Step Of Heme Biosynthesis Research Articles

Indispensable function for embryogenesis, expression and regulation of the nonspecific form of the 5‐aminolevulinate synthase gene in mouse

The first step of heme biosynthesis in animals is catalyzed by 5-aminolevulinate synthase (ALAS), which controls heme supply in various tissues. To clarify the roles that the nonspecific isoform of ALAS (ALAS-N) plays in vivo, we prepared a green fluorescent protein (GFP) knock-in mouse line in which the Alas1 gene (encoding ALAS-N) is replaced with a gfp gene. We found that mice bearing a homozygous knock-in allele (Alas1(GFP/GFP)) were lethal by embryonic day 8.5, demonstrating that ALAS-N is essential for early embryogenesis. Fluorescence microscopic and flow cytometric analyses of heterozygous mouse (Alas1(+/GFP)) tissues showed that the Alas1 expression level differs substantially in tissues; Alas1 is highly expressed in testis Leydig cells, exocrine glands (including submandibular and parotid glands), endocrine glands (such as adrenal and thyroid glands) and hematopoietic lineage cells (including neutrophils and eosinophils). Quantitative analyses of GFP mRNA and ALAS-N mRNA in various tissues of Alas1(+/GFP) mice suggested that the destabilization of ALAS-N mRNA was not uniform in the various tissues. These results thus lay bare that elaborate control of the endogenous heme supply operates in various mouse tissues through regulation of the ALAS-N expression level and that this control is essential for heme homeostasis in animals.

A Role for the Transcriptional Coactivator PRC1 in Mitochondrial Biogenesis During Erythroid Development.

Abstract 3642Poster Board III-578Red cell development requires unparalleled mitochondrial metabolic and biosynthetic capacities to support hemoglobin formation. In addition to meeting the energetic requirements of the erythroblast and reticulocyte, mitochondria are the site of multiple steps in heme biosynthesis and are essential for iron utilization. As such, perturbation of mitochondrial function in hematopoietic cells appears to differentially affect red cell development. For instance, we show that the drug chloramphenicol (an inhibitor of mitochondrial protein synthesis) preferentially inhibits erythroid colony formation with relatively little effect on other types of myeloid progenitors. Similarly, we show that knockout of the mitochondrial superoxide dismutase (Sod2) preferentially inhibits BFU-E, CFU-E and GEMM colonies. Clinically, several mutations in mitochondrial DNA or in nuclear encoded mitochondria-localized proteins preferentially disrupt erythroid development resulting in sideroblastic anemia. In order to better understand regulation of mitochondrial activity during red cell development/differentiation, we have investigated expression of transcription factors and coactivators implicated as regulators of mitochondrial biogenesis in other tissues including PGC-1 α and β, ERR proteins, NRF 1 and NRF 2 in normal and Sod2 deficient murine erythroblasts. Microarray and qPCR data suggest that neither of the classic transcriptional coactivators PGC-1 α or β are expressed at appreciable levels in erythroblasts. However, a PGC-1 related coactivator, PRC1 is detectable at both the transcript and protein level in developing erythroid cells. In the Sod2 deficiency model, one of the most prominent transcriptional changes we have observed in erythroblasts is a broad down regulation of expression of nuclear encoded mitochondria localized protein transcripts including multiple components of the electron transport chain, TCA cycle enzymes and mitochondrial ribosomal proteins. We show that PRC1 expression is down regulated in Sod2 deficient cells—suggesting that this coactivator plays a key role in mitochondrial biogenesis during erythroid development. Further, using the MEL cell line, we show that PRC1 expression is turned on following induction of differentiation with DMSO. These results identify PRC1 as a novel, and perhaps exclusive regulator of mitochondrial biogenesis during erythroid development. A better understanding of regulation of PRC1 expression and identification of transcriptional targets of PRC1 during erythroid development will improve our understanding of how perturbations in mitochondrial physiology affect the red cell compartment. Disclosures:No relevant conflicts of interest to declare.

Mitochondria and Hematologic Disorders.

