Stages in iron storage in the ferritin of Escherichia coli (EcFtnA): analysis of Mössbauer spectra reveals a new intermediate.

Iron uptake into the nonheme ferritin of Escherichia coli (EcFtnA) and its site-directed variants have been investigated by Mössbauer spectroscopy. EcFtnA, like recombinant human H chain ferritin (HuHF), oxidized Fe(II) at a dinuclear ferroxidase center situated at a central position within each subunit. As with HuHF, Mössbauer subspectra observed between 1 min and 24 h after Fe(II) addition were assigned to Fe(III) monomers, "c", mu-oxo-bridged dimers, "b", and clusters, "a", the latter showing magnetically split spectra, "d", at 4.1 K. Like those of HuHF, the mu-oxo-bridged dimers were formed at the ferroxidase centers. However, the analysis also revealed the presence of a new type of dimer, "e" (QS1 = 0.38 mm/s, IS1 = 0.51 mm/s and QS2 = 0.72 mm/s, IS2 = 0.50 mm/s), and this was also assigned to the ferroxidase center. Dimers "b" appeared to be converted to dimers "e" over time. Subspectra "e" became markedly asymmetric at temperatures above 90 K, suggesting that the two Fe(III) atoms of dimers "e" were more weakly coupled than in the mu-oxo-bridged dimers "b", possibly due to OH- bridging. Monomeric Fe(III), giving relaxation spectra "c", was assigned to a unique site C that is near the dinuclear center. In EcFtnA all three iron atoms seemed to be oxidized together. In contrast to HuHF, no Fe(III) clusters were observed 24 h after the aerobic addition of 48 Fe(II) atoms/molecule in wild-type EcFtnA. This implies that iron is more evenly distributed between molecules in the bacterial ferritins, which may account for its greater accessibility.

Iron in parkinsonian and control substantia nigra--a Mössbauer spectroscopy study.

We used Mössbauer spectroscopy to study the iron content, the redox state, and the binding site of iron in substantia nigra (SN) from parkinsonian (PD) and control brains. Measurements performed on fresh-frozen, formalin-fixed, and lyophilized samples demonstrated the presence of ferric (Fe3+) iron only, both in PD and control SN. Ferrous iron, if present at all, may represent at most 5% of the total iron. We found no difference in the total amount of iron in SN between PD and control brains. The Mössbauer spectra observed at 4.1 K in fresh (frozen or lyophilized) samples were different from those obtained in formalin-fixed (frozen or lyophilized) samples. In the fresh samples, only ferritin-like iron was observed, whereas in the samples frozen or lyophilized from formalin, non-ferritin iron was detected.

Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli.

Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores. Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation. Mössbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin. Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN). A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III). Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex. Mössbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins. Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components. This provides further evidence that the ferritin protein shell can influence the structure of its iron core.

[Mossbauer spectroscopy of iron in substantia nigra in Parkinson disease and controls]. / Spektroskopia Mössbauerowska zaelaza istoty czarnej w chorobie Parkinsona i kontroli.

Mössbauer spectroscopy was used to study iron content, its redox state and binding sites in substantia nigra from parkinsonian and control brains. Measurements performed on fresh frozen samples demonstrated the presence of ferric iron only, both in disease and control. We found no difference in the total amount of iron in substantia nigra between the disease and control. Mössbauer spectra observed at 4.1 K in fresh frozen samples were different from those obtained in formalin fixed samples. In the fresh frozen samples only ferritin like iron was observed, whereas in the formalin fixed samples also non-ferritin iron was detected. It seems that in formalin fixed brains, during years, iron is released from ferritin and bound to an iron chelator or formalin.

Iron (II) oxidation and early intermediates of iron-core formation in recombinant human H-chain ferritin.

The paper describes a study of Fe(II) oxidation and the formation of Fe(III)-apoferritin complexes in recombinant human H-chain ferritin and its variants. The effects of site-directed changes in the conserved residues associated with a proposed ferroxidase centre have been investigated. A change in any of these residues is shown to reduce the rate of Fe(II) oxidation, confirming the importance of the ferroxidase centre in the catalysis of Fe(II) oxidation. Mössbauer and u.v.-difference spectroscopy show that in the wild-type protein Fe(II) oxidation gives rise to Fe(III) monomers, dimers and larger clusters. The formation of Fe(III) mu-oxo-bridged dimers occurs at the ferroxidase centre and is associated with fast oxidation: in three variants in which Fe(II) oxidation is especially slow, no Fe(III) dimers are seen. Within the time scale 0.5-20 min in wild-type human H-chain ferritin, dimer formation precedes that of the monomer and the progression dimer-->monomer-->cluster is observed, although not to completion. In a preliminary investigation of oxidation intermediates using a stopped-flow instrument, an Fe(III)-tyrosine complex reported by Waldo et al. (1993), is attributed to Tyr-34, a residue at the ferroxidase centre. The Fe(III)-Tyr-34 complex, forms in 0.5 s and then decays, as dimer absorbance increases. The relationship between Fe(III)-tyrosinate and the formation of Fe(III) dimers is uncertain.

