A comparison of an undecairon(III) complex with the ferritin iron core

The iron core of ferritin is comprised of up to 4,500 Fe(III) atoms as Fe2O3.nH2O, which is maintained in solution by a surrounding, spherical coat of protein. Organisms as diverse as bacteria and man use the ferritin iron-protein complex as a reservoir of stored iron for other essential proteins. To extend studies of the steps in polynuclear iron core formation, a recently characterized undecairon(III) oxo-hydroxo aggregate [Fe11 complex] (Gorun et al., J. Am. Chem. Soc. 109, 3337 [1987]) was examined by x-ray absorption spectroscopy as a model for an intermediate. The results, which are comparable to the previous x-ray diffraction studies, show near neighbors (Fe-O) at 1.90 A that are distinct from those in ferritin and a longer distance of 2.02 A. However, contributions from neighbors (Fe-C) known to exist at ca. 2.7 A were obscured by a highly ordered Fe-Fe interaction and were not detectable in the Fe11 complex in contrast to a previously characterized Fe(III) cluster bound to the protein coat. Of the two Fe-Fe interactions detectable in the Fe11 complex, the shortest, at 3.0 A is particularly interesting, occurring at the same distance as a full shell (CN = 6) in ferritin, but having fewer Fe neighbors (CN = 2-3) characteristic of an intermediate in core formation. The incomplete Fe-Fe shell is much more ordered than in ferritin, suggesting that the disorder in ferritin cores may be associated with the later steps of the core growth. Differences between the Fe11 complex and the full core of ferritin indicate the possibility of intermediates in ferritin iron formation that might be like Fe11.     

Fe(III).ATP complexes. Models for ferritin and other polynuclear iron complexes with phosphate

Polynuclear iron complexes of Fe(III) and phosphate occur in seawater and soils and in cells where the iron core of ferritin, the iron storage protein, contains up to 4500 Fe atoms in a complex with an average composition of (FeO.OH)8FeO.OPO3H2. Although phosphate influences the size of the ferritin core and thus the availability of stored iron, little is known about the nature of the Fe(III)-phosphate interaction. In the present study, Fe-phosphate interactions were analyzed in stable complexes of Fe(III).ATP which, in the polynuclear iron form, had phosphate at interior sites. Such Fe(III).ATP complexes are important not only as models but also because they may play a role in intracellular iron transport and in iron toxicity; the complexes were studied by extended x-ray absorption fine structure, EPR, NMR spectroscopy, and measurement of proton release. Mononuclear iron complexes exhibiting a g' = 4.3 EPR signal were formed at Fe:ATP ratios less than or equal to 1:3, and polynuclear iron complexes (Fe greater than or equal to 250, EPR silent at g' = 4.3) were formed at an Fe:ATP ratio of 4:1. No NMR signals due to ATP were observed when Fe was in excess (Fe:ATP = 4:1). Extended x-ray absorption fine structure analysis of the polynuclear Fe(III).ATP complex was able to distinguish an Fe-P distance at 3.27 A in addition to the octahedral O at 1.95 A and 4-5 Fe atoms at 3.36 A. The Fe-O and Fe-Fe distances are the same as in ferritin, and the Fe-P distance is analogous to that in another metal-ATP complex. An observable Fe-P environment in such a large polynuclear iron cluster as the Fe(III).ATP (4:1) complex indicates that the phosphate is distributed throughout rather than merely on the surface, in contrast to earlier models of chelate-stabilized iron clusters. Complexes of Fe(III) and ATP similar to those described here may form in vivo either as normal components of intracellular iron metabolism or during iron excess where the consequent alteration of free nucleotide triphosphate pools could contribute to the observed toxicity of iron.     

A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Similarity of the structure of ferritin and iron . dextran (imferon) determined by extended X-ray absorption fine structure analysis.

Ferritin, a natural complex of iron oxide encased in protein, and iron . dextran, a synthetic complex of iron oxide coated with dextran, have the similar properties of maintaining high concentrations of iron in solution at physiological pH and releasing iron relatively slowly in vivo. Extended x-ray absorption fine structure (EX-AFS) analysis was performed on each complex and compared to see if the structures of the iron cores were similar. The results obtained from the extended x-ray absorption fine structure technique show that the near-neighbor environment around the average iron atom in ferritin and iron . dextran is identical, within experimental uncertainty, for the first three shells. The similarity of the iron cores in both complexes may explain the similarity of iron release in vivo. Ferritin has a protein coat which is composed of 24 subunits arranged in a hollow sphere with six channels through which the iron may move during deposition and release. However, little is known about the requirements of the protein structure in ferritin for the maintenance of high concentrations of iron in a soluble, nontoxic form or about the role of the protein in the release of iron from ferritin. The results suggest that iron . dextran will be a useful model compound in studies of the relation of the iron core and protein in ferritin to function.     

Initial iron oxidation in horse spleen apoferritin. Characterization of a mixed-valence iron(II)-iron(III) complex.

