Insertion Of Iron Research Articles

Tetra-crowned porphyrin as P450 biomimetic model for carbamazepine oxidation

A substituted porphyrin bearing four crown ether units, H(2)(TCP), was synthesized from the reaction between (5,10,15,20-tetra(o-aminophenyl) porphyrin) and the acyl derivative of the ether (4-carboxy-18-crown-6). The free-base porphyrin was characterized by C, N, and H elemental analysis; UV-vis and IR spectroscopies; and (1)H NMR. The corresponding ironporphyrin, Fe(TCP)Cl, was obtained via iron insertion into H(2)(TCP). Fe(TCP)Cl was employed as catalyst for carbamazepine (CBZ) oxidation by iodosylbenzene (PhIO), 3-chloroperoxybenzoic acid (m-CPBA) or sodium hypochlorite (NaOCl), in methanol or in a biphasic water/dichloroethane system. The crowned ironporphyrin proved to be a highly efficient and selective catalyst for CBZ epoxidation even in the biphasic dichloroethane /H(2)O system, with no need for an additional phase transfer agent.

Iron–ceria–zirconia fluorite catalysts for methane selective oxidation to formaldehyde

Ceria–zirconia catalysts modified by partial substitution of zirconium by iron were investigated for the reaction of the selective oxidation of methane to formaldehyde. The insertion of iron was ensured by the preparation method, based on decomposition of the mixed precursors. The crystalline structure was studied by XRD and the iron state was determined by Mössbauer spectroscopy. The insertion of Fe3+ into the lattice of the mixed oxide and the partial substitution of zirconium ions with iron cations up to 25% have not resulted in any phase rejection of the formed iron oxide. The reducibility of Ce4+ in the modified ternary oxide is enhanced by doping with iron. Despite their low specific surface area, the catalysts are efficient at activating the methane independently of the iron content. The selectivity to formaldehyde strongly depends on the amount of the iron inserted in the mixed oxide.

A homozygous mutation in the ferrochelatase gene underlies erythropoietic protoporphyria associated with palmar keratoderma

Conflicts of interest:none declared. M.M. and P.P.‐G. contributed equally to this manuscript. J.F. and R.E.deS. share senior authorship. Erythropoietic protoporphyria (EPP) (OMIM 177000) is one of the nonacute, cutaneous porphyrias. The disease results from an inherited partial deficiency of ferrochelatase (FECH; EC 4.99.1.1), the eighth and ultimate enzyme in haem biosynthesis, that is located in the mitochondria and catalyses the insertion of iron into protoporphyrin IX to form haem.1, 2 Although EPP is a rare disease overall, it is the most common type of porphyria manifesting in childhood with an estimated prevalence of one in 130 000.3, 4 Clinically overt disease usually commences in infancy or early childhood and is characterized by moderate to severe cutaneous photosensitivity affecting body sites exposed to ultraviolet (UV) radiation. Following sun exposure, pain, burning, itching, erythema and swelling can develop within minutes to hours.1–4 Petechiae, purpura and vesicles may be seen, but are uncommon.5 Chronic UV exposure might lead to lichenification, subtle scarring and hyperpigmentation, particularly over the knuckles and on the nose.1, 3 These clinical signs in association with the age at onset are quite characteristic for EPP and the tentative diagnosis is confirmed by biochemical and enzymatic analyses.1–4 In 2–5% of the patients, the disease can be complicated by severe liver failure with progressive hepatic dysfunction.3

Tailoring the properties and the reactivity of the spinel cobalt oxide

Pulsed spray evaporation chemical vapor deposition (PSE-CVD) was employed for the synthesis of cobalt-based spinel oxide thin films, Co(3-x)Fe(x)O4 with x = 0-1.56. XRD, Raman scattering and FTIR emission spectroscopy show that the normal spinel structure was retained for 0 < or = x < or = 0.65 by the selective insertion of Fe3+ in the octahedral sites. The spinel inversion was noticed above this range, whereas the insertion of Fe2+ was first indicated with x > or = 1. The room-temperature electrical resistivity of the thin films was controlled between 9 and 0.007 Omega cm by the adjustment of iron doping concentration. Furthermore an improvement of the thermal stability of the spinel was noticed upon doping by iron. The reducibility of the spinel in the presence of molecular hydrogen was efficiently adjusted by a shift of the reduction temperature by up to 110 degrees C upon the controlled insertion of iron in the octahedral sites of the spinel. The investigation of the catalytic oxidation of CO and ethanol over Co(3-x)Fe(x)O4 films with controlled structural modification enabled the confirmation of the Mars-van Krevelen mechanism for the oxidation of CO and the tight correlation between the selectivity of the conversion of ethanol to acetaldehyde and the abundance of surface basic sites. The controlled iron doping was demonstrated to be an efficient strategy to tune the reactivity and the selectivity of the cobalt-based spinel oxide. The doping-induced transition from normal to inverse spinel was observed to induce a clear discontinuity in the trend of all investigated physicochemical properties.

