JBIC (1997) 2 :411–417 Q SBIC 1997

MINIREVIEW

Harry A. Dailey

Enzymes of heme biosynthesis

Received: 12 March 1997 / Accepted: 8 May 1997

H.A. DaileyDepartment of Microbiology, Department of Biochemistry andMolecular Biology, and the Center for Metalloenzyme Studies,The University of Georgia, Athens, GA 30602-7229, USAFax: c1-706-542-2674; e-mail: Dailey6bscr.uga.edu

Abstract Heme is a necessary component in a varietyof oxygen-binding proteins and electron-transfer pro-teins, and as such it occupies a central role in cellularand organismal metabolism. With only rare exceptions,organisms that utilize heme possess the entire biosyn-thetic pathway to produce this tetrapyrrole compound.The enzymes involved catalyze a variety of interestingreactions and utilize both common and unique cofac-tors and metals. Aminolevulinate dehydratase from allorganisms and ferrochelatase from higher animals areboth metalloenzymes, while 5-aminolevulinate synthasecontains pyridoxal phosphate, and porphobilinogendeaminase possesses a unique dipyrrole cofactor. Twopathway enzymes catalyze multiple decarboxylationsand yet have no cofactors, and one enzyme catalyzes asix-electron oxidation with a single FAD. To add addi-tional scientific interest there exist biochemically andclinically distinct human genetic diseases for every stepin this pathway.

Key words Heme 7 Iron-sulfur cluster 7 Porphyria 7Ferrochelatase

The ability of organisms to utilize and synthesize hemeand other tetrapyrrole compounds represents an evolu-tionary milestone. Heme as an oxygen carrier in hemo-globin has allowed the continued evolution of animalspast the acoelomatic state, and heme as a redox com-pound in a variety of cytochromes has allowed in-creased energy generation from substrates via a pletho-ra of electron transport chains. Indeed, heme is the cor-

nerstone of aerobic life as we know it, and its value toorganisms is emphasized by the fact that essentially allheme-containing organisms possess a complete biosyn-thetic pathway for this compound. Since several com-prehensive reviews of tetrapyrrole biosynthesis areavailable [1–3], this mini-review will concentrate onmore recent discoveries and in particular on the termi-nal enzyme of the pathway ferrochelatase.

One driving force for research of heme biosynthesishas been the biomedical significance of this pathway.Genetic (or chemically induced) defects in any one ofthe enzymes after the first pathway enzyme, 5-aminole-vulinate synthase (ALAS), in mammals results in a dis-ease state commonly named porphyria [1, 4]. A defectin the first step results in sideroblastic anemia ratherthan a porphyria. There are biochemically and clinical-ly distinct porphyrias for each enzymatic step, althoughit has been popular to group the diseases into thosecausing acute episodic neurological manifestations andthose that are non-acute and possess cutaneous mani-festations (Table 1). None of the porphyrias routinelyexhibit anemia as a symptom. While most porphyriasare dominantly inherited genetic disorders, one of themore famous in the popular press, congenital erythro-poietic porphyria (CEP), is recessively inherited and isextremely rare. Because of the extreme manifestationsof CEP, suggestions have been made that individualssuffering from this disorder were the basis of the were-wolf legend [5, 6]. In addition there has been recentpopularity in ascribing porphyrias as the basis for vam-pires. However, this is not only unfounded but patentlyinaccurate, since porphyrics do not crave blood, are notkilled by exposure to sunlight, and do not possess anyspecial powers [6]. Unfortunately, inaccurate entries inpopular biochemistry texts, popular television shows,and public pronouncements by some individuals con-tinue to give life to this false myth.

The current body of experimental data for heme-synthesizing organisms suggests that all proceed via thesame pathway intermediates once the first committedcompound, 5-aminolevulinate (ALA), is formed

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Table 1 Genetic defects of heme biosynthesis

Porphyria Affected Enzyme Clinical Comment

Acute porphyrias

Acute intermittent porphyria (AIP) PBG deaminase No cutaneous involvement. Degree of clinical expres-sion highly variable.

Variegate porphyria (VP) Protoporphyrinogen oxidase Cutaneous involvement and acute attacks.

Coproporphyria (HCP) Coproporphyrinogen oxidase Photocutaneous lesions occur but rarely in the ab-sence of acute attacks.

