Heme Biosynthesis

Heme Biosynthesis M R O’Brian, State University of New York, Buffalo, NY, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Overview of Tetrapyrrole Synthesis Gene Nomenclature

Glossary decarboxylase An enzyme that removes a carboxyl group from a substrate. oxidase An enzyme that catalyzes the oxidation of a substrate. prokaryotes Single celled organisms belonging to the Eubacterial or Archaeal kingdoms.

Abbreviations ALA AdoMet eALAS GSA GTR HRM

-aminolevulinic acid S-adenosyl-L-methionine erythroid cell-specific glutamate-1-semialdehyde glutamyl-tRNA reductase heme regulatory motif

Defining Statement Hemes are involved in many metabolic processes, and recently have been found to also play important roles in regulation and cell signaling. The biosynthetic pathway leading to heme formation is, with a few interesting exceptions, well-conserved, and is controlled in accordance with cellular function. There has been significant progress in understanding the structures and catalytic mechanisms of the heme biosynthetic pathway enzymes over the last several years. In addition, the availability of whole genome sequences has shed light on the gaps in our understanding of heme biosynthesis in prokaryotes.

Introduction Anyone who has taken a college course in biochemistry probably learned about heme as the prosthetic group of hemoglobin, cytochromes, catalase, and peroxidases. In these capacities, heme is involved in electron transfer,

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The Steps in Heme Biosynthesis Regulation of Heme Biosynthesis Alternative Heme Biosynthetic Pathways Further Reading

prosthetic group A non-protein moiety covalently or non-covalently bound to a protein that is needed for function of that protein. pyrrole A five membered cyclic compound containing a nitrogen and four carbons. They are the building blocks of hemes, chlorophylls and other tetrapyrroles.

Irr PBG PCT ROS SAM XLSA

iron response regulator porphobilinogen porphyria cutanea tarda reactive oxygen species S-adenosylmethionine Sideroblastic X-linked anemia

O2 delivery, and detoxification. However, heme is now known to have diverse roles in prokaryotic and eukaryotic cells in which it acts as a signal or a sensor to modulate cellular activities. Whereas respiration and oxidative phosphorylation are synonymous with O2-based metabolism in eukaryotes, many prokaryotes carry out oxidative respiration in the absence of O2, using nitrate, sulfate, or other oxidants. Under those conditions, cytochromes are required, nevertheless, for electron transfer and the final reduction of the terminal electron acceptor as occurs with O2-based respiration. In addition, O2 is an obligatory substrate for heme biosynthesis in eukaryotes, which presents an important difference in prokaryotes where heme may be needed under anaerobic or hypoxic conditions. Thus, many prokaryotes must have O2-independent mechanisms for heme biosynthesis. Heme proteins have regulatory functions for signal transduction, and the sensing of diatomic gases such as oxygen, carbon monoxide, and nitric oxide. These heme-based sensors bind the gas ligand directly to ultimately control diverse

Physiology | Heme Biosynthesis 195

processes such as adaptation to anaerobiosis, vascular function, and circadian rhythms. In addition to serving as the active moiety of proteins, heme is also a regulatory molecule that mediates cellular responses to environmental, metabolic, and homeostatic cues. In these cases, heme appears to exert its effect by binding to proteins transiently or reversibly, and does not necessarily serve as a prosthetic group. In this capacity heme is a regulator at the level of transcription, translation, protein targeting, protein stability, and differentiation. It is also an effector that controls circadian rhythms. Heme refers to protoheme, the prosthetic group of hemoglobins and b-type cytochromes, and also to modified protoheme derivatives found in cytochrome oxidases and the covalently bound prosthetic group of cytochrome c (Figure 1). These modifications of heme, in the context within which they associate with the protein, increase their versatility by altering their oxidation–reduction potential and ligand-binding capability. Protoheme is ubiquitous in organisms that make heme, but the other hemes are more

specialized. Cytochrome c is particularly interesting because, unlike the other hemes, heme C is covalently linked to the apoprotein via thioether linkages. Thus, cytochrome c biogenesis requires the translocation of heme from the inner membrane where it is synthesized to the periplasmic space in concert with the ligation reactions. The modifications of protoheme are not described herein. This article describes the biosynthesis of protoheme (Figures 3 and 4), with an emphasis on enzymology, structure, and regulation. Although the focus is on prokaryotes, heme synthesis is also studied intensely in eukaryotes, and we will refer to that literature where it sheds light on understanding heme formation as a whole.

Overview of Tetrapyrrole Synthesis Hemes belong to a larger class of molecules called tetrapyrroles, which include chlorophylls, chlorins, corrins, and bilins. The first universal tetrapyrrole precursor

S-Cys-

α

2

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Protoheme (heme b)

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Heme d

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HOHC

N

N

N

Fe N

N

Fe N

N

N

HC O

HOOC

COOH

Heme o

HOOC

COOH

Heme a

Figure 1 Protoheme formation and its modifications. The numbering of protoheme carbons is by the Fischer system.

196 Physiology | Heme Biosynthesis Chlorophyll Bacteriochlorophyll Glycine + succinyl-CoA

ALA

Protoporphyrin IX

Uroporphyrinogen III

Glutamate

Vitamin B12 Other corrins

Protoheme

Heme a Heme c Heme d Heme o

Precorrin 2

Siroheme Heme d1

Bilins

Coenzyme F430

Figure 2 Outline of tetrapyrrole synthesis.

-aminolevulinic acid (ALA) is synthesized either from glutamate by the C5 pathway or from glycine and succinyl coenzyme A via ALA synthase (Figure 2). The synthesis of uroporphyrinogen III from ALA requires three enzymatic steps with conserved intermediates. Uroporphyrinogen III is the final common precursor for all tetrapyrroles. It is methylated to form precorrin 2, a precursor for vitamin B12, siroheme, and coenzyme F430 (Figure 2). Vitamin B12 is a cofactor in two known methyl transfer reactions. Siroheme is a prosthetic group of sulfite reductase and nitrite reductase. Coenzyme F430 is the prosthetic group of methyl coenzyme M reductase, an enzyme required for methanogenesis. For heme and chlorophyll synthesis, uroporphyrinogen III is metabolized by three successive enzymatic steps that modify the side groups of the macrocycle to yield protoporphyrin. Protoporphyrin chelates magnesium and iron catalyzed by different chelatases for chlorophyll and protoheme formation, respectively. Protoheme is covalently modified further for c-, d-, o- and a-type cytochromes (Figure 1), or is linearized for bilin formation. (We note here that the letter designation for a type of heme does not correlate with a gene designation. For example, heme C refers to a specific heme in cytochrome c ultimately derived from protoheme, whereas the hemC gene encodes an enzyme of the biosynthetic pathway leading to protoheme formation.)

Gene Nomenclature Table 1 lists the names of the genes encoding the enzymes of the heme biosynthetic pathway. Unfortunately, the hemA designation has been ascribed to both the gene encoding ALA synthase and that encoding the C5 pathway glutamyl-tRNA reductase. Although both enzymes are involved in ALA formation, they are structurally dissimilar proteins that have different substrates and products,

and therefore should not have the same gene name. This confusing nomenclature is based on the original assumption that bacteria as a whole possess ALA synthase because of studies with animals and on work with Rhizobium and Rhodobacter, where the presence of ALA synthase was demonstrated. Thus, the mutated genetic loci of Escherichia coli and Salmonella typhimurium ALA auxotrophs presumed to encode ALA synthase were designated hemA, and the bona fide cloned ALA synthase gene isolated from Sinorhizobium meliloti was named hemA as well. The subsequent molecular characterization of the enteric bacterial genes revealed that hemA encodes glutamyl-tRNA reductase, and made clear that the C5 pathway is not unique to plants or to chlorophyll synthesis. We suggested previously that the gene encoding glutamyl-tRNA reductase be renamed gtrA, a designation used for the plant enzyme, and that hemA designate the bacterial gene encoding ALA synthase. hemG and hemY encode distinct protoporphyrinogen oxidase proteins, but the porphyrin substrate and product are the same for both enzymes. The oxygen-dependent and oxygenindependent coproporphyrinogen oxidases are dissimilar proteins encoded by hemF and hemN, respectively. A second O2-independent coproporphyrinogen oxidase gene homologue has been identified in numerous bacteria and is called hemZ in Rhodobacter sphaeroides and Bacillus subtilis. There is no evidence to suggest that the hemZ genes are functionally similar to each other, and thus the hemZ designation may be confusing. This is confounded by the occasional hemZ designation for the gene encoding ferrochelatase, which is not correct.

