingly,Klein and Senger39 detected both a 'classical'ALA-synthase enzyme and a second ALA-formingmechanism in the same cells of an algal mutant. Onbalance, it appears that plants have evolved aseparate mechanism for the synthesis of theirporphyrins, but whether this separate mechanism isin addition to or replaces the mammalian ALAsynthase is not known with certainty.The enzymatic condensation of two molecules of

ALA to form porphobilinogen (PBG) is well charac-terised. The soluble enzyme involved, PBG synthase(E.C.4.2.1.24), is present in mammalian and aviancells, fungi, bacteria, algae, and higher plants.25 Firstobserved in avian erythrocytes,40 it has subsequentlybeen found that the enzymes from different organismshave different co-factor requirements; potassium insome photosynthetic bacteria, zinc in mammalianerythrocytes, and magnesium or manganese inhigher plants. However, the molecular weight of theenzyme (subunit mass about 35 000) appears to besimilar in all organisms. The enzyme is present inextraplastidic fractions in plants,41 but there isuncertainty as to the location of the enzyme withinthe plastids, whether soluble or bound to membranes.Four molecules of porphobilinogen condense to

form the first porphyrinogen in a two-step reaction

involving the co-operation of the enzyme porpho-bilinogen deaminase (also known as uroporphyrino-gen I synthase, E.C. 4.3.1.8.) and uroporphyrinogenIII co-synthase. A review describing these steps hasbeen provided by Battersby and McDonald.42 Inthis complex sequence of condensations there are anumber of details to be cleared up.27 However, thebasic mechanism involves the polymerisation offour PBG units by the deaminase to form a transientintermediate, which in the absence of co-synthaseforms uroporphyrinogen I, but in the presence ofco-synthase forms isomer III, this being the normalproduct. The evidence suggests that essentially thesame mechanism exists in human erythrocytes as inhigher plants.27 The two enzymes are present onlyin the cytosol in animals but the precise location inplants has yet to be shown clearly, though onelocation must be within the plastids.Uroporphyrinogen III is a substrate for at least

three separate pathways:4 x PBG

Fe Uroporphyrinogen III Co) ( _

Sirohaem Coproporphyrinogen Cobalamin

Sirohaem is structurally closely related to uropor-phyrinogen (fig 4). Its relationship to uropor-phyrinogen and its function in reducing environmentshas attracted considerable interest as a possibleprimitive cytochrome (see Granick and Beale27 andbelow). Sirohaem is the prosthetic group of sulphitereductases in several bacteria and fungi and nitratereductases in fungi and higher plants.4345The second compound closely related to uropor-

phyrinogen III is cobalamin or vitamin B12. Thebiosynthetic pathway and its unresolved problemshave been discussed46 with further informationprovided by Horig et al.47 The conversion of uro-porphyrinogen III to the intermediate cobyrinicacid involves cobalt insertion, and no less than sevenmethylation steps and the elimination of the 6-mesocarbon.46 The remaining steps involve seven amida-tions of the acidic peripheral groups, bonding ofdeoxyadenosine to the cobalt atom, and incorpora-tion of dimethylbenzimidazole and ribose-5-phos-phate. The final structure has been described as themost complex non-polymeric biomolecule.27 Vita-min B12 is synthesised by a wide range of prokaryo-tes, including the reportedly haemless anaerobeClostridium tetanomorphum, photosynthetic bacteria,blue-green algae, and the nitrogen fixing bacteriumRhizobium in higher plant root-nodules.The third and principal pathway for uroporphy-

rinogen III utilisation is in the formation of the major

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CH2 CH2

CH2 CH2

COOH COOHUroporphyrinogen

COOH

CH2 CH2

CH2 CH2

COOH COOHSirohcern ( proposed)

FIG 4 Structure of uroporphyrinogen III, a porphyrinprecursor, and of sirohaem, the prosthetic group of apossible primitive cytochrome.