Doss porphyria ALA dehydratase No cutaneous involvement. Acute symptoms only.Similar presentation to lead poisoning.

Non-acute porphyrias

Porphyria cutanea tarda (PCT) Uroporphyrinogen decarboxylase Cutaneous involvement only. No acute attacks.

Erythropoietic protoporphyria (EPP) Ferrochelatase Only cutaneous involvement. Degree of clinical ex-pression highly variable.

Congenital erythropoietic porphyria Uroporphyrinogen III synthase Severely photomutilating.

(Fig. 1). Formation of ALA is now known to occur viatwo distinct means [3]. In animals and some bacteria,this step involves the condensation of glycine with suc-cinylCoA to form ALA and CO2 [7, 8]. The enzyme ca-talyzing this step, ALA synthase (ALAS) (E.C.2.3.1.37)is a pyridoxal phosphate containing homodimer with amolecular weight of around 100 Kd. The enzyme is sol-uble and is found in the cytoplasm of bacteria and inthe mitochondrial matrix of animal cells. In eukaryotesit is nuclear encoded, synthesized in the cytoplasma as aprecursor and then translocated to the matrix in a steprequiring energy and proteolysis. Interestingly, in high-er animals there are two forms of ALAS that are en-coded on separate chromosomes and subject to differ-ent forms of regulation [9]. While the amino-terminalone-third of these two proteins differ considerably, thecarboxyl-terminal two-thirds, which comprise the cata-lytic portion of the enzymes, are highly homologous.One of these, the housekeeping form or ALAS-1, ispresent in all nonerythroid cell types, and its in vivolevel is sensitive to a variety of hormones, drugs, andxenobiotics. The protein is generally proposed to besubject to endproduct repression by heme, has a veryshort half-life (about 1 h), and its level may be induced100-fold under some circumstances. The second form ofALAS (ALAS-2) is expressed only during erythropoie-sis. Its regulation involves transcriptional erythroid-specific factors and translational control by an iron-re-sponsive element (IRE) [10]. It does not appear to besensitive to regulation by heme as is ALAS-1. Both reg-ulatory [9, 10] and protein structure-function features[11] of ALAS have been reviewed recently.

In plants and most bacteria, ALA is formed fromglutamate (actually glutamyl-tRNA) in a reaction in-volving three enzymes [3, 12]. This manner of formingALA is generally called the five-carbon pathway to dis-tinguish it from ALAS, which is called the four-carbonpath. Both structure-function features and regulatorymechanisms of the five-carbon pathway are currently

not well understood, but are being actively pursued [13,14].

Once ALA is formed, the next step is the condensa-tion of two ALA molecules to form the monopyrroleporphobilinogen (PBG), which is catalyzed by the en-zyme ALA dehydratase (E.C. 4.2.1.24) (also calledPBG synthase) [1, 3]. This is a metal-containing, multi-subunit (homo-octameric) enzyme in all organisms ex-amined, although the metal composition varies amongorganisms. In mammals and yeast, eight Zn ions arepresent in a homo-octamer, while in Escherichia coliand many bacteria there are eight Zn and eight Mg.Rhodobacter species, algae, and plants are reported tocontain 16 Mg (see [15]). The roles and positions ofthese metals in ALA dehydratase are incompletely re-solved, but the subject has been reviewed recently byJaffe [15]. One interesting observation has been madein the Bradyrhizobium japonicum ALAD which nor-mally requires Mg for activity. Alteration of four aminoacid residues in this enzyme can change the metal spe-cificity from Mg to Zn [16]. ALAD functions as a set ofcatalytically active homodimers (i.e., the native homo-octamer contains four active sites]. In the mammalianenzyme, data have been presented suggesting that fourZn (ZnA) ions are involved in catalysis, the other fourZn (ZnB) ions being protein-bound but not directly in-volved in catalysis. The Zn ions are proposed to bebound in a cys-, his-rich region similar in sequence to azinc finger motif. Models of these binding motifs basedupon EXAFS data have been presented, but final de-termination awaits crystallographic studies. For otherALADs, where a total of 16 metal ions are bound,there exist the same four active site ions with four non-catalytic ions (the A and B sites) plus an additional site(C site) to which Mg is bound. This additional Mg isnot required for activity, but does stimulate activity.

