M?ssbauer 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 M?ssbauer 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 M?ssbauer 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.     

M?ssbauer 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 M?ssbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic M?ssbauer 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).     

Ferritin: design and formation of an iron-storage molecule

Although essential for most forms of life, too much iron is harmful. To cope with these antagonistic phenomena an iron-storage molecule, ferritin, has evolved. The structure of horse spleen apoferritin, which has recently been refined, consists of 24 symmetrically related subunits forming a near-spherical hollow shell. In ferritin the central cavity is occupied by an iron core of 'ferrihydrite', a geologically ephemeral mineral found in hot or cold springs and in mine workings, or produced in the laboratory by heating solutions of ferric salts. Ferritin itself forms most readily from apoferritin, in the presence of dioxygen, from FeII, not FeIII. Access to its interior is through small intersubunit channels, and the protein influences both the rate of FeII-oxidation and the form of oxide produced.     

Lability of iron at the dinuclear centres of ferritin studied by competition with four chelators

Ferritin molecules contain 24 polypeptide chains folded as four-helix bundles and arranged as a hollow shell capable of storing up to 4500 Fe(III) atoms. H chains contain ferroxidase centres which lie within the bundle, about 12?Å (1.2?nm) from the outside surface and 8?Å from the inner surface of the protein shell. Catalysis of Fe(II) oxidation precedes storage of Fe(III) as ferrihydrite, with the formation of μ-oxo-bridged Fe(III) dimers as intermediates. Factors influencing the movement of μ-oxo-bridged Fe(III) from the ferroxidase centre to the ferritin cavity are uncertain. Assistance by small chelators is one possibility. The aim of this investigation was to determine whether iron at the dinuclear centres of three ferritins (human H chain homopolymer, HuHF, the non-haem ferritin of Escherichia coli, EcFTN, and horse spleen ferritin, HoSF) is accessible to chelators. Forty-eight Fe(II) atoms/molecule were added to the apoferritins followed, 2?min later, by the addition of chelator (1,10-phenanthroline, 2,2-bipyridine, desferrioxamine or 3,4-dihydroxybenzaldehyde). Iron species were analysed by Mössbauer spectroscopy or visible absorbance. Competition between chelators and apoferritin for Fe(II) was also investigated. The main conclusions of the study are that: (1) dinuclear iron and iron in small iron-cores in HuHF and EcFTN is mobilisable by all four chelators; (2) the chelators penetrate the shell; (3) 3,4-dihydroxybenzaldehyde is the most efficient in mobilising Fe(III) but the least successful in competing for Fe(II); (4) Fe(III) is more readily released from EcFTN than from HuHF; (5) 2,2′-bipyridine aids the movement of Fe(III) from ferroxidase centre to core.     

Role of phosphate in initial iron deposition in apoferritin

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe M?ssbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron(III) clusters bound to horse spleen apoferritin: an X-ray absorption and M?ssbauer spectroscopy study that shows that iron nuclei can form on the protein

Ferritin is a complex of a hollow, spherical protein and a hydrous, ferric oxide core of less than or equal to 4500 iron atoms inside the apoprotein coat; the apoprotein has multiple (ca. 12) binding sites for monoatomic metal ions, e.g., Fe(II), V(IV), Tb(III), that may be important in the initiation of iron core formation. In an earlier study we observed that the oxidation of Fe(II) vacated some, but not all, of the metal-binding sites, suggesting migration of some Fe during oxidation, possibly to form nucleation clusters; some Fe(III) remained bound to the protein. Preliminary extended X-ray absorbance fine structure (EXAFS) analysis of the same Fe(III)-apoferritin complex showed an environment distinct from ferritin cores, but the data did not allow a test of the Fe cluster hypothesis. In this paper, with improved EXAFS data and with M?ssbauer data on the same complex formed with 57Fe, we clearly show that the Fe(III) in the distinctive environment is polynuclear (Fe atoms with Fe-Fe = 3.5 A and TB = 7 K). Moreover, the arrangement of atoms is such that Fe(III) atoms appear to have both carboxylate-like ligands, presumably from apoferritin, and oxo bridges to the other iron atoms. Thus the protein provides sites not only for initiation but also for nucleation of the iron core. Sites commodious enough and with sufficient conserved carboxylate ligands to accommodate such a nucleus occur inside the protein coat at the subunit dimer interfaces. Such Fe(III)-apoferritin nucleation complexes can be used to study the properties of the several members of the apoferritin family.     

