Heme Regulatory Motifs Research Articles

A Novel Heme-Regulatory Motif Mediates Heme-Dependent Degradation of the Circadian Factor Period 2

Although efforts have been made to identify circadian-controlled genes regulating cell cycle progression and cell death, little is known about the metabolic signals modulating circadian regulation of gene expression. We identify heme, an iron-containing prosthetic group, as a regulatory ligand controlling human Period-2 (hPer2) stability. Furthermore, we define a novel heme-regulatory motif within the C terminus of hPer2 (SC(841)PA) as necessary for heme binding and protein destabilization. Spectroscopy reveals that whereas the PAS domain binds to both the ferric and ferrous forms of heme, SC(841)PA binds exclusively to ferric heme, thus acting as a redox sensor. Consequently, binding prevents hPer2 from interacting with its stabilizing counterpart cryptochrome. In vivo, hPer2 downregulation is suppressed by inhibitors of heme synthesis or proteasome activity, while SA(841)PA is sufficient to stabilize hPer2 in transfected cells. Moreover, heme binding to the SC(841)PA motif directly impacts circadian gene expression, resulting in altered period length. Overall, the data support a model where heme-mediated oxidation triggers hPer2 degradation, thus controlling heterodimerization and ultimately gene transcription.

Comparison of Apo- and Heme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2

Heme oxygenase (HO) catalyzes the first step in the heme degradation pathway. The crystal structures of apo- and heme-bound truncated human HO-2 reveal a primarily alpha-helical architecture similar to that of human HO-1 and other known HOs. Proper orientation of heme in HO-2 is required for the regioselective oxidation of the alpha-mesocarbon. This is accomplished by interactions within the heme binding pocket, which is made up of two helices. The iron coordinating residue, His(45), resides on the proximal helix. The distal helix contains highly conserved glycine residues that allow the helix to flex and interact with the bound heme. Tyr(154), Lys(199), and Arg(203) orient the heme through direct interactions with the heme propionates. The rearrangements of side chains in heme-bound HO-2 compared with apoHO-2 further elucidate HO-2 heme interactions.

Evidence That the Heme Regulatory Motifs in Heme Oxygenase-2 Serve as a Thiol/Disulfide Redox Switch Regulating Heme Binding

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.

Regulation of Globin Gene Transcription by Heme in Erythroleukemia Cells: Analysis of Putative Heme Regulatory Motifs in the p45 NF-E2 Transcription Factor

The function of the NF-E2 transcription factor, a p45/small Maf heterodimer, was analyzed in the erythroleukemia cell lines MEL and CB3. In contrast to MEL cells, CB3 cells are null for p45 and thus express only extremely low levels of adult globin transcripts upon induction by agents promoting erythroid differentiation. We investigated the response of erythroleukemia cells to hemin treatment. Hemin rapidly induces beta-globin gene transcript levels in MEL cells, but not in CB3 cells. Stable expression of the large p45 NF-E2 subunit in CB3 cells restores hemin mediated beta-globin gene transcription, suggesting that the presence of a functional NF-E2 is required for strong induction of beta-globin mRNA levels by hemin in erythroleukemia cells. We performed mutagenesis of two potential heme-regulatory motifs (HRMs) in p45 NF-E2 and found that the mutated versions are expressed and can still recognize a NF-E2 DNA binding element. In addition, we showed that p45 NF-E2 HRM mutants are able to restore beta-globin gene transcription in CB3 cells upon induction by hemin. Our results suggest that globin gene activation by heme appears to be independent of the putative HRMs in the p45 subunit of the NF-E2 transcription factor.

S1d2-1 Involvement of heme regulatory motif in iron-sensing, ubiquitination and degradation of IRP2(S1-d2: "Dynamical Structures and Signaling Mechanism of Sensor Metalloprotein",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1d2-1 Involvement of heme regulatory motif in iron-sensing, ubiquitination and degradation of IRP2(S1-d2: "Dynamical Structures and Signaling Mechanism of Sensor Metalloprotein",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

Involvement of Heme Regulatory Motif in Heme-Mediated Ubiquitination and Degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.