Reactions of Laser-Ablated Iron Atoms with Halomethanes: Infrared Spectra, Density Functional Calculations, and Structures of Simple Iron Insertion and Methylidene Complexes

Reactions of laser-ablated Fe atoms with halomethanes and ethane have been investigated through matrix infrared spectra and density functional calculations. Only insertion complexes are identified from reactions of Fe with methyl fluoride and ethane, parallel to the previously observed insertion complex CH3−FeH from the photochemical reaction with methane. However, Fe also forms methylidene complexes through α-X migration in the insertion products from Fe reactions with di-, tri-, and tetrahalomethanes. Calculated structures show no agostic distortion in these methylidene complexes. However, an interesting distortion and elongation of the C−Cl bond is observed in the Fe insertion products, such as H2ClC−FeCl, while no such distortion of the C−H or C−F bond is found in analogous calculated structures for H2FC−FeCl. This distortion is likely due to a Cl lone pair to Fe bonding interaction that ultimately leads to formation of the lower energy CH2═FeCl2 methylidene complex. This Cl atom transfer is reversibl...

Metal Ion Substrate Inhibition of Ferrochelatase

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

Four Novel Mutations in the Ferrochelatase (FECH) Gene in Patients with Erythropoietic Protoporphyria (EPP).

Erythropoietic protoporphyria (EPP) is an autosomal dominant disease with incomplete penetrance, due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the insertion of iron in the protoporphyrin molecule in the final step of the heme biosynthetic pathway. The disease is biochemically characterized by increased protoporphyrins in erythrocytes, plasma and faeces and normal levels in urine. The clinical manifestations have a childhood onset, characterized by mild to moderate cutaneous photosensitivity, mild anemia and, in 5–10% of the cases, by progressive hepatic failure. Diagnosis of EPP can be supported by the simple measurement with flow citometry of fluorescent erythrocytes in peripheral blood. The phenotype of EPP results from coinheritance of a mutated allele and a wild-type low expressed allele of the FECH gene that spans about 45 kb on the long arm of chromosome 18. The FECH gene encodes for a precursor of 423 amino acid residues that is targeted to the matrix side of the inner mitochondrial membrane via a 62 amino acid cleavable leader sequence. A single promoter directs both housekeeping and erythroid expression, but two polyadenylation sites produce two mRNAs of different length. The molecular analysis of FECH gene has allowed the identification of more than 100 different mutations responsible for EPP, showing a high genetic heterogeneity. Aim of this study was to search for molecular abnormalities in four patients with clinical and biochemical signs of EPP. Molecular analysis was performed by PCR and direct sequencing of the promoter, the entire coding region and the splicing junctions of the gene. In patients who remained negative for mutations, DNA analysis was completed by long-PCR and chromosome walking. In this study four different molecular defects have been identified: two missense mutations and two large deletions in the first region of gene. One A>G substitution at nucleotide 362 in exon 4 resulting in a Glu to Gly amino acid substitution at position 121; one change C>T in position 782 of exon 7 causing a Ser 261Phe substitution; the c.1-7887_67+2422del10376bp and the c.1-9628_67+2871del12566bp. Even though with different breakpoints, both deletions include a portion of upstream intergenic region, the promoter, the exon 1 and a portion of intron 1 of the FECH gene preventing the expression of the mutated allele. This deletions are probably caused by unequal intragenic recombination between ALU sequences which have been found close to the breakpoints. Recently others deletions involving ALU sequences were described suggesting that large deletions can affect the FECH gene and cause of EPP.

A π-Helix Switch Selective for Porphyrin Deprotonation and Product Release in Human Ferrochelatase

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein–protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved π-helix that is predicted to have a central role in product release.

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.