The Steps in Heme Biosynthesis 5-Aminolevulinic acid (ALA) is the first universal tetrapyrrole precursor (Figures 3 and 4). It is synthesized from glutamate in most prokaryotic species or with glycine and

Physiology | Heme Biosynthesis 197 Table 1 Prokaryotic enzymes and genes in heme biosynthesis Enzyme

Gene

-Aminolevulinic acid synthase Glutamyl-tRNA reductase Glutamate 1-semialdehyde aminotransferaseb -Aminolevulinic acid dehydratase (porphobilinogen synthase) Porphobilinogen deaminase (hydroxymethylbilane synthase) Uroporphyrinogen III synthase Uroporphyrinogen III decarboxylase Coproporphyrinogen III oxidase, O2-dependent Coproporphyrinogen III oxidase, O2-independent Protoporphyrinogen IX oxidase Protoporphyrinogen IX oxidase Ferrochelatase

hemA gtrA (hemA)a hemL hemB hemC hemD hemE hemF hemN hemG hemY hemH

a

Both glutamyl-tRNA reductase and ALA synthase have been designated hemA but are unrelated enzymes. We recommend hemA and GtrA as designated. b This enzyme is often annotated as glutamate semialdehyde 2, 1 aminomutase. The enzyme catalyzes an intermolecular amino transfer reaction between two glutamate 1-semialdehyde (GSA) molecules and therefore it is not a mutase reaction.

COOH

(a)

HOOC COOH

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ALA synthase (hemA) O

O

NH2

CoA-S

CO2

Succinyl-CoA

(b)

Glycine

ALA

COOH

COOH

Glutamyl-tRNA reductase (gtrA/hemA) H2 N

COOH

GSA aminotransferase (hemL) O

H 2N

CO

NADPH tRNAGlu

Glutamyl-tRNAGlu

H2N

NADP+ tRNA

CHO

Glutamate 1-semialdehyde

H2N

ALA

Figure 3 Two routes of ALA formation. (a) ALA synthesis from succinyl-coenzyme A and glycine via ALA synthase. (b) The C5 pathway for ALA synthesis from glutamate. The gene designations are shown in parenthesis. hemA denotes genes encoding both glutamyl tRNA reductase and ALA synthase, but they are unrelated proteins that catalyze different reactions. We suggest gtrA to denote the gene encoding glutamyl-tRNA reductase.

succinyl coenzyme A in a single taxonomic group (Figure 3). Two ALA molecules are subsequently condensed to form the monopyrrole porphobilinogen (PBG) (Figure 4). Four PBG molecules are then polymerized and cyclized to form uroporphyrinogen III (Figure 4), the tetrapyrrole from which all other tetrapyrroles are derived. The cyclization step involves inversion of one of the PBG molecules, which accounts for the asymmetry of

tetrapyrroles. Uroporphyrinogen III is modified in a series of steps that involve decarboxylation and oxidation reactions to yield protoporphyrin IX. Finally, an iron atom is chelated into the protoporphyrin to yield protoheme. For the most part, the heme biosynthetic pathway is highly conserved in terms of the intermediate precursors and the enzymes that catalyze each step, although important exceptions to this are noted herein. In recent years,

198 Physiology | Heme Biosynthesis HOOC

HOOC

HOOC

HOOC

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COOH

ALA dehydratase (hemB)

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PBG deaminase (hemC )

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Uroporphyrinogen synthase (hemD )

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Hydroxymethylbilane

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Ferrochelatase (hemH )

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Protoporphyrinogen oxidase (hemY or hemG)

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6H HOOC

COOH

Protoheme

HOOC

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Protoporphyrin IX

HOOC

COOH

Protoporphyrinogen IX

HOOC

COOH

Coproporphyrinogen III

Figure 4 Heme biosynthetic pathway from -aminolevulinic acid (ALA) to protoheme. The enzymes catalyzing each step are noted, and the genes encoding them are denoted in parentheses.

the X-ray crystal structure for each enzyme of the biosynthetic pathway has been resolved, and important mechanistic information uncovered. The sequencing of many prokaryotic genomes has made it possible to identify heme biosynthesis genes in many organisms, and to find both patterns and gaps in their distribution in prokaryotes. 5-Aminolevulinic Acid Synthase ALA synthase catalyzes the condensation of glycine and succinyl coenzyme A to form ALA using pyridoxal phosphate as a cofactor (Figure 3). The enzymology of mammalian ALA synthase is worked out in detail, and is generally applicable to the synthases from other organisms based on structural similarities. The crystal structure of Rhodobacter capsulatus ALA synthase has been determined, and it is shown to be a tightly interlocked homodimer. Each monomer contains three domains; the central catalytic domain contributes most of the dimer interface. Humans have erythroid cell-specific (eALAS) and housekeeping forms of the enzyme encoded by different genes, which are regulated differently. Sideroblastic X-linked anemia (XLSA) in humans is caused by mutation in the gene encoding eALAS, and mutations causing 37 different amino acid substitutions have been described. The bacterial crystal structure shows that mutations corresponding to the XLSA variants are found in the active site affecting

pyridoxal phosphate or substrate binding, but are also found on the protein surface and buried within the protein away from the active site. ALA synthase is found in nonphotosynthetic eukaryotes, where it is located in mitochondria. Among prokaryotes, it is restricted to the -proteobacteria, a bacterial taxonomic group that includes photosynthetic organisms, obligate intracellular pathogens of eukaryotes, and plant symbionts, and in general it encompasses a metabolically diverse group of organisms. The strict retention of ALA synthase and exclusion of the C5 pathway within the -proteobacterial group, despite the diversity of tetrapyrroles and ecological niches that it represents, suggests that the distribution of ALA synthase reflects primarily a common ancestry rather than an advantageous evolutionary adaptation in extant organisms. ALA synthase must have arisen in an organism that makes succinyl coenzyme A. However, the distribution of the synthase is restricted whereas the tricarboxylic acid cycle enzymes are widely distributed. Regardless, mitochondria are descended from the -proteobacteria, and eukaryotic ALA synthase likely has an antecedent from that group. Bacterial ALA synthase, encoded by hemA, has been studied most in Rhodobacter and in the rhizobia. R. sphaeroides contains two hemA homologues designated hemA and hemT, and genome information shows that two ALA synthase genes within an organism are not

Physiology | Heme Biosynthesis 199

uncommon, although it is not the norm. The purpose of two ALA synthase genes is not known in most cases. In R. sphaeroides, only hemA mRNA is expressed under aerobic or photosynthetic growth in wild-type cells, suggesting that the allele is sufficient for tetrapyrrole synthesis. However, hemT is expressed in a hemA mutant, which explains why both genes must be inactivated for ALA auxotrophy. A R. sphaeroides ALA auxotrophic mutant derived from chemical mutagenesis was deficient only in the hemA gene whereas the hemT coding region was unaltered. Furthermore, the auxotroph could be complemented by hemA in trans, but not by hemT even though hemT complements a hemA hemT double mutant. The basis for these observations is uncertain, but ALA synthase is a homodimer, and thus the nonfunctional hemA gene product in R. sphaeroides may form mixed dimers yielding a dominant-negative phenotype. Rhizobia occur as free-living organisms or as endosymbionts of certain legumes within specialized root organs called nodules. The interest in ALA synthase, and heme synthesis in general, in the rhizobia was originally founded on the hypothesis that plant nodule hemoglobin heme is derived from the bacterium. Although this idea is unlikely to be correct, the heme pathway in Rhizobium is highly regulated due to the metabolic diversity associated with its aerobic, microaerobic, and symbiotic growth. In the latter case, the bacteria carry out the energy-intensive process of nitrogen fixation in an O2-limited environment of a nodule, which requires de novo synthesis of cytochromes to accommodate that demand. ALA synthase mutants of Bradyrhizobium japonicum can establish a successful symbiosis on their soybean host because of an ALA uptake activity as described below. C5 Pathway for ALA Synthesis The C5 pathway synthesizes ALA from glutamate by a three-step mechanism that incorporates the carbon skeleton, intact, into ALA. The pathway was initially discovered in plants in the context of chlorophyll biosynthesis, but plants and algae use this pathway for the synthesis of all tetrapyrroles. Furthermore, the C5 pathway is the sole mechanism for ALA formation for archaea, and for bacteria as well, except for the -proteobacteria, which use ALA synthase. This taxonomic distribution is borne out in the hundreds of bacterial and archaeal genomes that have been sequenced. In the first step, glutamyl-tRNA synthetase charges tRNAGlu with glutamate, as occurs in protein synthesis; thus this step is not committed to ALA formation (Figure 3). Glutamyl-tRNAGlu is then reduced to glutamate-1-semialdehyde (GSA) by glutamyl-tRNA reductase, and finally GSA is converted to ALA by a transamination reaction. The tRNAGlu that activates glutamate has the