metalloporphyrins, including haems and chloro-phylls. In a series ofuroporphyrinogen modifications,four carboxyl carbons are removed from the aceticacid groups, one from each ring, by the enzymeuroporphyrinogen decarboxylase [E.C.4.1.1.37] toform the intermediate coproporphyrinogen M.48The enzyme has been detected in a wide range oforganisms including animals,49 photosynthetic bac-teria,50 and green algae.51 While detailed comparisonsare incomplete it appears that the enzyme is presentin the cytosol in higher plants and in animals25with an additional enzyme, perhaps, in the chloro-plast. The product, coproporphyrinogen isomer m,does not normally accumulate. It may be depositedin avian egg shells, and in millipedes it may bechelated with copper as an integument pigment. In

planarians coproporphyrin (and uroporphyrin)deposits have been implicated in phototacticresponses.'0The conversion of coproporphyrinogen III to

harderoporphyrinogen and then to protoporphy-rinogen IX involves the enzyme coproporphyrinogenoxidase [E.C.1.3.3.3.]. The first step in the conver-sion involves decarboxylation of the propionategroup at position 2 to a vinyl residue giving enzyme-bound harderoporphyrinogen. Harderoporphyrinhas been isolated from the Harderian gland of therat, and is present in small amounts in bile and bonemarrow,52 and in photosynthetic Euglena.55 Aninteresting point is that the oxidation of the vinylgroup of harderoporphyrinogen to a formyl group,and decarboxylation of the propionate at position 4to a vinyl group, would give rise to the porphyrinogencorresponding to compound haem s (or chloro-cruorin). I{aem s acts in place of haemoglobin inseveral polychaete worm families (see above).Harderoporphyrinogen itself does not normallyaccumulate in tissue, but is a transient intermediatebound to the coproporphyrinogen oxidase enzyme,where it may undergo a rapid rotation in situ so thatthe second propionate group (position 4) is de-carboxylated to a vinyl group.54 The product, proto-porphyrinogen, may undergo spontaneous oxidationto protoporphyrin IX, or a specific enzyme (proto-porphyrinogen dehydrogenase) may control thisstep.55 Such an enzyme has been found in severalorganisms including yeast and mammalian mito-chondria. Certainly the substrate for iron insertion,in the final step in protohaem (haem b) synthesis, isprotoporphyrin not protoporphyrinogen.

Protoporphyrin IX is, in one sense, the keycompound in porphyrin metabolism. It is the director indirect precursor of the major haem compoundsincluding haemoglobin, myoglobin, cytochromes a,b, c, and peroxidases and catalase, as well as thechlorophylls, and of bacteriochlorophyll in photo-synthetic organisms (bacteriocholorophylls c and dmay be exceptions, see Jbnes15). In healthy tissueprotoporphyrin IX is present in trace amounts only.Treatment by iron starvation, or porphyria-inducingdrugs, can lead to its accumulation in mammals,birds, fungi, plants, and bacteria. In all cases,throughout biological systems, only isomer IX isfound.

Ferrochelatase [E.C.4.99.1.1.] is a membrane-bound enzyme catalysing the insertion of iron (Fe2 +)into protoporphyrin IX, yielding protohaem (haemb). Protohaem does not appear to accumulate incells. Granick and Beale27 believe it is usual to findhaem bound in the ratio 1 haem: 1 apoprotein,implying a close regulation of apoprotein synthesiswith haem formation, and experimental evidence for

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Haems and chlorophylls: comparison offinction andformation

haem in controlling globin synthesis is now available.In fresh lysates of rabbit reticulocytes, globinsynthesis continues at a maximal linear rate forseveral minutes before ceasing abruptly in the absenceof haem. Haem inhibits the activity of a proteinkinase which in turn is an inhibitor of the initiationof protein synthesis. Exogenous haem inhibition ofthe kinase permits globin production to be main-tained.56 57Apart from haem b synthesis the biosynthetic

pathways for other haem compounds is less wellknown. Colleran and Jones58 showed that the haem-requiring slime mould Phycarum polycephalum usedprotohaem, not protoporphyrin IX, in the formationof cytochrome c. Sinclair et a159 showed the conver-sion of protohaem to haem a in a Staphylococcusmutant and similar results havc been obtained withhaem-requiring yeast mutant.60 It is presumed,though without conclusive evidence, that all cyto-chrome haems are synthesised from modifications ofprotohaem rather than directly from protopor-phyrin IX.The pathway leading to magnesium protopor-