A large body of work from Jordan’s group has de-monstrated that there exist two substrate (ALA) bind-ing sites per active site (see [3]). One, called the P site,

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Fig. 1 Heme biosyntheticpathway of animals. This dia-gram shows the enzymes andreactions that they catalyzeand the intracellular compart-mentation of the proteins inanimal cells

binds the first molecule of ALA via an enzyme-boundSchiff base. This ALA contributes the propionate sidechain of PBG. The second, or A, site binds the secondmolecule of ALA. Occupation of the A site by ALAoccurs only after the first molecule of ALA binds to theP site, and enzyme activity requires the presence of me-tal in the ZnA site in mammalian ALAD or Zna in E.coli [17].

Two additional features of ALAD deserve note.One of these is that ALAD, which is present in abun-dant levels in cells, has recently been suggested to alsofunction as an accessory protein in the ubiquitin-ATP-dependent protein degradation machinery of mammal-ian cells [18]. Such a bifunctional role for this proteinwould represent quite an interesting adaptation by na-ture to utilize a single protein for two such diverse

processes. The other item of note is that ALAD is inhi-bited by very low concentrations of lead [1, 15]. It isthis enzyme that is strongly inhibited by environmentallead, and it is suggested that the accumulation of itssubstrate, ALA, leads to the neurological manifestationseen in lead poisoning.

The next step in the pathway is the head-to-tail con-densation of four PBG molecules with concomitantdeamination to form a linear tetrapyrrole, hydroxyme-thylbilane. The enzyme catalyzing this step, PBG deam-inase (PBGD) (E.C. 4.3.1.8), is the most thoroughlystudied enzyme of the pathway [3, 19] and is the onlyone for which crystal structures are currently published[20, 21]. One interesting feature of this monomeric en-zyme molecule is that it contains a cofactor that it syn-thesizes itself [19, 22]. In PBGD’s first catalytic cycle,

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Fig. 2 Spiro model for uro-porphyrinogen III synthase.The figure shows the pro-posed mechanism for conver-sion of hydroxymethylbilanevia a spiro intermediate touroporphyrinogen III [23]. Itshould be noted that it is theD ring which is flipped

the apoprotein assembles six molecules of PBG into anenzyme-bound hexapyrrole before cleaving and releas-ing the terminal tetrapyrrole. A dipyrrole remains cov-alently bound to the enzyme to form the active holoen-zyme. The released linear tetrapyrrole will sponta-neously cyclize to form uroporphyrinogen I unless thenext enzyme of the pathway, uroporphyrinogen III syn-thase (E.C. 4.2.1.75), is present [3, 19]. This synthasetakes the linear tetrapyrrole and cyclizes it to uropor-phyrinogen III in an interesting process where the ter-minal pyrrole ring [ring D] is rearranged [e.g. “flip-ped”]. Although this enzyme remains the least wellcharacterized protein of the pathway, considerable ef-forts have been made to characterize the chemistry ca-talyzed by this enzyme (see [23]). From these studies, itis clear that the linear bilane produced by PBGD is thesubstrate for uroporphyrinogen III synthase, and thatthe reaction pathway involves a spiro mechanism torearrange ring D (Fig. 2).

Uroporphyrinogen decarboxylase (UroD)(E.C.4.1.1.37) catalyzes the stepwise decarboxylation ofthe four acetic acid side chains to methyl groups [24].UroD will use both the I and III isomers, the productbeing the corresponding isomer of coproporphyrinog-en. Interestingly, this enzyme utilizes no cofactors forthese decarboxylations. UroD has been the object ofconsiderable attention because of its role in sponta-neous porphyria cutanea tarda (PCT) and its apparentassociation with hemochromatosis [25]. Hepatic ironoverload may lead to inactivation of uroD in vivo, al-though the enzyme is unaffected by iron in vitro. Re-cent studies by Roberts et al. [26] showing that an in-creased frequency of a particular hemochromatosis(C282Y) mutation is common in sporadic PCT may bethe first step to understanding this disorder.