UUC anticodon, and is also used in protein synthesis. A point mutation in the T-loop of chloroplast tRNAGlu from Euglena gracilis affects chlorophyll formation but not protein synthesis. As expected from that phenotype, the altered tRNA was acylated by the synthetase, but GlutRNAGlu was a poor substrate for tRNA reductase. Thus, the tRNA has different specificity elements for the reductase than for the protein elongation factor Tu. Although glutamyl-tRNA synthetase is not committed to ALA formation, one of the two synthetases in Acidithiobacillus ferrooxidans is inhibited by heme. This bacterium synthesizes a large quantity of heme, with cytochromes comprising up to 10% of the total cellular protein. It is proposed that the major glutamyltRNA synthetase in A. ferrooxidans is committed to heme synthesis, and control by heme is consisitent with that idea. Since the discovery of a role for glutamyl-tRNA synthetase in a process unrelated to protein synthesis, noncanonical functions for tRNA synthetases, including involvement in transcription, RNA splicing, inflammation, apoptosis, cell proliferation, and DNA repair, have been described. These functions do not appear to involve aminoacyl-tRNA synthetase activity, but work in concert with other proteins. Thus, this class of proteins has functions well beyond the realm of translation. Glutamyl-tRNA reductase

Glutamyl-tRNA reductase uses NADPH in the reduction of glutamyl-tRNA to GSA in the first committed step in tetrapyrrole synthesis in organisms that express the C5 pathway (Figure 3). The structure of a prokaryotic glutamyl-tRNA reductase has been resolved, and detailed information on substrate recognition, catalytic mechanism, and intermediate channeling has been obtained. The Methanopyrus kandleri enzyme crystallized with a tRNAglu-like inhibitor is a V-shaped dimer, showing extensive tRNA–protein contacts. The N-terminal catalytic domain recognizes the glutamate moiety of the substrate. From these and enzymatic studies with the E. coli tRNA reductase, it was shown that a nucleophilic cysteine on the reductase attacks the esterl linkage between the amino acid and the tRNA to generate a thioester intermediate that is subsequently reduced by hydride transfer from NADPH to form GSA and release tRNAglu. GSA is unstable, with a half-life of about 4 min at physiological pH. The instability of this ALA intermediate is apparently resolved by substrate channeling between glutamyl-tRNA reductase and GSA aminotransferase. Molecular modeling shows that the aminotransferase dimer structure obtained from Synecococcus sp. fits well into the cavity created by the V-shaped architecture of the reductase. Moreover, the structures are compatible with a ternary structure that includes the substrate glutamyl-tRNAglu. In support of this model, glutamyl-tRNA reductase and GSA aminotransferase interact with each other in E. coli and in the alga Chlamodymonas reinhardtii.

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Glutamate-1-semialdehyde aminotransferase

GSA aminotransferase catalyzes the net transfer of the amino group from the 2-carbon to the 1-carbon position to form ALA (Figure 3). This enzyme is also referred to as GSA aminomutase, but the reaction involves intermolecular nitrogen transfer between two GSA molecules, and therefore it is not an aminomutase. The conversion of GSA to ALA occurs nonenzymatically in vitro at physiological pH, and probably does so in cells as well since an E. coli hemL mutant defective in GSA aminotransferase has a leaky growth phenotype, whereas gtrA/hemA mutants do not grow unless ALA is provided exogenously. GSA aminotransferse is a pyridoxal phosphate-dependent enzyme. The X-ray crystal structure of GSA aminotransferase from Synechococcus has been solved in the presence of the substrate analogue gabaculine, revealing that the active site includes residues from the N-terminal domain. The enzyme is a dimer with asymetry in pyridoxal phosphate cofactor binding and active site reactivity. Pyridoxal phosphate binds to Lys-273 of one of the subunits, and a region that is disordered on that subunit is ordered on the other. A separate study shows an active site gating loop in which conformational changes that are simultaneously open in one subunit and closed in the other, consistent with the asymmetry of the crystal structure, occur. Although the C5 pathway is not found in the -proteobacteria, many genomes from this taxonomic group have a gene annotated as GSA aminotransferase even though the glutamyl-tRNA reductase gene is absent. However, GSA aminotransferase is homologous to aminotransferases in general, which is the likely result of the misannotation. Furthermore, GSA aminotransferase activity could not be detected in extracts from the -proteobacteria B. japonicum or Azorhizobium caulinodans. 5-Aminolevulinic Acid Dehydratase (Porphobilinogen Synthase) ALA dehydratase catalyzes the asymmetric condensation of two ALA molecules to the monopyrrole porphobilinogen (Figure 4). There are two ALA binding sites, the so-called A and P sites corresponding to the ALA that contributes to the acetate and propionate portion of PBG, respectively. The P site ALA forms a Schiff’s base with a conserved lysine residue (Lys-247 in E. coli). Features of the A site probably differ among the different enzymes, depending on the metal requirement. The enzyme is a homooctomer in both eukaryotes and prokaryotes, and requires metal for activity. Three binding sites for divalent metals have been described. Animals and yeast have two zinc-binding sites (ZnA and ZnB), whereas prokaryotes have either ZnB- or no-zinc-binding site. ZnB is part of the active site proximal to the two lysine residues An allosteric magnesium site can also be found at the monomer–monomer interface of the bacterial and plant

enzymes. Furthermore, structures of Pseudomona aeruginosa ALA dehydratase bound to inhibitors make a very strong case for Mg2þ in the active site for Zn2þ-independent enzymes. B. japonicum ALA dehydratase is a Mg2þ-dependent enzyme. Amino acid substitutions that place three cysteines in positions normally found in the zinc enzymes of animal and yeast enzymes are sufficient to alter the metal specificity from Mg2þ to Zn2þ. Thus, it was possible to identify regions responsible for each metal in the respective enzymes. The modified enzyme also acquires sensitivity to lead, and has a pH optimum 3.5 units lower than in the wild type, as is observed for the naturally occurring Zn2þ ALA dehydratases. Similarly, the Zn2þindependent, Mg2þ-stimulated ALA synthase from P. aeruginosa was converted to a Zn2þ-dependent enzyme by introduction of cysteine residues corresponding to their positions in the Zn2þ-dependent enzyme from E. coli. In that study, a series of mutant combinations intermittent between the two types of enzymes were extensively characterized. These variants were similar to other naturally occurring ALA dehydratases from other organisms that vary in their dependence on Zn2þ or Mg2þ. Of the nine mutants constructed, five have no known natural equivalent, and four of those are inactive. Thus, a plausible evolution path of metal dependence could be deduced. Binding and kinetic studies indicate that B. japonicum ALA dehydratase contains four active site Mg2þ atoms per octomer, and eight allosteric Mg2þ ions. It is also stimulated by the monovalent cation Kþ. The Mg2þ enzyme from P. aeruginosa contains only four allosteric ions per octomer, but activity is not strictly metal-dependent. The crystal structure of the P. aeruginosa enzyme shows that it is composed of four asymmetric dimers. The monomers in each dimer differ by having a closed active site pocket shielded from the solvent by a ‘lid’ that is disordered in the open active site. The Mg2þ is 14 A˚ from the Schiff base lysine, which is too far to play a direct catalytic role. However, it is suggested that the open and closed forms of each monomer are governed by Mg2þ binding. Unlike the P. aeruginosa enzyme, E. coli ALA dehydratase is symmetric. Interestingly, crystal structures of P. aeruginosa mutants with cysteine substitutions show decreasing asymmetry with increasing cysteines. Although structural information on ALA dehydratases from many sources shows that the predominant form of the enzyme is a homo-octomer, alternate oligomeric states have been identified. Dr. Eileen Jaffe introduced the term morpheein to describe this phenomenon. A morpheein is a homo-oligomeric protein that exists in an equilibrium of functionally distinct, quaternary structural isoforms. The morpheein isoforms of human ALA dehydratase are an octomer, hexamer, and two different dimers. The interconversion of octomer to hexamer involves dissociation to dimer. The hexamer isoform was initially recognized in a rare human ALA dehydratase allele, whereby

Physiology | Heme Biosynthesis

rearrangement of the N-terminal arm caused by an F12L substitution is responsible for the oligomeric switch leading to a low-activity enzyme. The variant N-terminal arm structures vary among the morpheeins, and the relationship between the arm and the  -barrel domain dictates the isoforms. An R240A mutation in ALA dehydratase that affects this relationship stabilizes the hexamer, which can be converted to the octomer in the presence of substrate. The physiological relevance of these morpheeins is apparent in a study of ALA dehydratase mutants found in patients with ALAD porphyria. All eight known porphyria-associated variants shifted the morpheein isoform from the octomer toward the hexamer, which can explain the deficiency in those patients. Interestingly, ALA dehydratase from R. capsulatus is normally active as a hexamer and does not require any metal.