Defining the roles of the threefold channels in iron uptake, iron oxidation and iron-core formation in ferritin: a study aided by site-directed mutagenesis.

This paper aims to define the role of the threefold intersubunit channels in iron uptake and sequestration processes in the iron-storage protein, ferritin. Iron uptake, measured as loss of availability of Fe(II) to ferrozine (due to oxidation), has been studied in recombinant human H-chain ferritins bearing amino acid substitutions in the threefold channels or ferroxidase centres. Similar measurements with recombinant horse L-chain ferritin are compared. It is concluded that significant Fe(II) oxidation occurs only at the H-chain ferroxidase centres and not in the threefold channels, although this route is used by Fe(II) for entry. Investigations by Mössbauer and u.v.-difference spectroscopy show that part of the iron oxidized by H-chain ferritin returns to the threefold channels as Fe(III). This monomeric Fe(III) can be displaced by addition of Tb(III). Fe(III) also moves into the cavity for formation of the iron-core mineral, ferrihydrite. Iron incorporated into ferrihydrite becomes kinetically inert.

Further evidence for the interaction of the antimalarial drug amodiaquine with ferriprotoporphyrin IX.

Evidence for complex formation of the antimalarial drug amodiaquine (AD) with ferriprotoporphyrin IX (FP) in aqueous medium is presented, in addition to previous preliminary data. A mole ratio of one between the complex components is determined for the insoluble complex at pH 6.7-6.8. Mössbauer data obtained at pH 7-8 and at higher concentrations in the millimolar range confirm the interactions existing between the complex components. These data are considered to aid in removing previous objections to a mechanism of antimalarial action involving complexes of FP with AD and related drugs.

Structure and composition of ferritin cores from pea seed (Pisum sativum).

Iron cores from native pea seed (Pisum sativum) ferritin have been analysed by electron microscopy and Mössbauer spectroscopy and shown to be amorphous. This correlates with their relatively high phosphate content (Fe: P = 2.83; 1800 Fe, 640 P atoms/molecule). Reconstituted cores obtained by adding iron (2000 Fe atoms/molecule) in the absence of phosphate to pea seed apoferritin were crystalline ferrihydrite. In vitro rates of formation of pea-seed ferritin iron cores were intermediate between those of recombinant human H-chain and horse spleen apoferritin and this may reflect the amino-acid residues of its ferroxidase and putative nucleation centres. The high phosphate content of pea-seed ferritin suggests that this molecule could be involved in both phosphorus and iron storage. The high phosphate concentration found within plastids, from which the molecules were isolated, is a possible source of the ferritin phosphate.

Deferoxamine-induced iron mobilization and redistribution of myocardial iron in cultured rat heart cells: studies of the chelatable iron pool by electron microscopy and Mössbauer spectroscopy.

Iron mobilization by deferoxamine from iron-loaded rat heart cells in culture was studied by electron microscopy and Mössbauer spectroscopy to identify the chelatable iron pool. Studies in which iron 59 was used have shown a diminishing response to deferoxamine with increasing time intervals, which suggests a gradual transit from a more available to a less available storage iron compartment. Mössbauer spectroscopy showed that practically all iron mobilized by deferoxamine was derived from the small (less than 3.0 nm) recently acquired iron particles, which supports the "last-in, first-out" principle. Quantitation of cytosolic ferritin iron particles has shown a highly reproducible increase in cytosolic ferritin iron after deferoxamine treatment. This intracellular redistribution of iron stores is explained either by a reduced transfer of cytosolic ferritin into siderosomes or, more likely, by increased mobilization of membrane-bound iron deposits from insoluble polynuclear iron complexes in siderosomes and their subsequent incorporation into cytosolic ferritin. Thus the protective effect of deferoxamine on iron-loaded heart cells may be twofold: (1) net removal of excess iron by the formation of a stable complex of iron with deferoxamine and its secretion into the extracellular environment and (2) a shift of solubilized iron from membrane-bound deposits into the cytosol where iron is detoxified by its incorporation into the hollow shell of the ferritin protein.