Two Heme Binding Sites Are Involved in the Regulated Degradation of the Bacterial Iron Response Regulator (Irr) Protein

The iron response regulator (Irr) protein from Bradyrhizobium japonicum is a conditionally stable protein that degrades in response to cellular iron availability. This turnover is heme-dependent, and rapid degradation involves heme binding to a heme regulatory motif (HRM) of Irr. Here, we show that Irr confers iron-dependent instability on glutathione S-transferase (GST) when fused to it. Analysis of Irr-GST derivatives with C-terminal truncations of Irr implicated a second region necessary for degradation, other than the HRM, and showed that the HRM was not sufficient to confer instability on GST. The HRM-defective mutant IrrC29A degraded in the presence of iron but much more slowly than the wild-type protein. This slow turnover was heme-dependent, as discerned by the stability of Irr in a heme-defective mutant strain. Whereas the HRM of purified recombinant Irr binds ferric (oxidized) heme, a second site that binds ferrous (reduced) heme was identified based on spectral analysis of truncation and substitution mutants. A mutant in which histidines 117-119 were changed to alanines severely diminished ferrous, but not ferric, heme binding. Introduction of these substitutions in an Irr-GST fusion stabilized the protein in vivo in the presence of iron. We conclude that normal iron-dependent Irr degradation involves two heme binding sites and that both redox states of heme are required for rapid turnover.

Identification of Novel Heme-Regulated Genes during Erythroid Differentiation.

(Introduction) During erythroid differentiation, a large amount of heme is synthesized for hemoglobin formation. Besides its fundamental role as an oxygen carrier, heme is known to play a key role in transcriptional regulation of certain genes and in translational control of protein synthesis. Since it is known that both the level of erythroid-specific genes and heme content increase together during erythroid differentiation, heme may likely regulate the expression of erythroid-specific genes. It is also possible that heme might regulate even a wider variety of genes than hemoglobin synthesis in cells undergoing erythroid differentiation. With this view in mind, we have searched for novel genes that are under the control of heme. For this purpose, we used the wild-type and heme-deficient erythroblasts generated from the wild-type and ALAS2 (-) ES cells in vitro and compared their gene expression profiles. By this approach, we have identified and reported four novel erythroid-specific genes previously (ASH meeting 2003). In the present study, we have further investigated the mechanisms of heme-mediated regulation of these genes.(Methods) cDNA sequences of these genes were determined by 5′RACE and data-base search. In order to generate erythroblasts containing various amounts of heme, ALAS2 (-) ES subclones, which had been partially rescued by human ALAS2 cDNA driven under the erythroid-specific promoter, were established and induced to undergo erythroid differentiation. Correlation between the level of expression of these genes and intracellular heme content was examined. In addition, the promoter region of one of these genes, NuSAP, was cloned, and its cis-element, through which heme regulates the expression, was determined by promoter analysis and EMSA.(Results) The level of expression of all these genes was closely correlated with intracellular heme content in the partially rescued ALAS2 (-) erythroblasts, indicating that expression of these genes is clearly under the control of heme. The results of 5′-RACE and database search have allowed us to identify that these genes consist of uncoupling protein2 (UCP2), nucleolar spindle-associated protein (NuSAP) and two as yet uncharacterized genes (EST1 and 2). While EST1 consists of 110 a.a. with a GK motif which is characteristic of acetyltransferase, EST2 consists of 972 a.a, with nucleotide sequence indicative of a serine/threonine kinase with a putative transmembrane domain. In order to determine the heme regulatory motif, we also performed the promoter analysis of the NuSAP gene. The results showed that CCAAT box, located 34 nucleotides upstream from the transcription initiation site, was essential for the promoter activity, and the binding activity of a protein complex to this element was enhanced in DMSO-treated MEL cells, suggesting that heme regulates the expression of the NuSAP gene through this motif.(Conclusion) These results demonstrate that expression of a wide variety of genes, which may have quite different functions from hemoglobin formation, may also be regulated by heme in erythroid cells undergoing cell differentiation.