Coproporphyrinogen III (but not the I isomer) is ox-idatively decarboxylated to protoporphyrinogen IX bythe enzyme coproporphyrinogen oxidase (CPO)(E.C.1.3.3.3) [2]. In aerobic organisms there is a singleprotein (homodimer) that is located in the intramito-chondrial membrane space in higher eukaryotes andutilizes molecular oxygen in its conversion of the A andB ring propionates to vinyl groups and 2CO2. Althoughearly studies on the yeast CPO suggested the presenceof iron [27], and a more recent publication presenteddata suggesting copper was involved [28], a more thor-ough study of the purified recombinant human enzymeshowed no evidence for any metal ions [29]. Early stud-ies on a purified bovine CPO did suggest that tyrosylresidues were involved in catalysis [30]. In anaerobic

organisms where tetrapyrroles are synthesized this stepis carried out by an oxygen-independent enzyme [31].Although genetic and molecular biology approaches tothis step have been presented, there are still no clearexperimental data identifying putative gene products asthe actual oxygen-independent CPO.

The problem of aerobic vs anaerobic conversion alsoexists in the next step of the pathway, the conversion ofprotoporphyrinogen IX to protoporphyrin IX [2]. Thissix-electron oxidation to form the fully conjugated ma-crocyclic protoporphyrin is catalyzed by the homodim-eric FAD-containing enzyme protoporphyrinogen oxi-dase (PPO) (E.C.1.3.3.4) [32, 33], which is bound to theinner mitochondrial membrane in eukaryotes and thecytoplasmic membrane in bacteria. This enzyme utilizesthree molecules of molecular oxygen, which are re-duced to H2O2 as the porphyrin is formed. Other thanthe single noncovalently bound FAD, there are no oth-er redox-active cofactors or metals involved in the reac-tion.

This particular enzyme has enjoyed a recent increasein interest for two quite different reasons. PPO was thelast pathway enzyme to be cloned and sequenced, and,therefore, the disease variegate porphyria [VP], whichoccurs when PPO is deficient [1, 4], was the last por-phyria to be characterized at the molecular level. Thisporphyria is of particular interest since “South AfricanVP” is one of the best characterized of all porphyriasand is one of the prime examples of what is referred toas the founder effect in genetics [4, 34]. A second as-pect of PPO is that this enzyme has been demonstratedto be the site of action of a large class of herbicides; thediphenyl-ether herbicides [35]. These herbicides arehighly specific for PPO and highly efficient, and thefinding that PPO from Bacillus subtilis is resistant tothese herbicides [36] has encouraged certain agrichemi-cal companies to pursue plant PPO in an effort to findor design herbicide-resistant forms of PPO that may begenetically introduced into crop plants.

In anaerobic and facultative organisms, the PPOstep is catalyzed by a multimeric protein complex thatis connected with the cell’s respiratory chain [2, 37].Genetic and molecular approaches have tentativelyidentified two putative subunits of this complex (hemGand hemK gene products). However, while the identityof hemG gene product as bona fide subunit seems se-cure [38], the finding of hemK in microorganisms thatdo not produce heme and lack all other heme-biosyn-thetic enzymes casts doubt on this gene product as aPPO subunit.

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The terminal step in the heme-biosynthetic pathwayis the insertion of ferrous iron into protoporphyrin IXto form protoheme IX (heme). Ferrochelatase(E.C.4.99.1.1) is the enzyme that [2, 39] catalyzes thisstep. With the exception of the enzyme in B. subtilis,ferrochelatase is a membrane-associated enzyme. In eu-karyotes it is nuclear encoded, synthesized in the cytos-olic compartment and then translocated to the mito-chondrion. Since the terminal three enzymes are mem-brane associated, there has been speculation about thepossibility of a multi-enzyme complex involving theseproteins, but available data suggests that, if such a com-plex exists, it does not carry out true substrate channel-ing [40].

Ferrochelatase will catalyze the insertion of Fe2c,Co2c, and Zn2c into a variety of IX isomer porphyrinsincluding proto-, hemato-, meso- and deuteroporphyrin[39]. No known ferrochelatase will utilize Fe3c, Cd2c,Mn2c or Mg2c although the B. subtilis enzyme will useCu2c but not Co2c, and the Azospirillum brasilenseenzyme will catalyze the insertion of Ni2c. In general,the Kms for both substrates are around 10–5 M. For por-phyrin, the natural substrate protoporphyrin IX has thelowest measured Km of those porphyrins that can beutilized in vitro, but also has the lowest Vmax. Deutero-porphyrin, the most soluble of the porphyrin substrates,is usually found to have the highest Km and highestVmax. Ferrochelatases are inhibited by a variety of di-valent heavy metals such as Hg2c, Pb2c, Mn2c, andCd2c and, for enzymes other than A. brasilense, Ni2c isinhibitory. No monovalent or trivalent cations are inhi-bitory.

