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.

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.

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.

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.

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.

Erythroid 5-aminolevulinate synthase is required for erythroid differentiation in mouse embryonic stem cells.

We have examined the induction of the enzymes of the heme biosynthetic pathway during erythroid differentiation of mouse embryonic stem (ES) cells. Following transfer to appropriate medium all of the pathway enzymes are induced within three days. Unlike differentiating mouse erythroleukemia cells (Lake-Bullock, H. and Dailey, H.A. Mol Cell Biol 13:7122-7132, 1993), all of the enzymes appear to be induced simultaneously and not sequentially in differentiating ES cells. The role of erythroid 5-aminolevulinate synthase (ALAS-2) in this differentiation process was examined by disruption of the ALAS-2 gene. The targeting vector used for disruption replaced all of exons 4 to 6 with a selectable neomycin resistance gene. The resulting genetically modified (ALAS-2 knockout) cells, as well as normal ES cells were used to study induction of heme biosynthesis. Following 10 days of culture in methylcellulose media significant morphological differences between the embryoid bodies (EBs) of the two cell lines were observed. ES cells exhibited morphology of typical EBs with a dark field (blood island) in the center, while ALAS-2 knockout ES cells developed very poorly both in size and shape. At 8 days of differentiation, only 3% of all EBs contained visible erythropoietic cells (i.e., stained positively for hemoglobin) in the ALAS-2 knockout cell line, compared with 50% in ES cells. Most of the genes in the heme synthetic pathway were expressed to a stable level within 3 to 6 days after induction in normal ES cells, while the ALAS-2 knockout cell line failed to significantly increase the level of expression of these genes. Fetal beta-globin mRNA was not detectable in the differentiating ALAS-2 knockout cells, whereas mRNA for this gene was detected in normal ES cells within 3 days of differentiation. These results suggest that ALAS-2 is necessary for ES cell erythroid differentiation and that there is an interrelationship between heme and globin synthesis in differentiating ES cells.

Site-directed mutagenesis and spectroscopic characterization of human ferrochelatase: identification of residues coordinating the [2Fe-2S] cluster.

The five cysteines closest to the carboxyl terminus of human ferrochelatase have been individually mutated to serine, histidine, or aspartate residues in an attempt to identify the protein ligands to the [2Fe-2S] cluster. Mutations of cysteines at positions 403, 406, and 411 (C403D, C403H, C406D, C406H, C406S, C411H, and C411S mutants) all resulted in inactive enzyme that failed to assemble the [2Fe-2S] cluster as judged by whole-cell EPR studies. In contrast, mutation of the cysteines at positions 360 and 395 to serines (C360S and C395S mutants) did not affect the enzymatic activity, and the resulting enzyme assembled a [2Fe-2S] cluster that was spectroscopically indistinguishable from the wild-type enzyme. The results indicate that three of the conserved cysteines in the 30-residue C-terminal extension of mammalian ferrochelatase are involved in ligating the [2Fe-2S] cluster. Resonance Raman and variable-temperature magnetic circular dichroism studies of heme-free preparations of human ferrochelatase are reported, and the spectra are best interpreted in terms of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster. Such anomalous coordination could account for the cluster lability compared to similar clusters with complete cysteinyl ligation and hence may be intrinsic to the proposed regulatory role for this cluster in mammalian ferrochelatases.

Function of the [2FE-2S] cluster in mammalian ferrochelatase: a possible role as a nitric oxide sensor.

