Studies on the utilization of ferritin iron in the ferrochelatase reaction of isolated rat liver mitochondria.

The utilization of ferritin as a source of iron for the ferrochelatase reaction has been studied in isolated rat liver mitochondria. 1. It was found that isolated rat liver mitochondria utilized ferritin as a source of iron for the ferrochelatase reaction in the presence of succinate plus FMN (or FAD). 2. Under optimal experimental conditions, i.e., approx. 50 micromol/1 FMN, 37 degrees C, pH 7.4 and 0.5 mmol/l Fe(III) (as ferritin iron), the release process, as shown by the formation of deuteroheme, amounted to approx. 0.5 nmol iron/min per mg protein. 3. The release process could not be elicited by ultrasonically treated mitochondria, lysosomes, microsomes or cytosol, i.e., the release of iron from ferritin was due to mitochondria and was a function of the in situ orientation of the mitochondrial inner membrane. 4. The release of iron from ferritin by the mitochrondria might be of relevance not only for the in situ synthesis of heme in the hepatocyte, but also with respect to the mechanism(s) by means of which iron is mobilized for transport to the erythroid tissue.     

A human mitochondrial ferritin encoded by an intronless gene

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

Biophysical investigation of the ironome of human jurkat cells and mitochondria

The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with (57)Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K M?ssbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to S = 0 [Fe(4)S(4)](2+) clusters and low-spin (LS) Fe(II) heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be Fe(III) oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) Fe(II) species. A portion of these species may constitute the "labile iron pool" (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. Electron paramagnetic resonance spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS Fe(III) heme and non-heme species. The basal heme redox state of mitochondria within cells was reduced; this redox poise was unaltered during the anaerobic isolation of the organelle. Contributions from heme a, b, and c centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using inductively coupled plasma mass spectrometry. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells - the first "ironome" profile of a human cell.     

Mobilization of ferritin iron in erythroblasts by chelating agents

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.     

The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.     

Ferritin is not a required intermediate for iron utilization in heme synthesis

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.     

Mobilization of iron from ferritin by isolated mitochondria effects of species compatibility between ferritin and mitochondria and iron content of ferritin

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.     

Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain

Abstract : Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using ¹²⁵I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant ( K _D) of 4.65 × 10^-9 M and a binding site density ( B _max) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi     

Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.     

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

Kinetics of iron release from pig spleen ferritin with bare platinum electrode reduction

Several anaerobic electrochemical cells were employed to study the kinetics of iron release from pig spleen ferritin (PSF) at a bare platinum electrode. Controlled potential microcoulometry (CPM) is the principal technology used to investigate the kinetics in the absence of a mediator. A kinetic study of iron release by microcoulometry has revealed that ferritin undergoes direct electron transfer at the electrode in the absence of a mediator, indicating that ferritin is an electroactive protein. Several experiments failed to show that alpha'alpha-bipyridyl has the capacity to reduce hydrolyzed Fe(3+) within the ferritin core after it has been reduced by the electrode at -600 mV vs. NHE in the absence of mediator. PSF is known to bind heme to generate a hemeoprotein, named pig spleen hemeoferritin (PSF(ho)). The rate of iron release is accelerated by the heme binding to PSF(ho) without the need for small mediators. Under similar conditions, two kinetic processes for iron release from PSF and bacterial ferritin of Azoaobacter vinelandii (AvBF) were studied and both fit a zero-order law. In addition, the rate of iron release in PSF can be accelerated two-fold by a specific reduction system consisting of ascorbic acid (AA) and the bare platinum electrode at -600 mV. However, this kinetic process does not follow zero-, half-, first, or second-order rate laws. A model is proposed to explain a mechanism of direct electron transfer between ferritin and the electrode is derived to describe the kinetics of iron release.     

Physiological implications of mammalian ferritin-binding proteins interacting with circulating ferritin and a new aspect of ferritin- and zinc-binding proteins

Serum ferritin levels are relatively low (<1 µg/ml) and serum ferritin generally disappears rapidly from the circulation (t _1/2 < 10 min). There are various mammalian ferritin-binding proteins (FBPs) in the blood. Ferritin is cleared by direct uptake by ferritin receptors and by indirect receptor-mediated uptake of FBP complexed with ferritin. Mammalian ferritin binds both heme and iron, and binding occurs through two mechanisms: direct binding with ferritin to H-kininogen and anti-ferritin autoantibody, and indirect heme-mediated binding of fibrinogen and apolipoprotein B to ferritin. Anti-ferritin autoantibody and fibrinogen are proposed to be common mammalian FBPs, as is α₂-macroglobulin. FBP-ferritin binding may affect blood coagulation and influence iron metabolism, oxidative condition, angiogenesis, inflammatory condition and immune response. Aside from apolipoprotein B, FBPs bind zinc ion to form antioxidant and anti-inflammatory agents. The possible simultaneous uptake of zinc ion with FBP-ferritin complex is likely to attenuate iron- and/or heme-mediated oxidative damage and inflammatory response.     

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.     

Iron uptake and heme synthesis by isolated rat liver mitochondria. Diferric transferrin as iron donor and the effect of pyrophosphate

Isolated rat liver mitochondria accumulate iron from fully saturated transferrin at neutral pH. With 5 microM iron as diferric transferrin, accumulation at 30 degrees C amounts to approx. 40 pmol/mg protein per h. With access to a suitable porphyrin substrate, 70-80% of the amount of iron accumulated is recovered in heme. Mobilization of iron and synthesis of heme both depend on a functioning respiratory chain. Vacant iron-binding sites on mono- and apotransferrin compete with the mitochondria for iron mobilized from transferrin. Pyrophosphate at concentrations in the range 10-50 microM enhances mobilization of iron, counterbalances the inhibitory effect of mono- and apotransferrin and enhances metallochelatase activity. The results emphasize the putative suitability of pyrophosphate as an intracellular iron-transport ligand in situ.     

Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.