phyrins is not clearly established. There is nounequivocal proof for a magnesium chelataseactivity in extracts of either plants or photosyntheticbacteria. Evidence from mutants suggests that proto-porphyrin IX is the substrate for magnesiumchelation. The presumed product, Mg-protopor-phyrin, undergoes esterification of the propionategroup at position 6. The methyl donor is S-adenosylmethionine in a reaction catalysed by a methyl-transferase [E.C. 2.1.1.11.].The subsequent steps from Mg-protoporphyrin

monomethyl ester to protochlorophyllide have notyet been defined. The most likely sequence appearsto be that suggested by Griffiths and Jones6' andinvolves the following reactions.

(1) The esterified propionate group at position 6undergoos dehydrogenation, hydration, and oxi-dation to yield a r-oxo group.

(2) This modified propionate group is attached tothe -y-meso bridge carbon to form the cyclo-pentane ring E. The product, Mg-2,4-divinylphaeoporphyrin a5 monomethyl ester, has beendetected in photosynthetic bacteria62 and in theseed coat of marrow.63

(3) Reduction of the vinyl group at position 4 toethyl forms protochlorophyllide.

In most of the lower plants protochlorophyllide israrely detected. Thus, while higher plants (forexample grasses) remain yellow if deprived of light,pine seedlings, mosses, and seaweeds grow green (orbrown) with or without light. In higher plants (the

Angiosperms), the conversion of protochlorophyllide(a porphyrin) to chlorophyllide (a chlorin) involvesthe reduction of the carbon-carbon bond at positions7 and 8 in an NADPH-light dependent reaction bythe enzyme protochlorophyllide reductase.64The insertion of chlorophyllide a into the chloro-

plast membrane requires the conversion of chloro-phyllide to chlorophyll.65 The esterification ofchlorophyllide with a long chain fatty alcoholtransforms the slightly polar precursor into alipid-membrane soluble end-product. The enzymechlorophyllase [E.C.3.1.1.14.] may catalyse thisesterification. The consequence is a loss of polarsolubility and the early moments following chloro-phyll formation are usually associated with changesin absorption spectra linked probably to successiverearrangements of the cholorophyll melecules withinmembranes and their association with specificproteins and lipids. The membranes with their arraysof chlorophylls and other pigmcnts are calledthylakoids and are the physical location of thephotochemical acts of photosynthesis.The second chlorophyll of higher plants, chloro-

phyll b, is likely to be formed from chlorophyll a.Shlyk and his associates66 have reviewed much ofthis evidence.

Bacteriochlorophyll a is probably derived fromchlorophyllide a in a series ofreactions. The structureof the other bacteriochlorophylls is established butlittle is known of the biosynthetic pathways.

Just as haemoglobins and indeed all other haemproteins are associations of haem and protein,the chlorophylls are also associated with variousproteins within the lipid membrane. Some six orseven chlorophyll-protein complexes have beenpartially characterised, with molecular weightsranging from 8000 to 70 000. One chlorophyll-protein complex contains about 20 chlorophyll amolecules (and one carotenoid); another complexwhich has been crystallised has been shown to con-tain seven chlorophyll molecules on a single poly-peptide chain.67There is a considerable amount of restructuring

of the photosynthetic apparatus in the green plantduring the first few hours of exposure to light,whether as a seedling or as a plant initially grown inthe dark. The restructuring involves formation ofdiscoid membranous structures known as thylakoidswithin which the chlorophylls are sited. The thyla-koids, present usually as aggregates or grana, aresurrounded by an aqueous stroma limited by adouble layered envelope, the whole forming thechloroplast. The processes concerned with the bio-physical and chemical changes linked to the forma-tion of a functional photosynthetic unit are complex.The changes include the synthesis of sulpholipids and

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G A F Hendry andO T G Jones

galactolipids and additional amounts of a specialc-type cytochrome (known as cytochrome f), bcytochromes, carotenoids, plastocyanin and plasto-quinones.