Ferrochelatase (E.C. 4.99.1.1) is the terminal enzyme of the heme biosynthetic pathway, catalyzing the insertion of ferrous iron into protoporphyrin. In mammals the enzyme contains a labile [2Fe-2S] center. Although this cluster is absent in all prokaryotic, plant, and yeast ferrochelatases, its destruction or elimination from the mammalian enzyme results in loss of enzyme activity. In the current study we present data which clearly demonstrate that mammalian ferrochelatase is strongly inhibited by nitric oxide and that this effect is mediated via destruction of the [2Fe-2S] cluster. Carbon monoxide has no inhibitory effect, and yeast ferrochelatase, which lacks the [2Fe-2S] cluster, is not affected by NO (or CO). EPR and UV-visible absorption of purified recombinant human ferrochelatase provides evidence that NO is targeting the [2Fe-2S] center. UV-visible absorption spectroscopy of both human and murine recombinant ferrochelatase incubated with NO or the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), indicate a rapid loss of the visible absorption spectrum of the [2Fe-2S] cluster. EPR studies of the resulting samples reveal the characteristic axial S = 1/2 resonance, g perpendicular = 2.033, and g parallel = 2.014 of a cysteinyl-coordinated monomeric iron-dinitrosyl cluster degradation product. Parallel spectroscopic studies of spinach ferredoxin, which also contains a [2Fe-2S] cluster, gave no indication of NO-induced cluster degradation under the same experimental conditions. Exposure of DMSO-induced murine erythroleukemia cells exposed to SNAP results in an initial decrease in heme production, suggesting that in vivo the cluster is rapidly destroyed. The potential physiological relevance of these data to the anemias that are found in individuals with chronic infections is discussed.

Cloning, sequence, and expression of mouse protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin. A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria. We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase. The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library. The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp. This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870. The protein as expressed in E. coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen. Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

Generation of resistance to the diphenyl ether herbicide acifluorfen by MEL cells.

The diphenyl ether herbicide acifluorfen has been shown to act by inhibition of the terminal enzyme of the protoporphyrin biosynthetic pathway, protoporphyrinogen oxidase (E.C. 1.3.3.4) (PPO), in plant and animal cells. In the present study we show that long term maintenance of murine erythroleukemia (MEL) cells in acifluorfen, which is normally toxic to these cells at 5 microM concentration, results in cells that grow at a near normal rate in 100 microM acifluorfen. Acifluorfen resistant cells do not have increased levels of PPO activity, nor does the PPO made by these cells have increased resistance to acifluorfenin, but these cells accumulate porphyrin and have elevated levels of heme. Data is presented that suggests the resistance of these MEL cells to acifluorfen may be attributable to induction of a cytochrome P450(s).

Human ferrochelatase is an iron-sulfur protein.

Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed approximately 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40,000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase.

During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.

Serum-free, defined medium for the growth and differentiation of murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are frequently employed to study both cell growth and erythroid differentiation. Although these cells are easily cultured and induced to differentiate, they are routinely maintained in a medium that contains 10%-15% fetal bovine serum. Because of the variability between different lots and the cost of serum, it was desirable to define a serum-free medium in which to culture MEL cells. In the present work, a totally serum-free, defined medium is described that supports both normal cell growth and dimethyl sulfoxide induced differentiation in the two MEL cell lines examined (DS-19 and 270). A variety of hormones and biological compounds are examined in this medium to determine their effects on growth and differentiation. This medium does not support the growth of the mouse hepatoma cell line.

Multiple mechanisms for the regulation of haem synthesis during erythroid cell differentiation. Possible role for coproporphyrinogen oxidase.

Murine erythroleukaemia (MEL) cells are virus-transformed erythroid precursor cells that, when induced to differentiate by dimethyl sulphoxide (DMSO), will initiate haem biosynthesis by the induction and synthesis de novo of all of the enzymes of the haem-biosynthetic pathway. The activities of porphobilinogen (PBG) deaminase (EC 4.3.1.8), coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), ferrochelatase (EC 4.99.1.1) and NADH:ferric iron reductase, as well as the synthesis of the enzyme ferrochelatase and the levels of excreted porphyrins, were monitored during DMSO-induced differentiation of MEL cells in culture. The data demonstrate that PBG deaminase and protoporphyrinogen oxidase activities rise rapidly and early, in comparison with ferrochelatase activity, which rises more slowly, and coproporphyrinogen oxidase activity, which decreases by 60% within 24 h of induction before returning to initial levels by 72 h. NADH:ferric iron reductase activity increases slightly, but is always present at levels higher than needed for haem synthesis. Total immunoprecipitable ferrochelatase also rises slowly and parallels the increase in its activity, suggesting that it is not synthesized early in a slowly processed precursor form. Examination of culture media demonstrated that, whereas excretion of protoporphyrin and coproporphyrin occurs within 24 h of induction, coproporphyrin is excreted in amounts 4-15 times greater than protoporphyrin.