Substrate interactions with human ferrochelatase

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Human ferrochelatase is a homodimeric, inner mitochondrial membrane-associated enzyme that possesses an essential [2Fe-2S] cluster. In this work, we report the crystal structure of human ferrochelatase with the substrate protoporphyrin IX bound as well as a higher resolution structure of the R115L variant without bound substrate. The data presented reveal that the porphyrin substrate is bound deep within an enclosed pocket. When compared with the location of N-methylmesoporphyrin in the Bacillus subtilis ferrochelatase, the porphyrin is rotated by approximately 100 degrees and is buried an additional 4.5 A deeper within the active site. The propionate groups of the substrate do not protrude into solvent and are bound in a manner similar to what has been observed in uroporphyrinogen decarboxylase. Furthermore, in the substrate-bound form, the jaws of the active site mouth are closed so that the porphyrin substrate is completely engulfed in the pocket. These data provide insights that will aid in the determination of the mechanism for ferrochelatase.

Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells.

The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood , Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.

Iron Complexes of C- and N-Methylated 2-Aza-21-carbaporphyrin: NMR Studies

Insertion of iron(II) into methylated derivatives of N-confused porphyrins 2-aza-2-methyl-5,10,15,20-tetraphenyl-21-carbaporphyrin (MeCTPPH)H, 2-aza-5,10,15,20-tetraphenyl-21-methyl-21-carbaporphyrin (CTPPMe)H2, and 2-aza-2-methyl-5,10,15,20-tetraphenyl-21-methyl-21-carbaporphyrin (MeCTPPMe)H yielded N- or C-methylated high-spin iron(II) complexes (MeCTPPH)Fe(II)Br, (HCTPPMe)Fe(II)Br, and (MeCTPPMe)Fe(II)Br. One electron oxidation of (Me-CTPPH)Fe(II)Br using Br2, accompanied by deprotonation of a C(21)-H(21) fragment and formation of an Fe-C(21) bond, produces an intermediate-spin, five-coordinate iron(III) complex (MeCTPP)Fe(III)Br. Simultaneously, a high-spin complex [(MeCTPPH)Fe(III)Br]+ was formed which preserved the side-on interaction between the metal ion and the inverted pyrrole ring. &[(MeCTPPH)Fe(III)Br]+ was also obtained by titration of (MeCTPP)FeIIIBr with TFA due to the C(21) protonation. A titration of (HCTPPMe)Fe(II)Br and (MeCTPPMe)Fe(II)Br with Br2 yielded solely corresponding high-spin iron(III) species [(HCTPPMe)Fe(III)Br+ and [(MeCTPPMe)Fe(III)Br+. Dioxygen reacts cleanly with (MeCTPPH)Fe(II)Br carbaporphyrin to form solely (MeCTPP)Fe(III)Br. The 1H NMR spectra of paramagnetic iron(II) and iron(III) complexes were examined. The characteristic patterns of pyrrole, C-methyl, and N-methyl resonances were found diagnostic of the ground electronic state of iron and the coordinating nature of the N-confused pyrrole. The characteristic C-Me resonances occur in a unique window (520-420 ppm) for iron(III) C-methylated N-confused porphyrins which remains in contrast with relatively small values found for iron(II) C-methylated derivatives (50-80 ppm).

Iron Complexes of N-Confused Pyriporphyrin: NMR Studies

Insertion of iron(II) into 6,11,16,21-tetraaryl-3-aza-m-benziporphyrin (N-confused pyriporphyrin, (PyPH)H) yielded the high-spin iron(II) complex, (PyPH)Fe(II)Br. The coordination of iron(II) to the perimeter nitrogen atom of (PyPH)Fe(II)Br resulted in the formation of the diiron species. Oxidation and oxygenation of (PyPH)Fe(II)Br were followed by 1H NMR spectroscopy. The addition of Br2 to the solution of (PyPH)Fe(II)Br in the absence of dioxygen results in a one-electron oxidation yielding the high-spin iron(III) N-confused pyriporphyrin [(PyPH)Fe(III)Br]+ which preserves the side-on interaction between the inverted pyridine ring and metal ion. The reaction of (PyPH)Fe(II)Br with dioxygen ends up with the formation of a five-coordinate species (PyPO)Fe(III)Br] ((PyPOH)H = 3-aza-22-hydroxy-m-benziporphyrin, PyPO = the corresponding dianion) which is formed by oxygenation at the C(22) position. Coordination of a metal ion by 3-aza-22-hydroxy-benziporphyrin imposes a steric constraint on the geometry of the ligand. The halide ligand of (PyPO)Fe(III)Br coordinates on one of the two inequivalent faces of the macrocycle, leading to two distinct species: syn and anti. The (1)H NMR spectra of paramagnetic iron(II) and iron(III) N-confused pyriporphyrin complexes have been examined. The characteristic patterns of pyrrole and pyridine resonances have been found to be diagnostic of the ground electronic state of iron and the donor nature of the C(22)H and N(3) centers. The enormous downfield H(22) paramagnetic shift, determined for the iron(II) N-confused pyriporphyrin, provides a distinct resonance in a peculiar spectroscopic window (350-800 ppm) for a series of axial ligands which can be considered as a diagnostic sign of an agostic Fe(II)...{C(22)-H} interaction. Coordination of the pyridine moiety via the perimeter N(3) atom is reflected unambiguously by the H(2/4) resonance at 201 ppm.