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

The formation of ferritin from apoferritin. Catalytic action of apoferritin

The iron-storage protein ferritin consists of a protein shell and has an iron content of up to 4500 iron atoms as a microcrystalline ferric oxide hydrate. A study was made of the uptake of ferrous iron by apoferritin in the presence of an oxidizing agent at very low iron:protein ratios. At ratios of less than about 150 iron atoms per apoferritin molecule hyperbolic progress curves were obtained, whereas at higher ratios the curves became sigmoidal under the conditions used. A computer model, developed previously (Macara et al., 1972), was shown to account for this result. The experimental evidence indicates that apoferritin binds ferrous iron and catalyses the initial stage in the formation of the ferric oxide hydrate inside the protein shell. This stage involves the oxidation of sufficient iron within the protein molecule to form a stable nucleus on which the growth of the microcrystalline iron-core particles can proceed. A possible schematic mechanism for the action of apoferritin is suggested.     

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.     

Structural variations in soluble iron complexes of models for ferritin: an x-ray absorption and M?ssbauer spectroscopy comparison of horse spleen ferritin to Blutal (iron-chondroitin sulfate) and Imferon (iron-dextran)

Variations in the turnover of storage iron have been attributed to differences in apoferritin and in the cytoplasm but rarely to differences in the structure of the iron core (except size). To explore the idea that the iron environment in soluble iron complexes could vary, we compared horse spleen ferritin to pharmaceutically important model complexes of hydrous ferric oxide formed from FeCl3 and dextran (Imferon) or chondroitin sulfate (Blutal), using x-ray absorption (EXAFS) and M?ssbauer spectroscopy. The results show that the iron in the chondroitin sulfate complex was more ordered than in either horse spleen ferritin or the dextran complex (EXAFS), with two magnetic environments (M?ssbauer), one (80%-85%) like Fe2O3 X nH2O (ferritinlike) and one (15%-20%) like Fe2O3 (hematite); since sulfate promotes the formation of inorganic hematite, the sulfate in the chondroitin sulfate most likely nucleated Fe2O3 and hydroxyl/carboxyls, which are ligands common to chondroitin sulfate, ferritin and dextran most likely nucleated Fe2O3 X nH2O. Differences in the structure of the iron complexed with chondroitin sulfate or dextran coincide with altered rates of iron release in vivo and in vitro and provide the first example relating function to local iron structure. Differences might also occur among ferritins in vivo, depending on the apoferritin (variations in anion-binding sites) or the cytoplasm (anion concentration).     

Forming the phosphate layer in reconstituted horse spleen ferritin and the role of phosphate in promoting core surface redox reactions.

Apo horse spleen ferritin (apo HoSF) was reconstituted to various core sizes (100-3500 Fe3+/HoSF) by depositing Fe(OH)3 within the hollow HoSF interior by air oxidation of Fe2+. Fe2+ and phosphate (Pi) were then added anaerobically at a 1:4 ratio, and both Fe2+ and Pi were incorporated into the HoSF cores. The resulting Pi layer consisted of Fe2+ and Pi at about a 1:3 ratio which is strongly attached to the reconstituted ferritin mineral core surface and is stable even after air oxidation of the bound Fe2+. The total amount of Fe2+ and Pi bound to the iron core surface increases as the core volume increases up to a maximum near 2500 iron atoms, above which the size of the Pi layer decreases with increasing core size. M?ssbauer spectroscopic measurements of the Pi-reconstituted HoSF cores using 57Fe2+ show that 57Fe3+ is the major species present under anaerobic conditions. This result suggests that the incoming 57Fe2+ undergoes an internal redox reaction to form 57Fe3+ during the formation of the Pi layer. Addition of bipyridine removes the 57Fe3+ bound in the Pi layer as [57Fe(bipy)3]2+, showing that the bound 57Fe2+ has not undergone irreversible oxidation. This result is related to previous studies showing that 57Fe2+ bound to native core is reversibly oxidized under anaerobic conditions in native holo bacterial and HoSF ferritins. Attempts to bury the Pi layer of native or reconstituted HoSF by adding 1000 additional iron atoms were not successful, suggesting that after its formation, the Pi layer "floats" on the developing iron mineral core.     