The synthesis of murine ferrochelatase in vitro and in vivo.

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

Inhibition of ferrochelatase during differentiation of murine erythroleukaemia cells.

During dimethyl sulphoxide-induced differentiation of DS-19 murine erythroleukaemia (MEL) cells, the activity of the terminal enzyme of the haem-biosynthetic pathway, ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), is thought to be the rate-limiting step for haem production. Differentiation of induced MEL cells in the presence of exogeneously supplied protoporphyrin IX showed that total haem production was affected by added porphyrin only after 48 h. These data suggest that iron insertion, the terminal step, is rate-limiting during the first 48 h of differentiation. Addition of low levels of diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine to differentiating cultures resulted in decreased haem production and decreased ferrochelatase activity. N-Methylprotoporphyrin at nanomolar concentrations also strongly inhibited ferrochelatase activity, but had no inhibitory effect on cellular haem production. The bivalent cations Co2+, Cd2+ and Mn2+ were tested for their effect on haem production and ferrochelatase activity. All three metals were found to inhibit both haem formation and ferrochelatase activity, with Mn2+ being the strongest effector. These data, together with those previously published, suggest that the terminal step in haem biosynthesis is rate-limiting during the early stages of differentiation in MEL cells.

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media. The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively. Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate. Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques. The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.

Iron transport and its relation to heme biosynthesis in Rhodopseudomonas sphaeroides.

The uptake of iron supplied as ferric citrate or ferric parabactin was examined in aerobically grown whole cells and vesicles of Rhodopseudomonas sphaeroides. Inner and outer membrane fractions from R. sphaeroides contained no membrane proteins which were inducible by growth in low-iron medium. Vesicles composed of the inner membrane and devoid of outer membrane and periplasmic proteins were able to transport iron supplied as ferric citrate and ferric parabactin. This uptake required the presence of NADH. When the electrical component of the proton motive force was depleted in whole cells, the uptake of iron supplied as ferric parabactin was completely inhibited. The uptake of iron supplied as ferric citrate was inhibited by gallium citrate; however, Ga3+ was not transported. The relationship between iron uptake and heme synthesis was examined by treating whole cells with N-methylprotoporphyrin which inhibits ferrochelatase, the enzyme which inserts ferrous iron into protoporphyrin to form heme. This treatment reduced ferrochelatase activity by 82% but had no effect on iron uptake, indicating that iron uptake and heme synthesis are not directly coupled. The fate of transported iron was investigated by measuring intracellular concentrations of heme and nonheme iron. It was determined that newly transported iron exists primarily as nonheme iron.

Siderophore utilization and iron uptake by Rhodopseudomonas sphaeroides.

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.

Differential interaction of porphyrins used in photoradiation therapy with ferrochelatase.

The mechanism of porphyrin accumulation by tumours is not yet established. If metabolism aids porphyrin elimination, tumours, unlike normal tissues, may not metabolize porphyrins used clinically, such as proto-, haemato-, OO'-diacetyl-haemato- and monohydroxyethyl-monovinyl-deutero-porphyrin. Proto-, haemato- and monohydroxyethyl-monovinyl-deutero-porphyrin are substrates for the mitochondrial enzyme ferrochelatase (EC 4.99.1.1), which can form haem analogues from exogenous porphyrins. The Km values for proto-, haemato- and monohydroxyethyl-monovinyl-deutero-porphyrin are 11, 22 and 23 microM respectively. However, OO'-diacetyl-haematoporphyrin is an effective competitive inhibitor with Ki of 11 microM. Hepatic ferrochelatase specific activity is 5.9 and 5.5 nmol of haem/h per mg of protein respectively in normal Buffalo rat and in those bearing the extrahepatic Morris 7288C hepatoma, and is only 0.13 nmol/h per mg in the hepatomas. Therefore low ferrochelatase activity in cancerous cells may provide one means whereby some porphyrins accumulate in tumours, and the ability of certain porphyrins to act as ferrochelatase inhibitors may provide another.