Porphobilinogen Deaminase (Hydroxymethylbilane Synthase) PBG is polymerized to the open-chain tetrapyrrole 1-hydroxymethylbilane by the enzyme PBG deaminase (Figure 4). The hemC gene encoding PBG deaminase is clustered with the hemD in many bacterial genomes. E. coli PBG deaminase is a 34 kDa monomer that has been crystallized and characterized. PBG deaminase has a three-domain structure; two of them resemble transferrins and periplasmic binding proteins. Interestingly, PBG is both a substrate and a component of the cofactor for PBG deaminase. The dipyrromethane cofactor is a PBG dimer covalently linked to Cys-242 (E. coli numbering) through its free amino group. This explains the deficiency of PBG deaminase activity in ALA synthase and ALA dehydratase mutants. The dipyrromethane cofactor in the active site primes the sequential addition and deamination of the PBG tetrapolymerization reaction. Thus, there is a transient PBG hexamer of which the hydroxymethylbilane tetrapyrrole is hydrolyzed, leaving the cofactor intact to prime the subsequent tetrapolymerization reaction. Polymerization terminates after four units because the size of the active site cavity sterically prevents additional PBG molecules, or else the chain is pulled through the active site. A deficiency in porphobilinogen deaminase in humans results in acute intermittent porphyria. The autosomal dominant phenotype is due to haploinsufficiency, but most heterozygotes do not show symptoms of the disease.

Uroporphyrinogen III Synthase Uroporphyrinogen III synthase catalyzes the cyclization of hydroxymethylbilane and inversion of the D ring to the asymmetric III isomer of uroporphyrinogen (Figure 4). Uroporphyrinogen III is the final common intermediate for all tetrapyrroles. Hydroxymethylbilane substrate is

201

unstable and is nonenzymatically cyclized to the I isomer of uroporphyrinogen. This nonphysiological isomer accumulates in uroporphyrinogen synthase mutants. Human uroporphyrinogen synthase is a monomeric bilobed structure in which the two domains are attached by a twostrand antiparallel -sheet. The few residues that are conserved in uroporphyrinogen synthases correspond to an area within a large open cleft between the two domains, which likely contains the active site. Mutation in any one of the conserved residues does not abolish activity of the human enzyme. The low sequence homology among uroporphyrinogen synthases has made it difficult to identify the gene in some of the genomes, which is further complicated by the fact that it is expressed as a fusion protein in some prokaryotes (see below). I searched for uroporphyrinogen synthases in the completed genomes available at the MicrobesOnLine website (www.microbesonline.org). Not all organisms synthesize tetrapyrroles, and so we searched for hemD homologues within the 336 genomes that contain a hemB homologue since its product, ALA dehydratase, has a high sequence conservation and should be found in the genomes of all organisms that synthesize tetrapyrroles. The initial homology search used the BLAST algorithm with uroporphyrinogen synthases from E. coli, B. subtilis, Chlorobium tepidum, and Staphylococcus aureus since the genes or enzymes have all been characterized to some extent, and the organisms are phylogenetically diverse. None of these searches found hemD homologues within the -proteobacteria, for which there are many representatives in the list of sequenced genomes. However, a putative uroporphyrinogen synthase gene from Mesorhizobium loti (mll4221) was then used in the search; the product of that gene has only modest homology to the E. coli enzyme, but it is downstream of hemC, as occurs in many prokaryotes. Despite using numerous proteins in the homology searches, we were unable to find a hemD homologue in 45 prokaryotic genomes from 33 different species and 22 genera. The list included numerous taxonomic groups within archaea and bacteria, and there is no obvious evolutionary theme. Even among eukaryotes, which generally contain less diversity compared with prokaryotes, uroporphyrinogen synthases are dissimilar. The uroporphyrinogen synthase gene of Arabidopsis was not obvious based on homology, but was ultimately identified by functional complementation of a yeast mutant. Similarly, the uroporphyrinogen synthase gene from the malaria parasite Plasmodium falciparum cannot be found based on sequence similarity to known genes. Additional X-ray crystal data on several uroporphyrinogen synthases will be needed to see whether the threedimensional structures of the disparate enzymes are similar to each other in spite of the low conservation at the primary sequence level. Although the bacterium Leptospira interrogans contains no HemD homologue, a variant of hydroxymethylbilane

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synthase encoded by hemC appears to have uroporhyrinogen synthase activity. This protein is similar to porphobilinogen deaminase at the N-terminal side, but has a C-terminal extension that has no homology to known proteins. The L. interrogans hemC gene is able to complement an E. coli hemD mutant. In addition to exhibiting structural diversity, numerous prokaryotic hemD genes are found as fusions. This was first characterized in the strict anaerobe Clostridium josui, in which a fusion gene of hemD with cobA was identified. The cobA gene encodes uroporphyrinogen III methyltransferase, an activity involved in cobalamin and siroheme synthesis. The cobA/hemD fusion can complement an E. coli hemD mutant; thus it has uroporphyrinogen III synthase activity. This gene fusion is observed in prokaryotes from numerous taxonomic groups, and thus it appears that this fusion is an old gene. The CobA (uroporphyrinogen III methyltransferase) protein is functionally and structurally homologous to the C-terminal portion of siroheme synthase (CysG), a multifunctional enzyme that catalyzes the methylation reaction of CobA in addition to catalyzing two additional reactions necessary for siroheme formation from uroporphyrinogen III. Interestingly, C. josui contains a hemA/cysG gene fusion in which the 59 portion of cysG is found. Thus, cysG sequence is separated in two sections that are fused to hemA and hemD. Accordingly, the gene cluster containing hemA/cysG-hemC-cobA/hemD-hemB can complement a cysG mutant of E. coli, strongly indicating that the cysG domains of each protein can collectively carry out the reactions normally carried out by an intact cysG product. The fusion of HemD with other proteins has confounded its genomic annotation, resulting in cobA or cysG being identified as hemD and vice versa. For this reason, one cannot rely on gene annotation alone in identification of hemD homologues. Uroporphyrinogen III Decarboxylase Uroporphyrinogen decarboxylase catalyzes the sequential decarboxylation of the four acetate residues of uroporphyrinogen III to methyl groups, yielding coproporphyrinogen III (Figure 4). It is also capable of decarboxylating the other nonphysiological isomers of uroporphyrinogen. At high substrate concentrations, the decarboxylation sequence is random, but occurs in a specific sequence physiologically, beginning with the acetate on ring D, followed by A, B, and finally C. Analysis of yeast uroporphyrinogen decarboxylase protein mutants suggests that all four carboxylations occur at a single active site. The X-ray crystal structure of the human recombinant decarboxylase dimer supports a single active site. The enzyme crystallized in the presence of its tetrapyrrole substrate shows that uroporphyrinogen is domed, and that pyrrole nitrogen atoms form hydrogen bonds with the carboxylate oxygen atom of the invariant

Asp86 residue. The central coordination geometry of Asp86 allows uroporphyrinogen and the partially decarboxylated intermediates to be bound with equivalent activating interactions, and thereby explains how all four acetate groups can be decarboxylated at the same catalytic center. The B. subtilis uroporphyrinogen decarboxylase structure is similar to that of the human enzyme with respect to overall folding and the active site cleft. In humans, a deficiency in uroporphyrinogen decarboxylase activity results in porphyria cutanea tarda (PCT). Only about one-third of PCT cases (familial PCT) are due to a mutation in the uroporphyrinogen decarboxylase gene. Recently, it was found that PCT in the remaining cases, called sporadic PCT, is due to, or contributed by, accumulation of porphomethene. Porphomethene is derived from partial oxidation of uroporphyrinogen, and is an inhibitor of decarboxylase, which explains diminished activity without a decrease in protein levels in sporadic PCT patients. The genetic and environmental factors responsible for generating porphomethene are not known, but it is proposed that CYP1A2, a cytochrome P450, is likely to have a central role because cyp1A2/ mice are resistant to regimens that induce PCT in wild-type mice. Coproporphyrinogen III Oxidase Coproporphyrinogen III oxidases catalyze the oxidative decarboxylation of the propionate groups at positions 2 and 4 of coproporphyrinogen III to vinyl groups to form protoporphyrinogen IX (Figure 4). This reaction is carried out by an oxygen-dependent or -independent mechanism catalyzed by two structurally dissimilar enzymes. Eukaryotes possess only the oxygen-dependent oxidase, and our understanding of that enzyme is primarily from those sources. In prokaryotes, oxygen-dependent coproporphyrinogen oxidase is encoded by the hemF gene. Examination of genome data using online software (www.microbesonline.org) shows that hemF is found in numerous Gram-negative bacteria and cyanobacteria, and in a few species from other taxonomic groups such as the Bacteroidetes. This search did not find homologues in Gram-positive bacteria or in archaea. E. coli HemF is a dimer, with a strict dependence on O2 for catalysis, which produces H2O2. Metal chelators abolish activity, which is restored with Mg2þ. Metal binding likely involves four conserved histidines on HemF based on mutant analysis. Heme and oxygen are positively correlated with each other in eukaryotes since both are required for respiration and oxygen metabolism. Indeed, in the yeast Saccharyomyces cerevisiae, control of genes for oxygen is thought to be sensed through heme biosynthesis via this O2-dependent step of the pathway. Therefore, O2 requirement for a heme biosynthetic step poses no problems, and in fact has a regulatory advantage. Many prokaryotes, however, can carry out respiratory metabolism using nitrate, sulfate, or

