Disulfide-masked iron prochelators: Effects on cell death, proliferation, and hemoglobin production.

The iron metabolism of malignant cells, which is altered to ensure higher acquisition and utilization, motivates the investigation of iron chelation strategies in cancer treatment. In a prochelation approach aimed at increasing intracellular specificity, disulfide reduction/activation switches are incorporated on iron-binding scaffolds resulting in intracellularly activated scavengers. Herein, this strategy is applied to several tridentate donor sets including thiosemicarbazones, aroylhydrazones and semicarbazones. The novel prochelator systems are antiproliferative in breast adenocarcinoma cell lines (MCF-7 and metastatic MDA-MB-231) and do not result in the intracellular generation of oxidative stress. Consistent with iron deprivation, the tested prochelators lead to cell-cycle arrest at the G1/S interface and induction of apoptosis. Notably, although hemoglobin-synthesizing blood cells have the highest iron need in the human body, no significant impact on hemoglobin production was observed in the MEL (murine erythroleukemia) model of differentiating erythroid cells. This study provides new information on the intracellular effects of disulfide-based prochelators and indicates aroylhydrazone (AH1-S)2 as a promising prototype of a new class of antiproliferative prochelator systems.

Peroxidase activity of cytochrome C facilitates the protoporphyrinogen oxidase reaction.

Protoporphyrinogen oxidase (PPO) catalyzes the penultimate reaction in heme biosynthesis. The 'oxygen dependent' form of this enzyme can utilize three molecules of oxygen as electron acceptors in the reaction. In the current study, the ability of cytochrome c to serve as an electron acceptor for PPO was examined. Cytochrome c was found to enhance the catalytic rate of Drosophila melanogaster PPO under reduced oxygen conditions, and cytochrome c became reduced during PPO catalysis. Further kinetic analysis under anaerobic conditions revealed that hydrogen peroxide, a byproduct of the PPO reaction, is required for this rate enhancement to occur. This suggests that the generation of free radicals via the peroxidase activity of cytochrome c plays a part in this rate enhancement, rather than cytochrome c acting as an electron acceptor for the PPO reaction. Given the abundance of cytochrome c in the intermembrane space of mitochondria, the cellular location of PPO, this process may potentially impact on the synthesis of heme in vivo particularly in conditions of low oxygen or hypoxia.

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.

Terminal steps of haem biosynthesis.

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (coproporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.

Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription.

Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined. In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution. The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices. Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC'. This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter. Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site.

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.

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus.

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.

The 2.0 A structure of human ferrochelatase, the terminal enzyme of heme biosynthesis.

Human ferrochelatase (E.C. 4.99.1.1) is a homodimeric (86 kDa) mitochondrial membrane-associated enzyme that catalyzes the insertion of ferrous iron into protoporphyrin to form heme. We have determined the 2.0 A structure from the single wavelength iron anomalous scattering signal. The enzyme contains two NO-sensitive and uniquely coordinated [2Fe-2S] clusters. Its membrane association is mediated in part by a 12-residue hydrophobic lip that also forms the entrance to the active site pocket. The positioning of highly conserved residues in the active site in conjunction with previous biochemical studies support a catalytic model that may have significance in explaining the enzymatic defects that lead to the human inherited disease erythropoietic protoporphyria.

Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes.

Several porphyrinogenic xenobiotics elicit mechanism-based inactivation of cytochrome P450 (CYP) isozymes, leading to the formation of N-alkylprotoporphyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal enzyme in heme biosynthesis. Recognizing their role in experimental porphyria, our long term objective is the establishment of an appropriate in vitro system for the detection and quantification of N-alkylPPs, formed in human liver after the administration of potential porphyrinogenic compounds. In a previous study, we used a combination of thin-layer chromatography and UV-visible spectrophotometry to isolate and identify N-alkylPPs after incubating porphyrinogenic compounds with rat liver microsomes. However, the overall yield of N-alkylPPs was low, and it was concluded that in vitro systems, such as human lymphoblastoid microsomal preparations containing single cDNA-expressed human cytochrome P450 (CYP) isozymes, do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP. In the present study we demonstrate that purified recombinant human ferrochelatase (FC) provides an extremely sensitive bioassay system for N-alkylPPs and is capable of detecting N-alkylPP in the 10(-6) nmol range. Therefore, we propose that this bioassay system might allow the use of human lymphoblastoid microsomal preparations containing single cDNA-expressed human CYP isozymes to detect N-alkylPP produced after mechanism-based (catalysis-based) CYP inactivation. If this is found to be correct it will facilitate identification of potentially porphyrinogenic drugs prior to administration to humans.

Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases.

Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1). Human and Schizosaccharomyces pombe forms of ferrochelatase contain a [2Fe-2S] cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity. Truncation mutants of S. cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity. Chimeric mutants of human, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10% of the carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90% S. cerevisiae. Activity was assessed by rescue of Deltahem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV-visible and EPR spectroscopy were used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutants that were constructed produced active enzymes. HYB, which is predominately human with the last 40 amino acids being that of S. cerevisiae, is an active protein which does not contain a [2Fe-2S] cluster. The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verified by UV-visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzyme activity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.

N-Methylprotoporphyrin is a more potent inhibitor of recombinant human than of recombinant chicken ferrochelatase.

The potency of N-methylprotoporphyrin IX (N-methylPP) as a ferrochelatase (FC) inhibitor has been previously studied using crude chick embryo liver FC preparations. However, interactions between N-methylprotoporphyrin IX (N-methylPP) and impurities in the enzyme preparation may have compromised the results. The first objective of this study was to compare the potency of N-methylPP as an inhibitor of purified chicken FC and crude chick embryo liver FC. The EC(50) values of N-methylPP previously observed in crude chick embryo liver FC was 2.9 x 10(-3) nmol/mg protein, and with purified recombinant chicken FC was 2.07 x 10(-3) nmol/mg protein. The difference in EC(50) values was not statistically significant, and we conclude that interactions between N-methylPP and impurities in crude enzyme preparations did not affect the estimation of potency of N-methylPP. The second objective of this study was to compare the potency of N-methylPP between purified human and chicken FC. The EC(50) value of N-methylPP observed in the purified human FC preparation was 1.7 x 10(-6) nmol/mg protein (chicken FC 2.07 x 10(-3) nmol/mg protein). Thus, the potency of N-methylPP was much higher with purified human FC than with purified chicken FC. Because the porphyrinogenicity of several xenobiotics involves N-alkylprotoporphyrin IX formation, results on drug-induced porphyria obtained with avian species may underestimate the potential porphyrinogenicity in humans.

Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters.

Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.

Multiple regulatory steps in erythroid heme biosynthesis.

Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent down-stream rate-limiting steps.

Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe-2S] cluster. Gel filtration chromatography and dynamic light scattering measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453+/-10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a=93.5 A, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.

Purification, crystallization and preliminary X-ray analysis of Drosophila melanogaster ferrochelatase.

Ferrochelatase (protoheme ferrolyase, E.C. 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. In eukaryotes, the protein is associated with the inner surface of the inner mitochondrial membrane, and in higher animals the enzyme contains a [2Fe-2S] cluster. This cluster is highly sensitive to NO and is coordinated by four Cys residues whose spacing in the primary sequence is unique. Ferrochelatase from Drosophila melanogaster has been expressed in Escherichia coli with an amino-terminal six-histidine tag and purified to homogeneity. The protein has been crystallized with the [2Fe-2S] cluster intact. The crystals belong to space group I422, with unit-cell dimensions a = b = 158.1, c = 171.2 A and two molecules in the asymmetric unit, and diffract to 3. 0 A resolution.

Cloning and characterization of Gallus and Xenopus ferrochelatases: presence of the [2Fe-2S] cluster in nonmammalian ferrochelatase.