Role of the Heme Regulatory Motif in the Heme-Mediated Inhibition of Mitochondrial Import of 5-Aminolevulinate Synthase

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging.

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.

The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase

The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and 1H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H2O solutions and micellar environments. A well defined amphipathic α-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short α-helix, defined by A5–L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and 1H NMR study of hemin and psALAS indicated that a heme–peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.

Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs.

Heme oxygenase-2 (HO-2) degrades heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of heme regulatory motif (HRM) with a conserved core of Cys264-Pro265 and Cys281-Pro282. We examined interaction of HO-2 HRMs with Fe-PP, Zn-protoporphyrin IX (Zn-PP; HO-2 inhibitor), and protoporphyrin IX (PP IX). Spectral analyses, using 1:4 or 1:1 molar ratio of the heme to 10-residue peptides, corresponding to HRM containing HO-2 sequences, revealed specific interactions as indicated by a shift in the absorption spectrum of heme. Five residue peptides qualitatively produced similar results. Substitution of cysteine with alanine in either peptide eliminated interactions, and substitution of proline with alanine reduced the peptides' affinity for heme. Neither Zn-PP nor PP IX absorption spectrum was affected by HRM peptides. The circular dichroism spectra confirmed heme-HRM peptides interactions. An astounding 4,000-6,000-fold higher concentrations of KCN were required at pH 7.5 to displace HRM peptides from heme. Data suggest (a) each HRM can contribute to HO-2-heme interaction, (b) heme iron interacts with cysteine thiol, (c) charged residues upstream of Cys264-Pro265 result in its high-affinity heme binding, and (d) inhibition of HO-2 activity by synthetic metalloporphyrins does not involve HRMs. We suggest that heme bound to HRMs may serve as a binding site/reservoir for gaseous signal molecules.

The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different functions?

Heme oxygenase isozymes, HO-1, HO-2 and HO-3, are HSP32 protein cognates, with a known function of catalyzing the isomer specific oxidation of the heme molecule, including that of NO synthase. Unknown until recent years was that the system is a central component of the cellular defense mechanisms; this can be attributed to a combination of many factors. In biological systems HO activity is responsible for production of equimolar amounts of CO, biliverdin and free Fe. The serine/threonine kinase, biliverdin reductase, catalyzes reduction of biliverdin to bilirubin. Bilirubin is a potent antioxidant and CO is a signal molecule. Although both active HO isozymes catalyze the same reaction, HO-1 and HO-2 may function in a rather distinct fashion in protection against tissue injury. HO-1 is the stress responsive cognate that is rapidly induced by free and stable radicals as well as by hypoxia. Supra induction of HO-1 completely protects ischemic kidney against tissue pathology. This involves rapid inactivation of the pro-oxidant heme of denatured hemoproteins and converting it to bilirubin and CO. In the case of severe tissue injury, such as compression injury, HO-1 is induced and colocalizes with cGMP and pro-apoptotic oncogenes. HO-2, which is the constitutive form, in addition to maintaining cell heme homeostasis, inactivates NO derived radicals. The isozyme binds the free radical at its "heme regulatory motifs" and is "suicide" inactivated at the protein and transcript levels. Data are shown that provide evidence for role of the HO system in the cellular defense mechanism against free radical-mediated tissue damage, and are consistent with the forwarded concept that HO isozymes have common, as well as distinct, roles in cellular defense mechanisms.

Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein.

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse-chase and immunoprecipitation experiments showed that Irr degraded in response to 6 microM iron with a half-life of approximately 30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 microM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys(29) --> Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

A photosensitising adenovirus for photodynamic therapy.