At the present time no crystal structure informationis available for any ferrochelatase (although a diffract-ing crystal for the B. subtilis enzyme has been reported[41]) , so all information about catalysis must begleaned from kinetic studies and protein chemical mod-ification/site-directed mutagenesis approaches. The ba-sis of the current catalytic model was initially proposedalmost a decade ago [42, 43] and has been updated anddetailed most recently in 1996 [39]. In this model en-zyme first binds ferrous iron and then the porphyrinsubstrate. Metallation occurs when the macrocycle isbent, thereby allowing metal insertion concomitantwith proton release from the porphyrin. Once heme isformed it becomes planar and is released as product.The basis for this model stems originally from the ob-servation that N–methylprotoporphyrin is a tight-bind-ing competitive inhibitor with a Ki measured in nano-moles. This Ki is about 103 lower than is found withother non-substrate porphyrins. Since the major struc-tural distinction between the N–alkylporphyrins andporphyrins is that the alkylated ring of the tetrapyrroleis bent about 307 out of the plane of the porphyrin ring(which is otherwise planar), it has been suggested thatthis bent-ring porphyrin serves as a transition state ana-log for ferrochelatase. Thermodynamic support for thismodel comes from data showing a 105–fold increase inthe rate of metallation of a distorted porphyrin ring as

compared to a planar macrocycle. Additional biochem-ical support for the macrocycle distortion model comesfrom the observation that antibodies made againstN–methylmesoporphyrin have ferrochelatase-like cata-lytic activity [44].

A considerable body of data has been accumulatedfor a variety of individual isomers of N–alkyl porphy-rins using crude preparations of ferrochelatase (see [2,39] for review). These data show that alkylation of theporphyrin A or B rings results in compounds that aremuch more effective inhibitors than are C or D ringsubstituents [45]. Current studies on these compoundswith purified recombinant human ferrochelatase mayprovide additional details. Furthermore, key supportfor this model may be provided by resonance Ramanstudies on the enzyme-bound porphyrin complexes. Ifthe macrocycle distortion model is appropriate, thenthe question arises as to how the ring is bent. One sug-gestion would be via p-bond interaction with aromaticresidues such as tryptophan. Among all known se-quences [39], only one tryptophan is conserved, but twoadditional sites contain only aromatic residues andthese represent likely residues for such an action.

An additional area of interest for ferrochelatase isthe substrate iron-binding site. While suggestions havebeen made that a region containing conserved carboxy-lates could serve as ligands for the iron [39, 46], recentsite-directed mutagenesis studies on some of these resi-dues appear to indicate that they may not be directlyinvolved with substrate iron chelation [47]. One residuefor which there is evidence in support of a role in ironbinding is His 263 [48]. This residue is identical in allsequences, and site-directed mutagenesis of this toH 263A has a profound effect upon the Km for iron.One additional set of potential ligands that can be eas-ily overlooked since they are widely spaced through theprimary sequence consists of three conserved tyrosylresidues. Transferrin, which has similarly spaced tyrosylresidues, binds iron in part via these residues, and theobservation that bicarbonate affects the binding of ironto ferrochelatase (J. J. Kools and H. A. Dailey, unpub-lished) makes the similarity more interesting.

One of the more interesting features about ferroche-latase concerns the presence of a single (2Fe-2S) clusterin the enzyme from higher animals [39, 49–51]. Ferro-chelatase from prokaryotes is approximately 30–50 am-ino acid residues shorter than eukaryotic ferrochela-tases on the COOH terminal end and does not containa cluster (see [39]). The bacterial enzymes are activeand relatively stable in their purified form without anyadditional factors or proteins, which would suggest thatthe COOH extension is not necessary for catalysis.However, the removal of this extension from the yeast(which lacks the cluster) or mammalian enzymes resultsin total loss of activity [50]. The mammalian, but notyeast or plant, ferrochelatases contain the (2Fe-2S)cluster. It has been shown for human ferrochelatasethat three of the four ligands for this cluster are Cysresidues present in the COOH terminal extension [52].