Function of chlorophyll-protein complexes andhaemoglobin

Neither protohaem nor the chlorophylls are foundin cells in any appreciable concentration in the freeform; they are invariably complexed to proteins.68The structure of the protohaem complex, haemo-globin, is of course well known. In mammals it is atetramer consisting normally of two pairs of unlikepolypeptide globin chains (cx- and ,-chains), each ofwhich contains a protohaem molecule constantlymaintained in the ferrous state. Each haem is co-ordinated to a histidine nitrogen of a globin chainwhich is folded so that the haem groups lie in cleftson the surface of the haemoglobin molecule approxi-mately equidistant from each other.69 Although oneside of the iron atom of haem is co-ordinated to theimidazole of histidine, the other side is free and canbind a molecule of oxygen forming oxyhaemoglobin.It is believed that on binding to oxygen the atomicradius of the haem iron is diminished sufficiently toallow it to move into the plane of the porphyrinring; its location in deoxyhaemoglobin is displacedout of the plane by 0 06 nm. As the iron of the haemmoves, it pulls its co-ordinated histidine with it,causing a whole series of changes in tertiary structureand breaking salt bridges and hydrogen bonds. Theeffect is to change the configuration of the moleculeso that oxygen binds more readily to haemoglobinonce one haem group is oxygenated, so a plot ofoxygen saturation of haemoglobin versus oxygentension is sigmoidal. In contrast, in myoglobin,which is a monomer, no such sub-unit co-operativityis possible and the oxygen binding curve is hyper-bolic (fig 5). The details of these mechanisms aredescribed in most modern textbooks of biochemistryand will not be covered here. Suffice it to say that theiron in the centre of the tetrapyrrole ring plays acrucial role in the function of haemoglobin and,indeed, in the function of all haem proteins. It isconcerned in binding the haem to the appropriateprotein and in binding and releasing oxygen. Inthe case of the cytochromes the iron is concerned inaccepting and donating electrons and itself undergoesreversible changes from the ferrous to the ferric state.

Chlorophyll-protein complexes have two separatebut closely related functions, both concerned withthe absorption of light energy and its use in driving avariety of redox reactions which lead to the synthesisof ATP and assimilation of CO2. In order to partici-pate in these reactions chlorophylls (or bacterio-

100

C

60

i-

0 20 40 60 80 100PO (mmHg)

FIG 5 Saturation ofhaemoglobin *-* andmyoglobin 0-* exposed to varying partial pressuresof oxygen.

chlorophylls) must be complexed with protein,although the structure of these proteins and thenature of their binding to the chlorophylls is farfrom clear. The function of the first group of chloro-phyll-proteins is to absorb light energy and to trans-fer the energy to the second smaller group ofchlorophyll molecules which, when excited by thislight energy, eject an electron which reduces aprimary acceptor, leaving behind oxidised chloro-phyll. The transfer of the electron is organised acrossthe photosynthetic membrane so that this primarycharge separation is stabilised, and an array ofelectron carriers converts this form of conservedenergy into the free energy of the ATP/ADP couple(by mechanisms described by Mitchell70) and theredox potential of reduced NAD or NADP.Chlorophyll thus functions either in light-harvestingprotein complexes or in the reaction centre proteincomplexes.A representation of the photosynthetic electron

transport system of higher plants is given in fig 6.This shows the two reaction centres characteristic ofthe chloroplast membrane, photosystems I and II(PSI and PSII). The reducing electron lost from theexcited reaction centre of PSI passes on eventuallyto NADP and is available for the reduction of CO2.In the oxidised reaction centre chlorophyll (positivehole), the redox state of the central magnesium isunchanged and the electron is lost from thet-electron system. It is re-reduced by an electronsupplied by the excitation of the second reaction

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Haems and chlorophylls: comparison offunction and formation

Light energy

FIG 6 Schematic representation of therole of tetrapyrroles in higher plantphotosynthesis.

2H20 'XCO2

* function not yet resolved

centre, PSII. The two reaction centres are connectedby a chain of electron transport carriers (plasto-quinones, cytochrome f, and the copper proteinplastocyanin) which mediate this electron transfer.The oxidised PSII is itself re-reduced by electronsderived from the enzymic splitting of water:

2H2 '- ------------------- 02+4H+ + 4e-.