Abstract SCI-3Mitochondria have a special relationship with the erythroid lineage. Although RBC are devoid of mitochondria, during RBC development the mitochondria is the site of multiple steps in heme biosynthesis, and is essential for proper utilization of iron. As evidence of this special relationship, multiple mutations in both mitochondrial DNA (hereditary and acquired) and in nuclear genes encoding mitochondrial localized proteins (hereditary) result in sideroblastic anemia—where the hallmark pathologic lesion is intramitochondrial iron accumulation in erythroid progenitors. The erythroid-lineage specific readout of these mitochondrial genetic lesions raises the possibility that mitochondrial dysfunction is a contributor to anemia in other contexts as well. In this view, red cell development can be considered an early warning system for mitochondrial dysfunction in hematopoiesis. A focus of our laboratory is to investigate how increased mitochondrial-derived reactive oxygen species affect hematopoietic development. Gene expression and proteomic analyses of erythroblasts demonstrate that mitochondrial biogenesis during erythroid development is inhibited by oxidant stress. Transcriptional control of mitochondrial biogenesis in erythroid cells involves induction of the distinct transcriptional coactivator PRC1—perhaps helping to explain the erythroid specificity of phenotypes noted above. As has been elegantly demonstrated by Wallace and others, mitochondrial dysfunction is an important determinant of age-related decline in functional capacity of many tissues. This decline in function is accompanied by an increase in mitochondrial DNA mutations—both point mutations and deletions found primarily in post-mitotic cells. Modeling of this process through creation of mice with an error prone mtDNA polymerase accelerates the appearance of age-related tissue changes—including the development of anemia. Transplantation of murine hematopoietic stem cells harboring a large deletion of mtDNA also leads to anemia in reconstituted animals. Are these findings relevant for age-related hematologic abnormalities in people—and if so, for what disorders? There is considerable epidemiologic evidence indicating an increase in the frequency of anemia in the elderly, peaking at a prevalence of greater than 20% for individuals in their 80's. Approximately 1/3 of these elderly anemic cases are idiopathic—that is, no underlying disease process is identified. In studying this group with idiopathic anemia, we have investigated a number of hypotheses including the possibility of mitochondrial dysfunction. To date we have found altered mitochondrial DNA content and a higher mutation frequency in mtDNA isolated from peripheral blood cells when comparing anemic versus age/sex matched controls. However, these studies are correlative, and do not prove causality. Proving a direct role for specific acquired mitochondrial DNA lesions in development of anemia, myelodysplasia or hematologic malignancy remains a technical challenge because of the difficulty in introducing specific mutant mtDNA's into relevant cells or tissues. The development of more facile methods for evaluation of mitochondria in few or even single cells promises to expand our understanding of how mitochondrial functional changes impact diverse hematopoietic cells, in addition to the erythroid lineage effects highlighted above. DisclosuresNo relevant conflicts of interest to declare.

RETRACTED: Peroxisome Proliferator-Activated Receptor α Controls Hepatic Heme Biosynthesis Through ALAS1

Heme is an essential prosthetic group of proteins involved in oxygen transport, energy metabolism and nitric oxide production. ALAS1 (5-aminolevulinate synthase) is the rate-limiting enzyme in heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target for the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). In primary human hepatocytes and in HepG2 cells, PPARalpha agonists induced an increase in ALAS1 mRNA levels, which was abolished by PPARalpha silencing. These effects are mediated by two functional PPAR binding sites at positions -9 and -2.3 kb relative to the ALAS1 transcription start site. PPARalpha ligand treatment also up-regulated the mRNA levels of the genes ALAD (5-aminolevulinate dehydratase), UROS (uroporphyrinogen III synthase), UROD (uroporphyrinogen decarboxylase), CPOX (coproporphyrinogen oxidase) and PPOX (protoporphyrinogen oxidase) encoding for enzymes controlling further steps in heme biosynthesis. In HepG2 cells treated with PPARalpha agonists and in mouse liver upon fasting, the association of PPARalpha, its partner retinoid X receptor, PPARgamma co-activator 1alpha and activated RNA polymerase II with the transcription start site region of all six genes was increased, leading to higher levels of the metabolite heme. In conclusion, these data strongly support a role of PPARalpha in the regulation of human ALAS1 and of five additional genes of the pathway, consequently leading to increased heme synthesis.