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The fourth ligand remains unidentified at present, but,based upon variable temperature magnetic circulardichroism and resonance Raman data [52], it has beensuggested that it may be an oxygenic residue.

An interesting finding with regard to the cluster isthat it is quite labile as compared to other (2Fe-2S)clusters. It is rapidly destroyed by NO, and concomi-tantly the enzyme loses activity [53]. A possible role forthis NO sensitivity in the body’s overall immune re-sponse to infection vis-à-vis iron sequestration has beensuggested, but more data are required to support such aproposition. It is of note that the enzyme functions in-dependently of the redox state of the cluster and thatcluster iron does not function as substrate iron.

Outstanding questions surrounding the cluster arewhat is its in vivo function and how is enzyme inactiva-tion modulated by the cluster. The first of these ques-tions may be answered once we know at what stage inevolution the cluster appeared and how genetically en-gineered organisms lacking this cluster via substitutionof a non-cluster ferrochelatase function and survive.The structural role for the cluster in higher animalsmay reflect a refinement of the function that theCOOH terminal extension in yeast and plant ferroche-latases serves. It could be envisioned that this regionfolds back over the catalytic core of ferrochelatase andinteracts in a fashion to stabilize a functional active-siteconformation. Disruption of this “bridge” would thentrigger a conformational change that inactivates the en-zyme. While it will require structural determination ofthe cluster-containing enzyme to support or deny thispossibility, the observation that individual mutations inthe enzyme at a region distinct from the carboxyl ter-minus result in disruption of the cluster make this aninteresting possibility.

Acknowledgements This work was supported by grants for theNational Institutes of Health DK 32303 and DK35898 to H.A.D.and by the National Science Foundation Training Group Awardto the Center for Metalloenzyme Studies [DIR9014281].

References

1. Kappas A, Sassa S, Galbraith RA, Nordmann Y (1995) In:Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The meta-bolic and molecular basis of inherited disease. McGraw-Hill,New York, pp 2103–2159

2. Dailey HA (1990) In: Dailey HA (ed) Biosynthesis of hemeand chlorophylls. McGraw-Hill, New York, pp 123–161

3. Jordan PM (1991) In: Jordan PM (ed) Biosynthesis of tetra-pyrroles, vol 19. Elsevier Science, New York, pp 1–66

4. Nordmann Y, Deybach JM (1990) In: Dailey HA (ed) Bio-synthesis of heme and chlorophylls. McGraw-Hill, New York,pp 491–542

5. Illis L (1964) Proc Roy Soc Med 57 :23–266. Moore MR (1990) In: Dailey HA (ed) Biosynthesis of heme

and chlorophylls. McGraw-Hill, New York, pp 1–547. Gibson KD, Laver WG, Neuberger A (1958) Biochem J

70 :71–818. Kikuchi G, Kumar A, Talmage P, Shemin D (1958) J Biol

Chem 233 :1214–1219

9. May BK, Dogra SC, Sadlon TJ, Bhasker CR, Cox TC, Bot-tomly SS (1995) Prog Nucl Acid Res Mol Biol 51 :1–51

10. Ponka P (1997) Blood 89 :1–2511. Ferreira GC, Gong J (1995) J Bioenerg Biomemb

27:151–15912. Beale SI, Weinstein JD (1990) In: Dailey HA (ed) Biosynthe-

sis of heme and chlorophylls. McGraw-Hill, New York, pp287–392

13. Kumar AM, Soll D (1996) Ind J Biochem Biophys 33 :30–3414. Hori N, Kumar AM, Verkamp E, Soll D (1996) Plant Physiol

Biochem 34 :3–915. Jaffe EK (1995) J Bioenerg Biomemb 27 :169–17916. Chauhan S, O’Brian MR (1995) J Biol Chem

270 :19823–1982717. Spencer P, Jordan PM (1995) Biochem J 305 :151–15818. Guo GG, Gu M, Etlinger JD (1994) J Biol Chem