Ferrochelatase (EC 4.99.1.1) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. This membrane-bound enzyme has been cloned from a variety of bacteria, plants, mammals, and yeast. Interestingly, only in mammals has the enzyme been found to contain a [2Fe-2S] cluster. Since the presence of this feature only in mammals would have significant evolutionary implications and because there have been no nonmammalian animal ferrochelatases cloned, expressed, and characterized, we report here the cloning and characterization of ferrochelatase from chicken (Gallus gallus) and an amphibian (Xenopus laevis). The cDNAs for both of these ferrochelatases were cloned by complementation of an Escherichia coli DeltahemH strain. The expressed and purified enzymes were characterized biochemically and both were found to contain [2Fe-2S] clusters. These clusters have spectral characteristics essentially identical to those of human ferrochelatase, although their EPR spectra are recognizably distinct from the human one. The [2Fe-2S] clusters of both chicken and amphibian ferrochelatases are readily destroyed by NO. Sequence analysis of the 3' UTR of both chicken and amphibian cDNAs show that while both have poly(A) tails neither have a consensus polyadenylation signal. The 5' UTR of Xenopus as isolated contained 135 bp and possesses no identifiable stem-loop structure.

Molecular characterization of homozygous variegate porphyria.

Variegate porphyria (VP) is a low penetrance, autosomal dominant disorder that results from partial deficiency of protoporphyrinogen oxidase (PPOX) activity caused by mutation in the PPOX gene. The rare homozygous variant of VP is characterized by severe PPOX deficiency, onset of photosensitization by porphyrins in early childhood, skeletal abnormalities of the hand and, less constantly, short stature, mental retardation and convulsions. We have identified PPOX mutations on both alleles of five of the 11 unrelated patients with homozygous VP reported to date. Two patients were homoallelic for missense mutations (D349A and A433P), while three were heteroallelic. Functional analysis by prokaryotic expression showed that the D349A and A433P and one missense mutation in each of the three heteroallelic patients (G358R in two patients and A219KANA) preserved some PPOX activity (9.5-25% of wild-type). Mutations on the other allele of the heteroallelic patients abolished or markedly decreased activity. There was no relation between genotype assessed by functional analysis and the presence or severity of non-cutaneous manifestations. The mutations were absent from 104 unrelated patients with autosomal dominant VP. Our findings define the molecular pathology of homozygous VP and suggest that mild PPOX mutations occur in the general population but have very low or no clinical penetrance in heterozygotes.

Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis.

The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 microM, respectively. Polyclonal antibody to B. subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 microM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.

Evidence that the fourth ligand to the [2Fe-2S] cluster in animal ferrochelatase is a cysteine. Characterization of the enzyme from Drosophila melanogaster.

In a previous study, site-directed mutagenesis experiments identified three of the four ligands to the [2Fe-2S] cluster in animal ferrochelatase as conserved cysteines in the COOH-terminal extension, Cys-403, Cys-406, and Cys-411 in human ferrochelatase (Crouse, B. R., Sellers, V. M., Finnegan, M. G., Dailey, H. A. & Johnson, M. K. (1996) Biochemistry 35, 16222-16229). The nature of the fourth ligand was left unresolved, and spectroscopic studies raised the possibility of one noncysteinyl, oxygenic ligand. In this work, we report two lines of evidence that strongly suggest the fourth ligand is a cysteine residue. Cysteine at position 196 in human recombinant ferrochelatase when changed to a serine results in an inactive enzyme that is lacking the [2Fe-2S] cluster. Furthermore, whole cell EPR studies demonstrate that in the C196S mutant the cluster fails to assemble. Additionally, the cloning and expression of Drosophila melanogaster ferrochelatase has allowed the identification, by EPR and UV-visible spectroscopy, of a [2Fe-2S]2+ cluster with properties analogous to those of animal ferrochelatases. The observation that Drosophila ferrochelatase contains only four conserved cysteines at positions 196, 403, 406, and 411, is in accord with the proposal that these residues function as cluster ligands. In the case of the ferrochelatase iron-sulfur cluster ligands, NH2-Cys-X206-Cys-X2-Cys-X4-Cys-COOH, the position distant from other ligands may lead to a spatial positioning of the cluster near the enzyme active site or at the interface of two domains, thereby explaining the loss of enzyme activity that accompanies cluster degradation and reinforcing the idea that the cluster functions as a regulatory switch.