Oxygen is evolved as a by-product and the protonsserve to provide part of the proton motive force usedin ATP synthesis, as described by Mitchell.70The chlorophyll-protein complexes involved in

green plant photosynthesis have been partly purified.The complex corresponding to the PSI reactioncentre has a molecular weight of about 110 kDcomposed of three sub-units of roughly equal size,with about 40 chlorophyll a molecules per reactioncentre. No chlorophyll b is present. The light-harvesting complex has a molecular weight of about35 kD, contains three chlorophyll a molecules andthree chlorophyll b molecules per mole,7' andaccounts for about 50% of the total chlorophyllpresent. Purification of the PSII reaction centre isless advanced and it is not so well characterised.

Reaction centres from photosynthetic bacteriahave been extensively purified and have given muchinformation about the mechanism of photosynthesis.They are smaller than green plant reaction centres(molecular weight around 70 kD) but like them theyare extracted from membranes with the use of

detergent. Usually they are composed of three sub-units and contain four bacteriochlorophylls, twobacteriophaephytins (that is bacteriochlorophyllslacking magnesium), one iron atom, and two boundquinones. Two of the bacteriochlorophylls are inclose association (the 'special pair' or dimer) andfunction as the primary photochemical electrondonor. One of the two bacteriophaeophytins is theprimary electron acceptor; that is, the primary, veryrapid, charge separation occurs between these two.The electron is then further transferred to a boundquinone which is closely associated with the ironatom. There is reasonable evidence from EPRspectroscopy that a similar ' special pair' of chloro-phyll a molecules is present in the PSI reaction centreof higher plants although there is no evidence for thepresence of a phaeophytin or a quinone in thisreaction centre.

Photosynthetic bacteria do not catalyse a water-splitting reaction of the type associated with PSI1of green plants. When an electron is lost from thereaction centre and donated eventually perhaps toNAD, it is replaced, not from a second light reaction,but by electrons donated from substrates present inthe environment. These may be organic materials,such as succinate or glutamate, or inorganic sulphurcompounds, such as sulphide or thiosulphate. Thesedonors are connected to the oxidised bacterialreaction centre by a chain of electron transportcomponents, including quinones and cytochromes.72

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12

The membranes of photosynthetic bacteria usuallyalso contain light-harvesting pigment-protein com-

plexes, two of which have been purified. They are

named B850 and B870, where the postscript numbersindicate the wavelength maxima of their principalabsorption bands. The B850 complex contains threebacteriochlorophylls per 20 kD sub-unit, and theB870 sub-unit two bacteriochlorophylls per 20 kDsub-unit (see Cogdell and Thornber73 for a review).The ratio of B850 to B870 and to reaction centrevaries with growth conditions. In general, the lowerthe light intensity during growth, the more light-harvesting complexes are synthesised.There is one case in which an unusual light-

harvesting pigment protein complex (isolated fromthe photosynthetic bacterium Prosthecochloris aestu-arii) has been crystallised and its structure deter-mined by x-ray diffraction. The molecule containsseven molecules of bacteriochlorophyll a, each on

average separated from an adjacent bacterio-chlorophyll by 1 2 nm, and essentially completelysurrounded by protein.67 These chlorophyll mole-cules are held firmly in place with little or no freedomof movement. The magnesium atoms of six of thebacteriochlorophyll molecules interact, on one sideonly, with the protein and there is evidence from theelectron density pattern that, in some cases, the sidechain ligand could be histidine, just as it is in haemo-globin. Access of a sixth ligand to the magnesium isprevented by the presence of the phytyl side chaintraversing one face of the rings.

In both green plants and bacteria the bulk of thechlorophyll pigments serves a light-harvestingfunction. They absorb light and channel it byresonance energy transfer to the photochemicaltraps which, in the PSI reaction centre and bacterialreaction centres, contain the 'special pair' of pigmentmolecules. The special properties of the chlorophyllsin vivo, the positions of their absorption maxima,and their photochemical activity are dependent ontheir association with specific proteins. In this,chlorophylls and protohaem can be seen to havegreat similarity. Stripped of their proteins they losetheir desirable properties. Their spectra changedramatically and their redox properties are quitedifferent.