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other electron acceptors in place of O2. Thus, hypoxia or anaerobiosis is not necessarily associated with fermentative metabolism, and the demand for heme persists and may actually increase. Similarly, hypoxia is required for induction of chlorophyll synthesis for photosynthesis in bacteria such as Rhodobacter, which requires coproporphyrinogen oxidase activity. This problem is resolved by the expression of an O2-independent coproporphyrinogen oxidase in prokaryotes encoded by the hemN gene. Unlike hemF, hemN is nearly ubiquitous in bacteria and archaea that synthesize heme, and numerous bacteria contain both hemF and hemN. The O2-independent coproporphyrinogen oxidase was originally identified based on a R. sphaeroides mutant that could not synthesize bacteriochlorophyll but accumulated coproporphyrin. The subsequent identification of hemN genes in S. typhimurium and E. coli demonstrated that the O2-independent oxidase is not dedicated to chlorophyll synthesis. In S. typhimurium, which contains both hemF and hemN, a mutation in hemF shows no growth impairment whereas a hemN mutant is a heme auxotroph only under anaerobic growth. Similarly, a hemF hemN double mutant can be complemented by hemF only for aerobic growth, but hemN confers normal aerobic or anaerobic growth. Thus, the O2-independent coproporphyrinogen oxidase can function in the presence of O2, but does not use it for catalysis. An O2-independent coproporphyrinogen oxidase activity that apparently uses NADPþ in the presence of ATP and S-adenosylmethionine (SAM) has been detected in extracts of R. sphaeroide and B. japonicum. However, that activity cannot be detected from the S. typhimurium hemN product. In B. japonicum, hemN2 is required for anaerobic growth and symbiosis with soybean, whereas a hemN1 mutant has no phenotype. Furthermore, the B. japonicum hemN2, but not hemN1, was able to complement a S. typhimurium hemF hemN strain, indicating that the hemN1 product may not be an active protein. Similarly, a mutation in only one of two hemN homologues of B. subtilis affected aerobic and anaerobic growth. Dieter Jahn and colleagues have carried out detailed structural and enzymatic studies of the oxygen-independent coproprophyrinogen oxidase HemN. HemN is a monomer belonging to the S-adenosyl-L-methionine (AdoMet) radical protein family. HemN and other members of this family contain an iron–sulfur cluster coordinated by cysteine residues in the conserved context CXXXCXXC. In HemN, the iron–sulfur cluster is also coordinated by carboxyl and amino groups of AdoMet, and it uses AdoMet as a cofactor to initiate radical-based catalysis. Unlike other AdoMet radical members, HemN binds a second AdoMet molecule proximal to the first. This may allow decarboxylation of the second propionate of the substrate without rotating it after the first decarboxylation reaction. Although

a potential site for an electron acceptor can be surmised from the structure of HemN, the physiological acceptor remains unknown.

Protoporphyrinogen IX Oxidase Protoporphyrinogen oxidase catalyzes the six-electron oxidation of protoporphyrinogen to protoporphyrin in the penultimate step of the heme pathway, and the final common step of heme and chlorophyll formation (Figure 4). This oxidation results in conjugated double bonds in the tetrapyrrole macrocycle that makes protoporphyrin, hemes, and chlorophylls chromophoric. A protoporphrinogen oxidase deficiency is the genetic basis for variegate porphyria in humans. In plants, protoporphyrinogen oxidase is the target of diphenyl ether herbicides such as acifluorfen-methyl. The phytotoxicity of these herbicides is based on the accumulation of protoporphyrinogen, which diffuses from the chloroplast to other parts of the cell where it is then oxidized nonenzymatically to generate reactive oxygen species (ROS), thereby damaging cellular components. The deleterious effects of protoporphyrin deficiency in animals and plants underscores the physiological need for an enzyme to catalyze protoporphyrinogen oxidation despite the fact that the reduced porphyrinogen can be readily oxidized nonenzymatically. In strictly aerobic organisms, molecular oxygen is the electron acceptor in protoporphyrinogen oxidation, producing H2O2. In anaerobes or facultative aerobes, another electron acceptor is involved, and oxidation may be linked to the respiratory chain. Although unproven, this link may explain the accumulation of porphyrins and deficiency in protoporphyrinogen oxidase activity of mutants defective in cytochrome c biogenesis. The purified enzyme from the anaerobic bacterium Desulfovibrio gigas is composed of three dissimilar subunits; it cannot use molecular oxygen, but does use the artificial electron acceptor 2,6-dichlorophenol-indophenol in vitro. The molecular characterization of bacterial protoporphyrinogen oxidase has focused on two dissimilar genes, hemG and hemY, and their protein products. E. coli hemG gene locus was identified by analysis of a mutant that accumulates protoporphyrinogen and is defective in protoporphyrinogen oxidase activity. The hemG gene expressed in trans from a plasmid complements a hemG mutant with respect to activity in crude extract and membrane preparations. Enzyme activity of the purified 21 kDa product has not been demonstrated; therefore hemG may not be sufficient for protoporphyrinogen oxidation, and hemG could encode a subunit of a larger complex. The apparent requirement for electron transport in protoporphyrinogen oxidation in E. coli may make it difficult to unambiguously characterize the hemG product in pure form. In fact, direct evidence

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that the hemG product is a protoporphyrinogen oxidase protein is absent. HemY is a bacterial oxygen-dependent protoporphyrinogen oxidase and shares weak to moderate similarity to the eukaryotic enzyme. The enzyme from the Myxococcus xanthus is a membrane-bound dimer, contains FAD, and is inhibited by acifluorifen, as is found for the eukaryotic enzyme. The crystal structure of that enzyme has been elucidated. Although protoporphyrinogen oxidases show low overall homology, a conserved residue corresponding to Arg95 in M. xanthus is in the active site and likely interacts with ring C of the substrate. A similar situation is observed for the mitochondrial enzyme. Modeling protoporphyrinogen within the active site indicates that the substrate cannot rotate, indicating that all six electrons are lost from a single point in the macrocycle. HemY from B. subtilis differs substantially from the M. xanthus enzyme. It is a soluble monomer that is resistant to diphenyl ether herbicides. Rice plants that express the B. subtilis hemY gene are resistant to the diphenyl ether herbicide oxyfluorfen, with protein targeted at plastids showing a greater resistance than those targeted at the cytoplasm. However, the effect of the transgene may be due to a high level of enzyme activity in those plants rather than to the resistance of the protein per se since transgenic Arabidopsis plants that overexpress the tobacco protoporphyrinogen oxidase are also herbicide resistant. In addition, B. subtilis HemY can oxidize coproporphyrinogen as well as protoporphyrinogen, but there is no evidence that this occurs in cells. Many prokaryotic genomes lack an identifiable protoporphyrinogen oxidase gene homologue

The hemK gene was originally identified as encoding a protoporphyrinogen oxidase, and some genomes still misannotate hemK that way. However, it is now known that hemK encodes an N5 glutamine methyltransferase that modifies peptide release factors. The hemK gene is fairly ubiquitous in prokaryotes, which masked the fact that many prokaryotic genomes contain neither a hemG nor a hemY homologue. I reexamined this issue by carrying out similarity searches for HemG or HemY homologues in the complete genomes available at the MicrobesOnline website (www.microbesonline.org). Some organisms do not synthesize tetrapyrroles, and others synthesize tetrapyrroles but not heme, and therefore protoporphyrinogen oxidase gene homologues are not expected to be found in those organisms. In the former case, one would not expect to find any genes encoding heme synthesis enzymes, whereas in the latter case, genes encoding the early steps through uroporphyrinogen III synthesis for the formation of tetrapyrroles derived from precorrin 2 would be expected (see Figure 1). In order to search for hemY or hemG homologues specifically in organisms where a protoporphyrinogen oxidase gene is expected, the search