Mössbauer spectroscopic investigation of structure-function relations in ferritins.

Ferritin plays an important role in iron metabolism and our aim is to understand the mechanisms by which iron is sequestered within its protein shell as the mineral ferrihydrite. We present Mössbauer spectroscopic data on recombinant human and horse spleen ferritin from which we draw the following conclusions: (1) that apoferritin catalyses Fe(II) oxidation as a first step in ferrihydrite deposition, (2) that the catalysis of Fe(II) oxidation is associated with residues situated within H chains, at the postulated 'ferroxidase centre' and not in the 3-fold inter-subunit channels previously suggested as the initial Fe(II) binding and oxidation site; (3) that both isolated Fe(III) and Fe(III) mu-oxo-bridged dimers found previously by Mössbauer spectroscopy to be intermediates in iron-core formation in horse spleen ferritin, are located on H chains; and (4) that these dimers form at ferroxidase centres. The importance of the ferroxidase centre is suggested by the conservation of its ligands in many ferritins from vertebrates, invertebrates and plants. Nevertheless iron-core formation does occur in those ferritins that lack ferroxidase centres even though the initial Fe(II) oxidation is relatively slow. We compare the early stages of core formation in such variants and in horse spleen ferritin in which only 10-15% of its chains are of the H type. We discuss our findings in relation to the physiological role of isoferritins in iron storage processes.

Iron (III) can be transferred between ferritin molecules.

The iron-storage molecule ferritin can sequester up to 4500 Fe atoms as the mineral ferrihydrite. The iron-core is gradually built up when FeII is added to apoferritin and allowed to oxidize. Here we present evidence, from Mössbauer spectroscopic measurements, for the surprising result that iron atoms that are not incorporated into mature ferrihydrite particles, can be transferred between molecules. Experiments were done with both horse spleen ferritin and recombinant human ferritin. Mössbauer spectroscopy responds only to 57Fe and not to 56Fe and can distinguish chemically different species of iron. In our experiments a small number of 57FeII atoms were added to two equivalent apoferritin solutions and allowed to oxidize (1-5 min or 6 h). Either ferritin containing a small iron-core composed of 56Fe, or an equal volume of NaCl solution, was added and the mixture frozen in liquid nitrogen to stop the reaction at a chosen time. Spectra of the ferritin solution to which only NaCl was added showed a mixture of species including 57FeIII in solitary and dinuclear sites. In the samples to which 150 56FeIII-ferritin had been added the spectra showed that all, or almost all, of the 57FeIII was in large clusters. In these solutions 57FeIII initially present as intermediate species must have migrated to molecules containing large clusters. Such migration must now be taken into account in any model of ferritin iron-core formation.

Oxidative stress in newborn erythrocytes.

Phenylhydrazine (PHZ) exposure is used to study in vitro red cell aging mechanisms dependent on Hb oxidation. The effect of PHZ on normal neonatal red blood cells was studied in unseparated blood and after separation into light and heavy cells. PHZ caused more extensive morphologic changes in neonatal than in adult red blood cells. PHZ exposure of neonatal cells caused less reduced glutathione depletion than in adult cells. Although we found the same total amount of oxidized Hb in both cells, a well-defined oxidation product of Hb was demonstrated by Mössbauer spectra only in neonatal cells. This oxidation product was not methemoglobin but a trivalent, high-spin iron compound. All neonatal cell populations were more sensitive to PHZ than were adult ones, as demonstrated by the presence of Heinz bodies at low PHZ concentration, which did not affect adult cells. These studies demonstrate greater sensitivity of neonatal cells to PHZ in all density-separated populations.

Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates.

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).

Chemical and Mössbauer spectroscopic evidence that iron-containing concanavalin A is a ferritin.

We report here the preparation of iron-containing concanavalin A. It has a protein-to-iron ratio of 2.0, and the iron compound it contains is particulate with an average diameter of 85 A. Iron-containing concanavalin A interacts reversibly with dextran and with methyl alpha-D-glucoside. The molecular basis of these findings is discussed and a possible mechanism suggested where one of the molecular forms of concanavalin A has the structure of an apoferritin into which iron is deposited in the form of ferrihydrite.

Ultrastructural pathology of iron-loaded rat myocardial cells in culture.