Evolution

Attempts have been made in the past to unify theevolution of haems, haemoglobin, cytochromes, andchlorophylls.65 7 The basis of these attempts hasbeen largely speculative though with some support-ing evidence.65 The postulates assume that in thecourse of evolution, each of the intermediates inporphyrin biosynthesis was, at one time, an end


product of metabolic advantage to the cell. Subse-quent modifications to the end product improved itsproperties and led to abandonment of the direct useof intermediates which now are unable to functionper se in primary metabolism. The evolutionaryprocess therefore represents a progressive elaborationof mechanisms for carrying out the original primitivefunctions of porphyrins in a more efficient manner.75A problem arises in attempting to link today's endproducts (haems and chlorophylls) with the functionsof porphyrins in the primitive environmentalconditions of early life. This problem has beendiscussed by Granick75 and Mauzerall77 and severalhypotheses have been made.

Granick75 has shown that the monopyrrole PBG,when heated in neutral or alkaline conditions, formsuroporphyrinogen isomers I or III (but not isomersII or IV). Such a chemical event, it is postulated,may have provided, in low concentrations, a com-pound used to advantage in the primitive cell.Genes then evolved to synthesise the porphyrinmore efficiently. The first tetrapyrrole in the bio-synthetic pathway is uroporphyrinogen. This com-pound can be oxidised to yield uroporphyrin whichabsorbs light, is fluorescent, and can carry outphotosynthetic reactions. It can form a metalchelate to serve as a redox catalyst.65 The modifica-tions to uroporphyrin(ogen) III that subsequentlytook place in all organisms would have reflectedchanging environmental conditions. One suchchange would have been the development of photo-synthesis and oxygen release into the atmosphere.

Mauzerall65 postulates that the change from thefree porphyrin to the metalloporphyrin initiated thedevelopment of efficient photosynthesis. Mg-porphy-rins, in light-induced excited states, form stronglyreducing agents; coupling reducing reactions withelectron donation from water-splitting (resulting inelectron, proton, and oxygen release) led ultimatelyto modern photosynthesis. Increasing oxygen levelsin the environment of the metalloporphyrin wouldlead to the quenching of the photo-excited state ofchlorophyll. However, in a lipid environment thephoto-pigment would be protected sufficiently toallow electron ejection and so highly hydrophobicmetalloporphyrins would be favoured. Haem por-phyrins, as cytochromes, are able to act as electronacceptors and donors and may therefore have evolvedtogether with photosynthesis.

Iron porphyrins are relatively photo-inactive butmay readily complex oxygen. The metabolic advan-tage gained from exploiting this property wouldprovide for the development of haemoglobin (or amyoglobin precursor) in oxygen transport, storage,and release. When linked to cytochrome oxidasethese would provide the essential components of



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respiration in higher organisms. Such a respiratoryfunction presumably arose after the development ofa mechanism for the continuous production ofoxygen (photosynthesis) to complete the mechanismof modern respiration.

Porphyrins function in a number of different waysin biological systems, including oxygen carrying andstorage, light energy transfer, and light inducedelectron ejection. Even more variation occurs inporphyrin structure, with many side group modifica-tions and a limited number of central metal ionsubstitutions. Despite these variations in structureand function, the evidence suggests that porphyrinbiosynthesis is essentially similar in all biologicalsystems. While there is a possible difference in thesynthesis of the porphyrin precursor 5-aminolaevulinic acid in plants from animals and bacteria,the subsequent steps in the formation of proto-porphyrin IX are similar in all organisms. Thesimilarity is sufficiently great to support the conten-tion that porphyrins have arisen in the course ofevolution from a common origin. The variation infunction between the two great families, the haemsand the chlorophylls, is perhaps the result of theadaptability of the porphyrin structure to changingbiochemical requirements.

GAFH acknowledges financial support from theMedical Research Council and OTGJ from theScience Research Council.

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Requests for reprints to Dr G A F Hendry,Department of Biochemistry, The Medical School,University of Bristol, Bristol BS8 ITD.



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