Heme-dependent metalloregulation by the iron response regulator (Irr) protein in Rhizobium and other Alpha-proteobacteria

Perception and response to nutritional iron by bacteria is essential for viability, and for the ability to adapt to the environment. The iron response regulator (Irr) is part of a novel regulatory scheme employed by Rhizobium and other Alpha-Proteobacteria to control iron-dependent gene expression. Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to regulate gene expression, thus it responds to an iron-dependent process rather than to iron directly. Irr mediates this response by interacting directly with ferrochelatase, the enzyme that catalyzes the final step in heme biosynthesis. Irr is expressed under iron limitation to both positively and negatively modulate gene expression, but degrades in response to direct binding to heme in iron-sufficient cells. Studies with Rhizobium reveal that the regulation of iron homeostasis in bacteria is more diverse than has been generally assumed.

Altered Orientation of Active Site Residues in Variants of Human Ferrochelatase. Evidence for a Hydrogen Bond Network Involved in Catalysis

Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. The crystal structures of human ferrochelatase both with and without the protoporphyrin substrate bound have been determined previously. The substrate-free enzyme has an open active site pocket, while in the substrate-bound enzyme, the active site pocket is closed around the porphyrin macrocycle and a number of active site residues have reoriented side chains. To understand how and why these structural changes occur, we have substituted three amino acid residues (H263, H341, and F337) whose side chains occupy different spatial positions in the substrate-free versus substrate-bound ferrochelatases. The catalytic and structural properties of ferrochelatases containing the amino acid substitutions H263C, H341C, and F337A were examined. It was found that in the H263C and H341C variants, but not the F337A variant enzymes, the side chains of N75, M76, R164, H263, F337, H341, and E343 are oriented in a fashion similar to what is found in ferrochelatase with the bound porphyrin substrate. However, all of the variant forms possess open active site pockets which are found in the structure of porphyrin-free ferrochelatase. Thus, while the interior walls of the active site pocket are remodeled in these variants, the exterior lips remain unaltered in position. One possible explanation for this collective reorganization of active site side chains is the presence of a hydrogen bond network among H263, H341, and E343. This network is disrupted in the variants by alteration of H263C or H341C. In the substrate-bound enzyme, the formation of a hydrogen bond between H263 and a pyrrole nitrogen results in disruption of the network. The possible role of this network in catalysis is discussed.

Direct Measurement of Metal Ion Chelation in the Active Site of Human Ferrochelatase

The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.

Breast cancer resistance protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin. We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria. This protoporphyria is mild compared with ferrochelatase-dependent EPP, and in itself not sufficient to cause phototoxicity, but it might exacerbate the consequences of other porphyrias. In this study, we identified the mouse harderian gland as a novel expression site of Bcrp1. Because of its pronounced role in porphyrin secretion, the harderian gland presents a useful tool to study the mechanism of Bcrp1-related protoporphyria and transport of porphyrins. Bcrp1(-/-) harderian gland displayed a highly increased accumulation of PPIX glycoconjugates, and a similar shift was seen in Bcrp1(-/-) liver. Tear- and hepatobiliary excretion data suggest that Bcrp1 controls intracellular levels of PPIX by mediating high affinity transport of its glycoconjugates and possibly low-affinity transport of unconjugated PPIX. This mechanism may allow cells to prevent or reduce cytotoxicity of PPIX under excess conditions, without spillage under physiological conditions where PPIX is needed.

Histone acetyltransferase p300 regulates the transcription of human erythroid-specific 5-aminolevulinate synthase gene

Erythroid-specific 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis of erythroid cells. Here, we show that treatment of erythroid K562 cells with HDAC inhibitors sodium butyrate or Trichostatin A gave rise to a significant increase in ALAS2 gene transcripts, with a concurrent increase in acetylation level of histone H4 at the ALAS2 gene promoter. Histone acetyltransferase p300 bound with ALAS2 promoter and overexpression of p300 increased both the promoter reporter expression and endogenous mRNA level of ALAS2. Additionally, two functional Sp1 sites located in ALAS2 promoter were identified. Both of the GATA-1 sites and all the Sp1 sites at the ALAS2 promoter contributed to the transcription synergistic action with p300. These data implicated a close relationship between the acetylation modification of histone at the ALAS2 promoter and the regulation of this gene. Meanwhile, this work identified that ALAS2 is a novel target gene for p300/CBP action as histone acetyltransferases.