was limited to genomes that contain the ferrochelataseencoding gene hemH. I found 296 genomes representing 206 different species. The hemG gene has been characterized in E. coli, and therefore its gene product, , as well as the homologue from M. loti, which has not been characterized, was used in a BLAST search. For hemY, I used the protein from B. subtilis and M. xanthus, since they have been characterized and confirmed to be bona fide protoporphyrinogen oxidases. The distributions of hemG and hemY are somewhat skewed by the genomes chosen for sequencing, and not all taxonomic groups are equally represented. Nevertheless, patterns and trends can be ascertained. A hemG gene was identified in 43 genomes, 36 of them are within the -proteobacteria to which E. coli belongs. Thus, hemG is phylogenetically restricted. The other homologues are found in two cyanobacterial species, three -proteobacteria, and one each in a -proteobacterium and a -proteobacterium. The taxonomic distribution of hemY found in 89 genomes is more diverse than hemG, but there is a concentration in the Gram-positive groups, particularly the Actinobacteria, Chlaymidiae, and the Firmicutes. It is notable that, with two possible exceptions, hemG and hemY are not found in the same genome. Although HemG has not been rigorously shown to be a protoporphyrinogen oxidase (see above), the lack of overlap with HemY further supports the conclusion that it is involved in protoporphyrinogen oxidation. Among the 296 genomes examined, 160 of them lack a hemY or hemG homologue, which means that the penultimate step in heme biosynthesis is largely not understood in prokaryotes. Although whole genome sequences have been determined in many organisms in which little other biological information is available, it should be noted that a protoporphyrinogen oxidase gene has not been identified in many well-studied prokaryotes, including P. aeruginosa, Neisseria meningitidis, Helicobacter pylori, and the rhizobia. Furthermore, protoporphyrinogen oxidation is also required for chlorophyll synthesis, and the enzyme catalyzing that reaction in most photosynthetic cyanobacteria remains to be determined. The lack of evolutionary conservation of a protein to catalyze this step of the pathway suggests that the extant mechanism of protoporphyrinogen oxidation is newer than the other steps of the pathway. This oxidation can proceed nonenzymatically in vitro, formally raising the possibility that this enzyme is not required in organisms where the gene has not been found. However, the gene is essential for heme synthesis in E. coli and B. subtilis as seen by the hemedefective phenotypes and the accumulation of heme precursors. It seems more likely that a heretofore unidentified protoporphyrinogen oxidase exists in prokaryotes, or else a known protein has an additional activity. The oxygendependent coproporphyrinogen oxidase of E. coli (hemF) has protoporphyrinogen oxidase activity, and a secondary

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site suppressor mutant of a hemG strain has elevated hemF promoter activity. Furthermore, overexpression of hemF rescues a hemG mutant. However, HemF is taxononomically limited, and would account for the dearth of protoporphyrinogen oxidase in many prokaryotes. Ferrochelatase Ferrochelatase, encoded by the hemH gene in prokaryotes, catalyzes the insertion of ferrous iron into the protoporphyrin IX macrocycle to yield protoheme (Figure 4). Ferrochelatases are conserved among prokaryotes and eukaryotes, but few amino acid residues are strictly conserved in all of them. Interestingly, porphyrin metallation can be catalyzed by catalytic antibodies, DNA, and RNA. Animal ferrochelatases are [2Fe–2S] iron–sulfur proteins. Three of the four cysteines that coordinate the [2Fe–2S] cluster are found in the C-terminus; these cysteines are absent in plant ferrochelatases, and most prokaryotic ferrochelatases lack the C-terminus altogether, and are not iron– sulfur proteins. However, several species do have the C-terminus with atypical cysteine spacing for iron–sulfur proteins, but do contain a [2Fe–2S] cluster nevertheless. It is interesting to note that the latter group was characterized in the enzymes from Caulobacter crescentus and Mycobacterium tuberculosis, which are not only phylogenetically distant, but differ in cell localization and oligomerization. More recently, a new class of bacterial ferrochelatases that are iron–sulfur proteins, but do not contain a C-terminal extension, was identified. Rather, the liganding cysteines are within the common portion of the protein. Like the eukaryotic ferrochelatase, mutation of a liganding cysteine results in the loss of the iron–sulfur cluster. However, unlike the former, the mutant bacterial chelatase does not lose enzymatic activity. Finally, the ferrochelatase from the photosynthetic bacterium Synechocystis strain PCC6803 has a plant-like C-terminal extension necessary for dimerization and normal functioning in vivo, even though a mutant lacking the extension can complement an E. coli hemH strain. Eukaryotic ferrochelatase is membrane-associated in mitochondria and in chloroplasts of plants, and is associated with membranes in some bacteria. However, the enzyme from B. subtilis and M. tuberculosis is soluble. This is an interesting feature that needs to be reconciled with the lipophilic nature of the substrate and product of ferrochelatase. The X-ray crystal structures of human, Saccharomyces cereviciae, and B. subtilis ferrochelatases have been solved. The eukaryotic enzyme is a homodimer that is stabilized by interactions between the C-terminal extensions of the monomers. Accordingly, the B. subtilis ferrochelatase is a monomer and lacks that extension. A C-terminal extension per se does not govern the oligomeric state since the M. tuberculosis ferrochelatase is a monomer, whereas the C. crescentus enzyme is a dimer even though both enzymes have the extension. A conserved serine (S54 in B. subtilis)

sticks out from the enzyme, and substitution of that residue for alanine does not affect enzyme activity in vitro, but a mutant strain harboring that variant shows diminished growth and accumulation of oxidized heme precursors. A role for this part of the protein in substrate channeling has been suggested. Although the crystal structures of human and B. subtilis ferrochelatases share numerous similarities, important differences were reported with respect to substrate binding. The human enzyme clamps down on protoporphyrin IX so that the substrate is completely engulfed in the pocket. The substrate is also deeper in the active site and rotated 100 relative to the B. subtilis enzyme bound to N-methyl mesoporphyrin. Finally, the porphyrin substrate bends about 30 in the bacterial enzyme, but shows less distortion in the active site of human ferrochelatase. Iron in the environment is primarily in the ferric form, and thus it must be reduced prior to chelation. Other divalent ions such as Co2þ and Zn2þ can serve as a substrate in vitro whereas others such as Mn2þ, Cd2þ, and Pb2þ are competitive inhibitors. Mutational and physical analyses of ferrochelatases from several sources identified an invariant histidine in substrate metal binding, and this has been borne out in metallated crystal structures. B. subtilis ferrochelatase crystallized with the natural metal substrate Fe2þ reveals the conserved histidine (H183) and a conserved glutamate (E264) as the only liganding amino acids, along with three water molecules. A regulatory Mg2þ-binding site has also been implicated. Ferrochelatase is functionally similar to magnesium chelatase, the enzyme that inserts Mg2þ into protoporphyrin in the first committed step in chlorophyll (and bacteriochlorophyll) formation. However, the enzymology of the two proteins is quite different. Ferrochelatase is a monomer or homodimer, whereas Mg-chelatase consists of three distinct subunits. Furthermore, ferrochelatase activity requires no ATP, in contrast to Mg-chelatase that requires ATP in a two-step activation and chelation process. The basis of metal specificity of the various types of chelatases is unclear. However, it has been proposed that chelatase-induced distortion of the porphyrin increases the reaction rate not only by lowering the activation energy of the reaction, but also by modulating which divalent metal is incorporated into the substrate.

Regulation of Heme Biosynthesis Regulation of Heme Biosynthesis by Iron Ferrous iron is inserted into protoporphyrin IX in the final step of heme biosynthesis catalyzed by ferrochelatase. Iron can be a limiting nutrient, and therefore coordination of heme biosynthesis with iron availability prevents porphyrin synthesis from exceeding iron availability. In B. japonicum, the iron response regulator (Irr) protein

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coordinates the heme biosynthetic pathway with iron availability to prevent the accumulation of toxic porphyrin precursors under iron limitation. Loss of function of the irr gene is sufficient to uncouple the pathway from irondependent control, as discerned by the accumulation of protoporphyrin under iron limitation. Irr accumulates to a high level in iron-limited cells to negatively regulate heme biosynthesis at hemB, the gene encoding the heme biosynthetic enzyme ALA dehydratase. Irr is a conditionally stable protein that degrades in cells exposed to iron, allowing derepression of heme biosynthesis. Iron-dependent degradation of Irr is mediated by heme. Heme binds directly to Irr at a heme regulatory motif (HRM), which is necessary for its degradation. Accordingly, Irr persists in heme synthesis mutant strains in the presence of iron, and a mutation in the HRM stabilizes Irr in the presence of the metal. Thus, heme is an effector molecule in Irr degradation that reflects the availability of iron for heme synthesis. Heme-mediated protein degradation has also been described in several eukaryotic systems. Degradation of Irr by heme is caused by redox activity of heme, which likely results in the reduction of O2 to ROS, thereby oxidizing Irr. Irr has different binding sites for oxidized and reduced forms of heme; mutation of either site stabilizes the protein in the presence of iron. Ferrochelatase catalyzes the insertion of iron into protoporphyrin to form heme in the final step of the heme biosynthetic pathway. Irr interacts directly with ferrochelatase and responds to iron via the status of heme and protoporphyrin localized at the site of heme synthesis. In the presence of iron, ferrochelatase inactivates Irr, followed by Irr degradation to derepress the pathway. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. Thus, metabolic control of the heme pathway involves the regulatory function of a biosynthetic enzyme that affects gene expression. Furthermore, heme can serve as a signaling molecule without accumulating freely in cells. The latter point is important because heme is lipophilic and toxic, and a free heme pool that is often invoked to explain the regulator effects of heme is not tenable in our view. This appears to be the only example in which a regulatory role for heme can be reconciled with its cellular toxicity. Although the regulatory input to Irr is the status of heme biosynthesis, Irr is now known to be a global regulator of iron homeostasis. Irr is both a positive and negative effector of gene expression, and in at least some cases the control is direct. Loss of normal iron responsiveness of those genes in an irr mutant, as well as a lower total cellular iron content, suggests that Irr is required for the correct perception of the cellular iron status. Accordingly, control of Irr-regulated genes by iron is also aberrant in a heme-defective strain, and iron-replete mutant cells