We have developed a new approach to photodynamic therapy based on adenoviral transduction of the rate-limiting enzyme in heme synthesis. Conventional phototherapy uses porphyrin-based chemical photosensitisers, including delta-aminolaevulinic acid (ALA) which is converted to protoporphyrin IX (PpIX) by the enzymes of the heme biosynthetic pathway. The lack of a specific mechanism for targeting chemical photosensitisers and PpIX to tumour cells means that therapeutic irradiation can damage normal tissue and exposure to sunlight following treatment can cause severe burns. The rate limiting enzyme in PpIX synthesis is ALA-synthase (ALA-S). We have developed a new yeast vector system for manipulation of the adeno- virus genome and used it to construct a virus expressing a mutant form of ALA-S lacking the iron response elements which regulate ALA-S translation and the heme regulatory motifs which regulate import of ALA-S into mitochondria. The virus induces a large increase in PpIX expression and confers photosensitivity on cultured cells. Unlike conventional photodynamic therapy, a viral approach makes it possible to restrict photosensitivity by biological rather than purely physical or chemical means. As with HSV thymidine kinase, ALA-S expression is a general mechanism for sensitisation to a therapeutic agent which can easily be adapted to whatever means of gene delivery is most effective.

Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.

Comparison of the beluga whale ( Delphinapterus leucas) expressed genes for 5-aminolevulinate synthase with those in other vertebrates

The cDNA and inferred amino acid sequences were determined for beluga whale ( Delphinapterus leucas) erythroid (E) and housekeeping (H) forms of 5-aminolevulinate synthase (ALS), and they were compared with known sequences for five other vertebrates with particular attention to regulatory features. The cDNAs for whale ALS-E and -H encode, respectively, proteins of 582 and 640 amino acids. Sequence alignments suggest that the whale ALS-H, like those for rat and chicken, has an N-terminal mitochondrial targeting sequence of 56 amino acids. There is a high degree of amino acid conservation between the beluga whale proteins and those of other vertebrates, including regulatory elements and functional residues that have been defined in other ALSs. Both whale proteins contain three heme regulatory motifs suggesting that mitochondrial uptake may be regulated by heme. The ALS-E mRNA contains an iron responsive element in its 5′-untranslated region indicating that its expression may be post-transcriptionally regulated by cellular iron. This extensive structural similarity and the presence of the same regulatory elements found in other ALSs indicate that regulation of ALS in beluga whale is similar to that in other vertebrates.

Heme Oxygenase-2 Is a Hemoprotein and Binds Heme through Heme Regulatory Motifs That Are Not Involved in Heme Catalysis

The heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron. In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2. The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein. The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 --> Ala/Ala) suggest a role of the HRMs in heme binding. While the His151 --> Ala mutation inactivates HO-2, Cys264 --> Ala and Cys281 --> Ala mutations individually or together (HO-2 mut) do not decrease HO activity. Also, Pro265 --> Ala or Pro282 --> Ala mutation does not alter HO-2 activity. Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme. The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell.

5-Aminolevulinate synthase and the first step of heme biosynthesis.

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate. In animals, fungi, and some bacteria, 5-aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway. Mutations on the human erythroid 5-aminolevulinate synthase, which is localized on the X-chromosome, have been associated with X-linked sideroblastic anemia. Recent biochemical and molecular biological developments provide important insights into the structure and function of this enzyme. In animals, two aminolevulinate synthase genes, one housekeeping and one erythroid-specific, have been identified. In addition, the isolation of 5-aminolevulinate synthase genomic and cDNA clones have permitted the development of expression systems, which have tremendously increased the yields of purified enzyme, facilitating structural and functional studies. A lysine residue has been identified as the residue involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor, and the catalytic domain has been assigned to the C-terminus of the enzyme. A conserved glycine-rich motif, common to all aminolevulinate synthases, has been proposed to be at the pyridoxal 5'-phosphate-binding site. A heme-regulatory motif, present in the presequences of 5-aminolevulinate synthase precursors, has been shown to mediate the inhibition of the mitochondrial import of the precursor proteins in the presence of heme. Finally, the regulatory mechanisms, exerted by an iron-responsive element binding protein, during the translation of erythroid 5-aminolevulinate synthase mRNA, are discussed in relation to heme biosynthesis.