Candidate gene mutation analysis in idiopathic acquired sideroblastic anemia (refractory anemia with ringed sideroblasts)

Background For most cases of idiopathic acquired sideroblastic anemia (IASA), the molecular pathogenesis is unknown, despite the consistent morphological signature of abundant pathological ringed sideroblasts with their characteristic iron-engorged mitochondria. Moderately elevated free erythrocyte protoporphyrin (FEP) levels have been described in IASA, suggesting that the activity of ferrochelatase, the enzyme that catalyzes the final step in heme biosynthesis (incorporation of ferrous iron into protoporphyrin), might be diminished in erythroid progenitor cells from IASA patients. Methods We confirmed FEP elevation in IASA, then pursued a candidate gene approach that included screening the gene encoding ferrochelatase, FECH, for promoter and coding region mutations and mRNA expression changes in bone marrow from 37 patients with IASA. Results The analytical techniques employed detected mutations in a test cohort of previously undiagnosed patients with biochemical evidence for erythropoietic protoporphyria, a condition resulting from germline mutations in FECH, but somatic missense mutations of FECH and its promoter were not observed in IASA patients. FECH was modestly overexpressed in progenitor cells from patients with IASA, compared with MDS patients without sideroblasts and healthy controls. In addition, we analyzed ABCB7 and PUS1, genes implicated in congenital sideroblastic anemia syndromes, but again found no coding mutations in acquired cases. Conclusion We conclude that acquired mutations in the factors currently known to cause inherited sideroblastic anemias are uncommon in IASA.

Remodeling the regulation of iron metabolism during erythroid differentiation to ensure efficient heme biosynthesis

Terminal erythropoiesis is accompanied by extreme demand for iron to ensure proper hemoglobinization. Thus, erythroblasts must modify the "standard" post-transcriptional feedback regulation, balancing expression of ferritin (Fer; iron storage) versus transferrin receptor (TfR1; iron uptake) via specific mRNA binding of iron regulatory proteins (IRPs). Although erythroid differentiation involves high levels of incoming iron, TfR1 mRNA stability must be sustained and Fer mRNA translation must not be activated because iron storage would counteract hemoglobinization. Furthermore, translation of the erythroid-specific form of aminolevulinic acid synthase (ALAS-E) mRNA, catalyzing the first step of heme biosynthesis and regulated similarly as Fer mRNA by IRPs, must be ensured. We addressed these questions using mass cultures of primary murine erythroid progenitors from fetal liver, either undergoing sustained proliferation or highly synchronous differentiation. We indeed observed strong inhibition of Fer mRNA translation and efficient ALAS-E mRNA translation in differentiating erythroblasts. Moreover, in contrast to self-renewing cells, TfR1 stability and IRP mRNA binding were no longer modulated by iron supply. These and additional data stemming from inhibition of heme synthesis with succinylacetone or from iron overload suggest that highly efficient utilization of iron in mitochondrial heme synthesis during normal erythropoiesis alters the regulation of iron metabolism via the IRE/IRP system.

Expression of the heme biosynthetic pathway genes hemCD, hemH, hemM, and hemA of Escherichia coli.

Little is known about the control of latter steps of heme biosynthesis in Escherichia coli. In this study we examined the transcriptional regulation of genes that encode two intermediate heme pathway enzymes, porphobilinogen deaminase (hemC) and uroporphyrinogen III cosynthase (hemD), and the final enzyme of the pathway, ferrochelatase (hemH). We also reexamined the regulation of hemA and the gene located immediately upstream of hemA, hemM. The regulatory regions of hemC, hemH, hemA and hemM were fused to lacZ. The resultant operon fusions were inserted into the E. coli chromosome in single copy and expression monitored under conditions of oxygen and heme limitation. Expression of hemM appeared constitutive under the conditions tested here. In contrast, expression of hemCD, hemH and hemA were shown to be mildly regulated in response to heme availability. Thus, transcription of four of the nine genes of the E. coli heme pathway appears to be only mildly regulated in response to heme limitation.