behave as if they are iron-limited. Thus, B. japonicum senses iron via the status of heme biosynthesis in an Irr-dependent manner to regulate iron homeostasis and metabolism. This differs from other bacterial systems where iron is sensed directly through the Fur protein. Regulation of Heme Biosynthesis by Heme Heme is the end product of its biosynthetic pathway, and thus regulation by heme makes sense. Even though there are several interesting regulatory mechanisms that coordinate heme biosynthesis as described below, there is no mechanism that is known to be generally applicable to many prokaryotic systems. Heme serves as a signaling molecule in Irr degradation for iron control of heme synthesis in B. japonicum, as described above, and allows coordination with heme transport, degradation, and other facets of iron metabolism. However, heme likely reflects the availability of iron for heme synthesis, which is probably not an autoregulatory mechanism. Nevertheless, control of heme synthesis by heme at the level of transcription, protein stability, and allosteric inhibition have been described. The gtrA (hemA) gene from Corynebacterium diphtheriae is negatively regulated by heme through the actions of the two-component regulatory systems ChrA-ChrS and HrrA-HrrS. These regulatory systems were originally discovered in studies to elucidate the mechanism of positive control of the heme oxygenase (hmuO) gene by heme and hemoglobin. Heme oxygenase catalyzes the degradation of heme for use as an iron source, and positive control of hmuO by heme makes physiological sense. ChrA is a response regulator and ChrS is the sensor histidine kinase that responds to the heme status. Mutations in genes encoding those proteins diminish, but do not abolish, activity, which led to the search and discovery of HrrA and HrrS. Loss of function of both systems abolishes control of hmuO and hemA. These findings show that heme biosynthesis and its subsequent metabolism are coordinately controlled. Although mechanistically different, this story is similar to Irr’s, where a regulator of heme biosynthesis also controls other types of heme metabolism to coordinate numerous cellular processes. R. capsulatus is a facultative phototrophic bacterium, and thus it synthesizes both heme and chlorophyll. Numerous tetrapyrrole synthesis genes are negatively regulated by exogenous heme in that organism by HbrL, a LysR-type transcriptional regulator. HbrL binds to the promoters of hemA, hemB, and hemZ (encoding an O2-independent coproporphyrinogen oxidase) and is enhanced in the presence of heme. In S. typhimurium, there is excellent evidence that heme regulates its own synthesis at the level of stability of the first committed step catalyzed by glutamyl-tRNA reductase (GTR). In experiments where heme content was

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controlled by exogenous supplementation in heme synthesis mutants, GTR levels were increased up to 25-fold in heme-starved cells, with only a twofold increase in gtr (hemA) promoter activity. The reductase is a conditionally stable protein with a half-life of about 20 min in the presence of heme, which increases to greater than 5 h under heme starvation. Degradation was energydependent and was blocked in mutants defective in the proteolytic enzymes Lon or ClpAP. A GTR-LacZ fusion shows heme-dependent degradation as in native protein, and the N-terminal 18 amino acids of GTR are sufficient to destabilize LacZ as well. However, degradation of a fusion containing only the N-terminal 18 amino acids is no longer heme-dependent. It is suggested that the 18-amino acid degradation tag is accessible to protease digestion when GTR is bound to heme, but is always accessible in a GTR(1–18)-LacZ fusion. Substitution of two adjacent leucine residues for lysine in the degradation tag of GTR abrogates heme-dependent turnover. Heme inhibits GTR activity in cell extracts from several bacterial species by an allosteric feedback mechanism. Recombinant GTR from Chlorobium vibrioforme is a homodimer that is purified from E. coli cells containing one tightly bound heme per monomer. This preparation was not inhibited by heme in vitro, but GTR purified from cells grown in the presence of the heme biosynthesis inhibitor gabaculine contained less heme, which was inhibited by exogenous heme in vitro. A Synechocystis PCC6803 strain with diminished ferrochelatase activity accumulates protoporphyrin and has elevated ALA synthesis activity. This mutant grows normally and synthesizes phycobilins that are derived from heme. It is likely that the diminution in heme synthesis in the mutant is not severe enough to limit heme for most cell functions, but is below a level needed to allosterically inhibit ALA synthesis, presumably at GTR. Regulation of Heme Biosynthesis by Oxygen Oxygen can be either a positive or a negative effector in heme-dependent processes. The effect of O2 may be direct, or it may be the indirect effects of O2 metabolism that produce H2O2 and other reactive forms of oxygen. Control by reactive oxygen species

The generation of ROS is a natural consequence of aerobic respiration as a result of the partial reduction of molecular oxygen and the subsequent reactions of those products with transition metals and other compounds. The environment too can be a source of ROS given the permeability of bacterial membranes to hydrogen peroxide. Superoxide (O2 – ) and hydrogen peroxide (H2O2), the initial partial reduction products of oxygen, can damage cell components but only to a limited extent. However, O2 – is capable of destroying exposed

iron–sulfur clusters with the release of free iron and the released iron subsequently reacts with H2O2 via the Fenton reaction, generating extremely reactive hydroxyl radicals (HO). Hydroxyl radicals can directly attack most macromolecules such as DNA, lipids, and proteins, which is the basis of oxygen toxicity. Bacteria have multiple defense strategies against oxidative stress, including the direct detoxification of ROS by catalase, peroxidases, and superoxide dismutase. Oxidative stress responses require the activation of regulatory proteins and the induction of genes under their control. In many bacteria, the transcriptional regulator OxyR senses hydrogen peroxide and induces numerous genes whose products are involved in peroxide defense, redox balance, and other factors. In B. subtilis, PerR is the major peroxide regulator and represses a large PerR regulon. The OhrR family of antioxidant regulators is responsible for organic hydroperoxide resistance. Catalases and peroxidases are heme proteins that detoxify H2O2 and peroxides, respectively. Regulation of genes encoding heme biosynthesis enzymes in response to oxidative stress has been reported in several bacteria, and this is presumably due to a need for heme for detoxification, although that causal relationship has not been established. The B. subtilis PerR protein mediates the induction of hemAXCDBL operon encoding enzymes for the early steps of heme synthesis. In E. coli and Salmonella, the hemH gene encoding the heme biosynthetic enzyme ferrochelatase is induced in response to H2O2 in an OxyR-dependent manner. In Salmonella, a mutant defective in the gene encoding the heme synthesis enzyme glutamyl-tRNA reductase is hypersensitive to H2O2. However, this was attributed to an increase in reducing equivalents to drive the Fenton reaction rather than to a decrease in catalase activity. In B. japonicum, H2O2 promotes the degradation of Irr in the presence of iron, which derepresses hemB expression. Control by O2 limitation

In animals and fungi, hypoxia or anaerobiosis is associated with fermentative metabolism since oxidative phosphorylation is strictly dependent on O2. Thus, oxygen and heme are functionally correlated. Furthermore, two heme biosynthetic enzymes, coproporphyrinogen III oxidase and protoporphyrinogen IX oxidase, use O2 as a substrate. In fact, O2 control of genes involved in aerobic metabolism occurs indirectly through heme in S. cereviciae. For many prokaryotes, however, this correlation is not observed because they can use electron acceptors other than O2 to support oxidative respiration. In addition, some bacteria have cytochrome oxidases with a very high affinity for O2, conferring on them the ability to grow under iron limitation. Thus hypoxia or anaerobiosis does not necessarily trigger a switch to fermentative metabolism, and may actually