Mossbauer effect studies in the fungus Phycomyces.

Mossbauer spectra of 57- Fe have been observed from different parts (mycelia, spores, sporangiophores) of the fungus Phycomyces blakesleeanus grown in an agar medium isotopically enriched with 57- Fe. The spectra indicate that the iron within Phycomyces exists primarily in two chemical states: one which is the same as that of the iron in the growth medium and the other in the form of ferritin, an iron-storage protein. The amount of iron in the former state is observed to decrease relative to the amount of iron in the latter state in going from mycelia to the sporangiophores to the sporangia themselves. Thus, the conversion of iron from the chemical state of the nutrient to ferritin has been monitored for different parts of the phycomyces. In addition, our spectra indicate that at low temperatures the iron atoms clustered within a ferritin molecule are antiferromagnetically coupled. The size of these clusters is inferred from their superparamagnetic behavior at low tempertures and comparison with horse ferritin indicates that the phycomyces ferritin iron clusters are smaller by a factor of two.     

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

Iron uptake into the nonheme ferritin of Escherichia coli (EcFtnA) and its site-directed variants have been investigated by M?ssbauer 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, M?ssbauer 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.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.     

Effects of Ceruloplasmin on Superoxide-Dependent Iron Release from Ferritin and Lipid Peroxidation

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin. suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum. and inhibited at higher concentrations. However. the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.     

Identification of catalytic residues involved in iron uptake by L-chain ferritins

When either horse spleen apoferritin (containing more than 90% of L chains) or recombinant horse L apoferritin are modified with glycineamide or taurine in the presence of a water-soluble carbodiimide, a total of 11 to 12 carboxyl groups per subunit are modified, and iron incorporation is effectively abolished. In contrast, when horse spleen ferritin (containing on average 2500 atoms per molecule) is modified under similar conditions, seven to eight carboxyl groups are modified. When apoferritin is prepared from this modified ferritin, it retains full iron incorporation activity. Apoferritin in which seven to eight carboxyls per subunit have been modified by glycineamide can subsequently be modified by taurine; a total of three to four carboxyl groups are modified accompanied by total loss of iron incorporation. Additional studies confirm that three carboxyl groups per subunit are protected from modification by glycineamide by Cr(III) inhibition of iron incorporation. Using tandem mass spectroscopy we have looked for taurine-labelled peptides in tryptic digests of succinylated apoferritins after taurine modification. In the sample where the residues involved in iron uptake have been modified with taurine, we have identified the peptide: This corresponds to residues 53–59 of the L subunit, where it is part of a region of the B-helix which is directed towards the inside of the apoferritin protein shell. The same peptide was identified using classical protein sequencing techniques after (1,2-³H)-taurine modification. We conclude that in L-chain apoferritins the Glu residues at positions 53, 56 and 57 are involved in the mechanism of iron incorporation. Glu 53 and 56 are conserved in L but not in H ferritins, and are located in close proximity to each other within the three-dimensional structure. There is ample room for rotation of Glu 57 to join with the other two to form an iron-binding site. This may represent a site of iron incorporation (most probably involving nucleation) unique to L-chain ferritins, and may explain the predominant L-chain involvement in conditions of iron overload.     

Effect of extracellular hemin on hemoglobin and ferritin content of erythroleukemia cells

Mouse (MEL) and human (K-562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme-linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K-562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe-hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 X 10(-14) g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron-saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K-562 cells were induced by 57Fe-hemin in the presence of 56Fe-transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe-hemin in the presence of 57Fe-transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K-562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra- or intracellular iron sources such as transferrin or ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.     

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.