The pathological changes induced by in-vitro iron-loading or cultured rat myocardial cells were studied. Cells were exposed to 59Fe-labelled ferric ammonium citrate for up to 24 h followed by 24-72 h chase experiment. After 24 h exposure 29% of the total cellular radioactivity was found in ferritin, 10% in non-ferritin heat supernatant and 61% in an insoluble heat-precipitable form. Mössbauer spectroscopy showed a gradual shift from intracellular iron particles less than 1.8 nm in diameter, through particles of intermediate size, to ferritin-like aggregates over 3.0 nm in diameter, reaching about 20% of total iron by 24 h. Ultrastructural studies showed premature damage such as mitochondrial abnormalities and excessive autophagocytosis. Small, 2.0-5.0 nm electron-dense cytosolic particles were noticed at 3 h of iron loading and reached maximal concentrations at 6 h. This was followed by accumulation of the small particles and of typical iron-rich ferritin cores within siderosomes. Because of the limited duration of iron loading and the high concentrations of non-transferrin inorganic iron employed, the present model is more relevant to acute than chronic iron overload. The efficient incorporation of large amounts of iron within ferritin molecules and its subsequent segregation, together with other smaller particles, within membrane-bound bodies, may represent a defence mechanism limiting iron toxicity in the face of advanced cytosiderosis.

The malarial pigment in rat infected erythrocytes and its interaction with chloroquine. A Mössbauer effect study.

Mössbauer studies of rat erythrocytes infected by Plasmodium berghei malaria parasites, using 57Fe-enriched rat red blood cells, were carried out in order to determine the physical parameters which characterize the malarial pigment iron and to test the effect of the widely used antimalaria drug, chloroquine, on these parameters. The iron in the malarial pigment which is derived from hemoglobin digestion by the intracellular parasite was found to be trivalent, high spin, with Mössbauer parameters which are significantly different from those of any known iron porphyrin containing compound. No difference was found between the parameters obtained in erythrocytes infected by drug-sensitive and drug-resistant strains of P. berghei, both before and after the treatment with chloroquine. The iron compound consists of microaggregates, about 30 A in diameter. These are somewhat larger in chloroquine-resistant strains and tend to increase in size in chloroquine-sensitive strains upon treatment with the drug. Mössbauer spectra of erythrocytes infected by a chloroquine-resistant strain revealed pigment iron in relative amounts invariable of those found in chloroquine-sensitive strains, demonstrating that drug-resistant parasites indeed digest hemoglobin.

Magnetosome dynamics in magnetotactic bacteria.

Diffusive motions of the magnetosomes (enveloped Fe3O4 particles) in the magnetotactic bacterium Aquaspirillum magnetotacticum result in a very broad-line Mössbauer spectrum (T approximately 100 mm/s) above freezing temperatures. The line width increases with increasing temperature. The data are analyzed using a bounded diffusion model to yield the rotational and translational motions of the magnetosomes as well as the effective viscosity of the material surrounding the magnetosomes. The results are [theta 2] l/2 less than 1.5 degrees and [x2] 1/2 less than 8.4 A for the rotational and translational motions, respectively, implying that the particles are fixed in whole cells. The effective viscosity is 10 cP at 295 K and increases with decreasing temperature. Additional Fe3+ material in the cell is shown to be associated with the magnetosomes. Fe2+ material in the cell appears to be associated with the cell envelope.

Iron storage in ferritin following intracellular hemoglobin denaturation in erythroleukemic cells.

Murine erythroleukemia (MEL) and human K-562 cell lines were cultured in the presence of 57Fe, and the quantities of cellular iron-containing compounds were determined with the aid of Mössbauer spectroscopy. Upon induction of differentiation, both ferritin-iron and hemoglobin (Hb) iron could be detected. Treatment of the cells with 0.01%-0.02% acetylphenylhydrazine (APH) resulted in gradual denaturation of Hb and incorporation of the released Hb-iron into ferritin. Following treatment with APH, the ratio of Hb-57Fe to ferritin-57Fe decreased from 2.6 to 0.2 in MEL cells and from 0.56 to 0.12 in K-562 cells. No change was observed in the total intracellular iron. Using fluorescence ELISA, an increased level of immunologically detectable ferritin was found in hemoglobinized K-562 cells treated with APH, as compared to the amount of ferritin found in untreated cells. Ferritin may thus function not only as an intermediate during Hb synthesis, but also as storage protein for iron released during Hb denaturation.