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require an increase in tetrapyrrole synthesis. Also, low O2 is a cue to induce photosynthesis-related genes in bacteria. Photosynthesis requires tetrapyrrole synthesis enzymes to make chlorophyll, and also to make the heme prosthetic group of electron transfer components. As stated above, bacteria that respire in the absence of O2 have an O2independent coproporphyrinogen III oxidase and (presumably) protoporphyrinogen oxidase not found in eukaryotes. The gtrA (hemA) gene of E. coli is modestly induced under anaerobic growth via the transcriptional regulators Fnr and ArcA, which act as negative and positive regulators, respectively. However, in some other organisms, O2 status is a major regulatory factor in control of tetrapyrrole biosynthesis. Regulation of tetrapyrrole synthesis genes by oxygen in R. sphaeroides and R. capsulatus is under numerous regulatory circuits for photosynthetic and anaerobic metabolism. The fnrL locus was identified in a mutant screen for elevated hemA (ALA synthase) gene expression under aerobic conditions, and is required for photosynthetic growth and anaerobic growth with dimethylsulfoxide. FnrL binds to both promoters of hemA for anaerobic induction of the gene. Morover, FnrL affects numerous other genes in heme biosynthesis in both R. sphaeroides and R. capsulatus. In addition to FnrL, the two-component response regulator PrrA (RegA) has been shown to control heme synthesis gene expression in Rhodobacter. PrrA/RegA responds to the cellular redox state to control anaerobic metabolism in numerous bacterial species. PrrA binds to both promoters of R. sphaeroides hemA under aerobic conditions, but anaerobic activation requires binding to the P2 promoter. The PpsR/CrtJ is a repressor of photosystem development under aerobic conditions, and this transcriptional regulator also controls numerous heme synthesis genes. R. sphaeroides contains two hemN gene homologues encoding O2-independent coproporphyrinogen oxidase designated hemN and hemZ. hemN is negatively regulated by the outer membrane protein TspO, apparently by a posttranscriptional control mechanism. TspO negatively regulates aerobic production of carotenoid and bacteriochlorophyll. . Introduction of multiple copies of hemN in trans induces the formation of bacteriochlorophyll, a phenotype similar to a tspO mutant. The two coproporphyrinogen oxidase isozymes must have different functions because expression of hemZ in trans did not alter gene expression. It was proposed that TspO attenuates HemN activity resulting in the accumulation of a tetrapyrrole precursor, which in turn acts as a corepressor of bacteriochlorophyll and carotenoid gene expression. Rhizobia can establish symbiosis with leguminous plants, which culminates with the development of a specialized plant organ called a root nodule. The bacteria convert atmospheric nitrogen to ammonia to fulfill the nutritional nitrogen requirement of the plant host. The oxygen tension within a nodule is very low, and this serves as a signal

for the induction of genes necessary for nitrogen fixation and microaerobic metabolism. De novo heme biosynthesis for synthesis of cytochrome cbb3 oxidase and other cytochromes is required for microaerobic metabolism, hence a need to control heme biosynthesis. The RegRS system in B. japonicum is homologous to the RegAB/PrrAB proteins in Rhodobacter. Although heme synthesis is regulated by O2 in B. japonicum, and is phylogenetically related to Rhodobacter, the RegR regulon does not appear to include heme synthesis genes as judged from transcriptional profiling data. Rather, O2 control of heme synthesis and cytochrome production occurs by a regulatory cascade initiated by another two-component regulatory system, FixLJ. FixL senses O2 by binding directly to the protein via a heme moiety. Deoxygenated FixL phosphorylates FixJ to activate target genes, including fixK2, encoding An Fnr family regulator. FixK2 positively controls hemA, hemB, and hemN, and is also needed to down regulate cyotchrome aa3 levels, which are not needed in microaerobic nodules. B. japonicum has two hemN genes, designated hemN1 and hemN2. Neither gene is expressed aerobically, but both are strongly induced under microaerobic or anaerobic conditions. A hemN1 mutant does not have a discernible phenotype, but, a hemN2 mutant is unable to grow anaerobically on nitrate and does not elicit functional nodules on its plant host. Furthermore, hemN2, but not hemN1, was capable of complementing a S. typhimurium hemF hemN double mutant in trans, indicating that hemN1 does not encode a functional protein. The presence of multiple hemN homologues and control by Fnr-like proteins is not confined to the -proteobacteria Rhodobacter and Rhizobium. A hemN gene from P. aeruginosa is under control of two Fnr-type regulators Dnr and Anr, both of which are needed for anaerobic induction. The hemN gene in that organism is also expressed aerobically, which is dependent only on Anr. Surprisingly, the hemF gene encoding the O2-dependent oxidase is also strongly induced by anaerobiosis, which is mediated by both Anr and Dnr, which if fully expressed suggests an alternative function for that enzyme.

Alternative Heme Biosynthetic Pathways The absence of identifiable heme synthesis genes in an organism known to contain heme can suggest the possibility of an alternative synthetic pathway. In the cases where a single enzyme cannot be identified by homology (see above), it is likely that a similar reaction is carried out by a structurally disparate enzyme since both the substrate and product of the missing step can be accounted for. However, genome sequencing projects reveal cases where numerous enzymes catalyzing successive steps of heme formation cannot be found in organisms that express heme enzymes. A comparative genome analysis

Physiology | Heme Biosynthesis

revealed that numerous archaea are known or predicted to contain heme proteins, but lack the genes encoding enzymes of the latter part of the pathway that synthesize heme from uroporphyrinogen III. There is ample evidence for heme synthesis from uroporphyrinogen III that involves methylation reactions using S-adenosylmethionine as the methyl donor in the bacterium Desulfovibrio vulgaris and in the archaeon Methanosarcina barkeri. In the conventional pathway, the four methyl groups of the porphyrin ring are derived from decarboxylation of the acetate groups of uroporphyrinogen III to yield coproporphyrinogen III via uroporphyrinogen III decarboxylase. Thus, the methyl groups and all carbon atoms are derived from ALA. In the alternate scheme, it is proposed that uroporphyrinogen III is first methylated at positions C-1 and C3 (Fischer numbering system) to yield precorrin 2, a precursor for siroheme and vitamin B12. The methyl donor is SAM. The acetate groups at those carbon positions are removed and the remaining acetates are decarboxylated to methyl groups, yielding coproporphyrinogen III. Thus, the methyl groups at positions 5 and 8 are derived from ALA, but the 1 and 3 methyl groups originate from SAM. The enzymes that carry out these steps have not been identified. Furthermore, the subsequent steps from coproporphyrinogen III to heme have not been characterized, but require decarboxylation, oxidation, and iron chelation. The pathway described in D. vulgaris is not generally applicable to anaerobic tetrapyrrole synthesis however. Bollivar and colleagues showed that the methyl groups of protoporphyrin synthesized anaerobically in the strict anaerobe Chlorobium vibrioforme or in the facultative anaerobe R. sphaeroides are derived from ALA and not from L-methionine. Furthermore, a S. typhimurium cysG mutant that cannot make precorrin 2 is capable of synthesizing heme anaerobically. See also: Coenzyme and Prosthetic Group Biosynthesis; Energy Transduction Processes; Methanogenesis; Pigments, Microbial

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Further Reading Al-Karadaghi S, Franco R, Hansson M, Shelnutt JA, Isaya G, and Ferreira GC (2006) Chelatases: Distort to select? Trends in Biochemical Sciences 31: 135–142. Breinig S, Kervinen J, Stith L, et al. (2003) Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase. Nature Structural Biology 10: 757–763. Buchenau B, Kahnt J, Heinemann IU, Jahn D, and Thauer RK (2006) Heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions. Journal of Bacteriology 188: 8666–8668. Dailey HA, Dailey TA, Wu CK, et al. (2000) Ferrochelatase at the millennium: Structures, mechanisms and [2Fe-2S] clusters. Cellular and Molecular Life Sciences 57: 1909–1926. Erskine PT, Senior N, Awan S, et al. (1997) X-ray structure of 5-aminolaevulinate dehydratase, a hydrid aldolase. Nature Structural Biology 4: 1025–1031. Koch M, Breithaupt C, Kiefersauer R, Freigang J, Huber R, and Messerschmidt A (2004) Crystal structure of protoporphyrinogen IX oxidase: A key enzyme in haem and chlorophyll biosynthesis. The EMBO Journal 23: 1720–1728. Layer G, Moser J, Heinz DW, Jahn D, and Schubert WD (2003) Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes. The EMBO Journal 22: 6214–6224. Luer C, Schauer S, Mobius K, et al. (2005) Complex formation between glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1aminomutase in Escherichia coli during the initial reactions of porphyrin biosynthesis. The Journal of Biological Chemistry 280: 18568–18572. Mense SM and Zhang L (2006) Heme: A versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Research 16: 681–692. O’Brian MR and Tho¨ny-Meyer L (2002) Biochemistry, regulation and genomics of haem biosynthesis in prokaryotes. Advances in Microbial Physiology 46: 257–318. Phillips JD, Bergonia HA, Reilly CA, Franklin MR, and Kushner JP (2007) A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda. Proceedings of the National Academy of Sciences of the United States of America 104: 5079–5084. Phillips JD, Whitby FG, Kushner JP, and Hill CP (2003) Structural basis for tetrapyrrole coordination by uroporphyrinogen decarboxylase. The EMBO Journal 22: 6225–6233. Qi Z and O’Brian MR (2002) Interaction between the bacterial iron response regulator and ferrochelatase mediates genetic control of heme biosynthesis. Molecular Cell 9: 155–162.

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