269 :12399–1240219. Shoolingin-Jordan PM (1995) J Bioenerg Biomemb

27:181–19520. Louie GV, Brownlie PD, Lambert R, Cooper JB, Blundell

TC, Wood SP, Warren MJ, Woodlcock SC, Jordan PM (1992)Nature 359 :33–39

21. Hadener A, Matzinger PK, Malashkevich VN, Louie GV,Wood SP, Oliver P, Alefounder PR, Pitt AR, Abell C, Bat-tersby AR (1993) Eur J Biochem 211 :615–624

22. Warren MJ, Jordan PM (1988) Biochemistry 27 :9020–903023. Leeper FJ (1994) In: The biosynthesis of the tetrapyrrole pig-

ments. Ciba Foundation Symposium 180, Wiley, Chichester,USA, pp 111–130

24. Elder GH, Roberts AG (1995) J Bioenerg Biomemb27:207–214

25. Lundvall O, Weinfeld A, Lundin P (1970) Acta Med Scand188 :37–53

26. Roberts AG, Whatley SD, Morgan RP, Worwood M, ElderGH (1997) Lancet 349 :321–323

27. Camadro JM, Chambon H, Jolles J, Labbe P (1986) Eur JBiochem 156 :579–587

28. Kohno H, Furukawa T, Tokunaga R, Taketani S, YoshinagaT (1996) Biochim Biophys Acta 1292 :156–162

29. Medlock AE, Dailey HA (1996) J Biol Chem271 :32507–32510

30. Yoshinaga T, Sano S (1980) J Biol Chem 255 :4727–473131. Coomber SA, Jones RM, Jordan PM, Hunter CN (1992) Mol

Microbiol 6 : 3159–316932. Dailey HA, Dailey TA (1996) J Biol Chem 271 :8714–871833. Dailey TA, Dailey HA (1996) Prot Sci 5 : 98–10534. Meissner PN, Dailey TA, Hift RJ, Ziman M, Corrigall AV,

Roberts AG, Meissner DM, Kirsch RE, Dailey HA (1996)Nature Genetics 13 :95–97

35. Matringe M, Camadro JM, Labbe P, Scalla R (1989) BiochemJ 260 :231–235

36. Dailey TA, Meissner P, Dailey HA (1994) J Biol Chem269 :813–815

37. Klemm DJ, Barton LL (1987) J Bacteriol 169 :5209–521538. Sassarman A, Letowski J, Czarka G, Ramirez V, Nead MA,

Jacobs JM, Morais R (1993) Can J Microbiol 39 :1155–116139. Dailey HA (1996) In: Hausinger RP, Eichorn GL, Marzelli

LG (eds) Mechanisms of metallocenter assembly. VCH, NewYork, pp 77–98

40. Proulx KL, Woodard SI, Dailey HA (1993) Prot Sci2 :1092–1098

41. Hasson M, Al-Karadaghi S (1995) Proteins 23 :607–60942. Lavallee DK (1988) In: Liebman JF Greenberg A (eds) Me-

chanistic principles of enzyme activity. VCH, New York, pp279–314

43. Dailey HA, Jones CS, Karr SW (1989) Biochim Biophys Acta999 :7–11

44. Cochran AG, Schultz PG (1990) Science 249 :781–78345. Kimmett SM, Whitney RA, Marks GS (1992) Mol Pharmacol

42 :307–310

417

46. Labbe-Bois R, Camadro J-M (1994) In: Winkelmann G, Winge DR (eds) Metal ions in fungi. Dekker, New York, pp413–453

47. Gora M, Grzybowska E, Rytka J, Labbe-Bois R (1996) J BiolChem 271 :11810–11816

48. Kohno H, Okuda M, Furukawa R, Taketani S (1994) BiochimBiophys Acta 1209 :95–1

49. Dailey HA, Finnegan MG, Johnson MK (1994) Biochemistry33 :403–407

50. Dailey HA, Sellers VM, Dailey TA (1994) J Biol Chem269 :390–395

51. Ferreira GC, Franco R, Lloyd SG, Pereira AS, Moura I,Moura JJG, Huynh BH (1994) J Biol Chem 269 :7062–7065

52. Crouse BR, Sellers VM, Finnegan MG, Dailey HA, JohnsonMK (1996) Biochemistry 35 :16222–16229

53. Sellers VM, Johnson MK, Dailey HA (1996) Biochemistry35 :2699–2704