Density-Functional Study of Mechanisms for the Cofactor-Free Decarboxylation Performed by Uroporphyrinogen III Decarboxylase

Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen III to yield coproporphyrinogen III. The enzyme acts by successively protonating each of the four pyrrole rings present in the substrate, thereby allowing decarboxylation of their side chains, but the identity of the proton donors has not been established yet. Tyr164 has been suggested as a proton donor, and Asp86 has been proposed to act either as a proton donor or as an intermediate-stabilizing residue. We have performed density-functional calculations to study this reaction mechanism, and found that the rate-limiting step is substrate protonation, rather than decarboxylation. Surprisingly, whereas Tyr164 is unable to protonate the substrate, this protonation can be effected by a nearby arginine residue (Arg37), with a free energy barrier of 21.4 kcal.mol(-1), in remarkable agreement with the experimental value of 19.5 kcal.mol(-1). The central positioning of this residue in close proximity to all four pyrrole rings in the substrate may play a key role in the sequential activation of each of these moieties.

Fusidic acid-resistant mutants of Salmonella enterica serovar typhimurium have low levels of heme and a reduced rate of respiration and are sensitive to oxidative stress.

Mutations in the translation elongation factor G (EF-G) make Salmonella enterica serovar Typhimurium resistant to the antibiotic fusidic acid. Fus(r) mutants are hypersensitive to oxidative stress and rapidly lose viability in the presence of hydrogen peroxide. We show that this phenotype is associated with reduced activity of two catalase enzymes, HPI (a bifunctional catalase-hydroperoxidase) and HPII (a monofunctional catalase). These catalases require the iron-binding cofactor heme for their activity. Fus(r) mutants have a reduced rate of transcription of hemA, a gene whose product catalyzes the first committed step in heme biosynthesis. Hypersensitivity of Fus(r) mutants to hydrogen peroxide is abolished by the presence of delta-aminolevulinic acid, the precursor of heme synthesis, in the growth media and by the addition of glutamate or glutamine, amino acids required for the first step in heme biosynthesis. Fluorescence measurements show that the level of heme in a Fus(r) mutant is significantly lower than it is in the wild type. Heme is also an essential cofactor of cytochromes in the electron transport chain of respiration. We found that the rate of respiration is reduced significantly in Fus(r) mutants. Sequestration of divalent iron in the growth media decreases the sensitivity of Fus(r) mutants to oxidative stress. Taken together, these results suggest that Fus(r) mutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity. The sensitivity of Fus(r) mutants to oxidative stress could be associated with loss of viability due to iron-mediated DNA damage in the presence of hydrogen peroxide. We argue that understanding the specific nature of antibiotic resistance fitness costs in different environments may be a generally useful approach in identifying physiological processes that could serve as novel targets for antimicrobial agents.

A heme-deficient strain of Escherichia coli has a three-base pair deletion in a “hotspot” in hemA

The key regulatory step in heme biosynthesis in Escherichia coli is at the level of glutamyl-tRNA reductase (GTR), an enzyme which is encoded by hemA. A strain, HU227, with a spontaneous in-frame mutation in hemA has no GTR activity. The mutation is shown to be a three-base deletion at a “hotspot” in the gene. The amino acid sequence in this region is highly conserved.

Hypoxic up-regulation of erythroid 5-aminolevulinate synthase

AbstractThe erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

The terminal steps of heme biosynthesis

The terminal steps of heme biosynthesis

Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400

A Pseudomonas fluorescens Tn 5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis. Since this mutant was leaky, a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor δ-aminolevulinate (ALA). Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth. A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin. The Δ hemH mutant was characterized and the corresponding hemH gene sequenced. Antibodies against P. fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant. The Δ hemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans. These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production.

Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400.

A Pseudomonas fluorescens Tn5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis. Since this mutant was leaky, a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor delta-aminolevulinate (ALA). Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth. A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin. The DeltahemH mutant was characterized and the corresponding hemH gene sequenced. Antibodies against P. fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant. The DeltahemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans. These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production.

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues.

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.