A new class of [2Fe-2S]-cluster-containing protoporphyrin (IX) ferrochelatases

Protoporphyrin (IX) ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin IX to form haem. These ferrochelatases exist as monomers and dimers, both with and without [2Fe-2S] clusters. The motifs for [2Fe-2S] cluster co-ordination are varied, but in all cases previously reported, three of the four cysteine ligands are present in the 30 C-terminal residues and the fourth ligand is internal. In the present study, we demonstrate that a group of micro-organisms exist which possess protoporphyrin (IX) ferrochelatases containing [2Fe-2S] clusters that are co-ordinated by a group of four cysteine residues contained in an internal amino acid segment of approx. 20 residues in length. This suggests that these ferrochelatases have evolved along a different lineage than other bacterial protoporphyrin (IX) ferrochelatases. For example, Myxococcus xanthus protoporphyrin (IX) ferrochelatase ligates a [2Fe-2S] cluster via cysteine residues present in an internal segment. Site-directed mutagenesis of this ferrochelatase demonstrates that changing one cysteine ligand into serine results in loss of the cluster, but unlike eukaryotic protoporphyrin (IX) ferrochelatases, this enzyme retains its activity. These data support a role for the [2Fe-2S] cluster in iron affinity, and strongly suggest convergent evolution of this feature in prokaryotes.

A Pool of Extramitochondrial Frataxin That Promotes Cell Survival

Frataxin is a mitochondrial protein involved in iron metabolism. Defective expression of frataxin causes Friedreich ataxia (FA), an inherited degenerative syndrome characterized by ataxia, cardiomyopathy, and high incidence of diabetes. Here we report that frataxin-deficient cells are more prone to undergo stress-induced mitochondrial damage and apoptosis, while the overexpression of frataxin confers protection to a variety of cell types. Moreover, we reveal the existence of an extramitochondrial pool of frataxin, which can efficiently prevent mitochondrial damage and apoptosis in different cellular systems. Remarkably, extramitochondrial frataxin can fully replace mitochondrial frataxin in promoting survival of FA cells.

Monomeric Yeast Frataxin Is an Iron-Binding Protein

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.

The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus)

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

The Conserved Active-Site Loop Residues of Ferrochelatase Induce Porphyrin Conformational Changes Necessary for Catalysis

Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin-binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase-bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode gamma(15), a saddling-like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate.

Mono-crowned substituded iron porphyrin: synthesis, analytical properties and catalytic activity

A substituted porphyrin bearing a crown ether unit, H 2( M 18 C 6 P ), was synthesized from the reaction between (5-mono(o-aminophenyl)10,15,20-triphenylporphyrin) and the acyl derivative of the ether (4-carboxy-18-crown-6). The corresponding iron porphyrin, Fe ( M 18 C 6 P ) Cl , was obtained through iron insertion into H 2( M 18 C 6 P ). The new free-base porphyrin was analyzed by C , N , and H elemental analysis, UV-vis and IR spectroscopies, electrospray mass spectrometry (MS) and 1 H NMR. It selectively extracts 18% of the K + ions present in aqueous medium to the organic phase (chloroform). UV-vis spectroscopy studies showed that H 2( M 18 C 6 P ) is also capable of forming aggregates with K +, probably the 1:1 complex, in methanol. This happens because the ionic radius of the potassium ion is appropriate for its accommodation inside the cavity of the crown ether, and also because of the associative effect between the porphyrin and the crown ether unit. The iron porphyrin Fe ( M 18 C 6 P ) Cl was used as catalyst for the epoxidation of cyclooctene by iodosylbenzene or sodium hypochlorite, in methanol or biphasic water/dichloroethane system, respectively. The results obtained with sodium hypochlorite show that Fe ( M 18 C 6 P ) Cl is a potential catalyst for selective oxidations, because the crown ether unit can select or orient substrates. This iron porphyrin can also be employed as a catalyst in a biphasic water/organic solvent system, with no need of a phase transfer agent.