In ferritin, iron is stored by oxidative deposition of the ferrous ion to form a hydrous ferric oxide mineral core. Two intermediates, formed during the initial stages of iron accumulation in apoferritin, have been observed previously in our laboratory and have been identified as a mononuclear Fe3(+)-protein complex and a mixed-valence Fe2(+)-Fe3(+)-protein complex. The physical characteristics of the mixed-valence Fe2(+)-Fe3+ complex and its relationship to the mononuclear Fe3+ complex in horse spleen apoferritin samples to which 0-240 iron atoms were added was examined by EPR spectroscopy. The results indicate that the mononuclear complex is not a precursor to the formation of the mixed-valence complex. Competitive binding studies with Cd2+, Zn2+, Tb3+, and UO2+(2) suggest that the mixed-valence complex is formed on the interior of the protein in the vicinity of the 2-fold axis of the subunit dimer. The mixed-valence complex could be generated by the partial oxidation of Fe2+ in apoferritin containing 120 Fe2+ or by the addition of up to 120 Fe2+ to ferritin already containing 18 Fe3+/protein molecule. The fact that the complex is generated during early Fe2+ oxidation suggests that it may be a key intermediate during the initial oxidative deposition of iron in the protein. The unusual EPR powder lineshape at 9.3 GHz of the mixed-valence complex was simulated with a rhombic g-tensor (gx = 1.95, gy = 1.88, gz = 1.77) and large linewidths and g-strain parameters. The presence of significant g-strain in the complex probably accounts for the failure to observe an EPR signal at 35 GHz and likely reflect considerable flexibility in the structure of the metal site. The temperature dependence of the EPR intensity in the range 8-38 K was modeled successfully by an effective spin Hamiltonian including exchange coupling (-2JS1.S2) and zero-field terms, from which an antiferromagnetic coupling of J = -4.0 +/- 0.5 cm-1 was obtained. This low value for J may reflect the presence of a mu-oxo bridge(s) in the dimer.     

Rapid reduction of iron in horse spleen ferritin by thioglycolic acid measured by dispersive X-ray absorption spectroscopy

Summary The release of iron from ferritin is important in the formation of iron proteins and for the management of diseases in both animals and plants associated with abnormal accumulations of ferritin iron. Much more iron can be released experimentally by reduction of the ferric hydrous oxide core than by chelation of Fe³⁺ which has led to the notion that reduction is also the major aspect of iron release in vivo. Variations in the kinetics of reduction of the mineral core of ferritin have been attributed to the redox potential of the reductant, redox properties of the iron core, the structure of the protein coat, the analytical method used to detect Fe²⁺ and reactions at the surface of the mineral. Direct measurements of the oxidation state of the iron during reduction has never been used to analyze the kinetics of reduction, although Mössbauer spectroscopy has been used to confirm the extent of reduction after electrochemical reduction using dispersive X-ray absorption spectroscopy (DXAS). We show that the near edge of X-ray absorption spectra (XANES) can be used to quantify the relative amounts of Fe²⁺ and Fe³⁺ in mixtures of the hydrated ions. Since the nearest neighbors of iron in the ferritin iron core do not change during reduction, XANES can be used to monitor directly the reduction of the ferritin iron core. Previous studies of iron core reduction which measured by Fe²⁺ · bipyridyl formation, or coulometric reduction with different mediators, suggested that rates depended mainly on the redox potential of the electron donor. When DXAS was used to measure the rate of reduction directly, the initial rate was faster than previously measured. Thus, previously measured differences in reduction rates appear to be influenced by the accessibility of Fe²⁺ to the complexing reagent or by the electrochemical mediator. In the later stages of ferritin iron core dissolution, reduction rates drop dramatically whether measured by DXAS or formation of Fe²⁺ complexes. Such results emphasize the heterogeneity of ferritin core structure.     

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).     

Crosslinks between intramolecular pairs of ferritin subunits: effects on both H and L subunits and on immunoreactivity of sheep spleen ferritin

Ferritin is a multisubunit protein, controlling iron storage, with a protein coat composed of 24 subunits (up to three distinct types) in different proportions depending on cell type. Little is known about the subunit interactions in ferritin protein coats composed of heterologous subunits, despite the relevance to ferritin structure and ferritin function (iron uptake and release). Synthetic crosslinking is a convenient way to probe subunit contacts. Crosslinks between subunit pairs in ferritin protein coats are also a natural post-translational modification which coincides with different iron content in ferritin from sheep spleen; ferritin from sheep spleen also contains H and L subunits. Crosslinks synthesized by the reaction of ferritin low in natural crosslinks with difluorodinitrobenzene (F2DNB) reproduced the effects of the natural crosslinks on iron uptake and release. We now extend our observations on the structural effects of natural and synthetic crosslinks to include immunoreactivity of the assembled protein, with monoclonal antibodies as a probe. We also demonstrate, for the first time, ferritin peptides involved in an apparent H- and L-subunit contact: two peptides decreased 4X in cyanogen bromide peptide maps after F2DNB crosslinking were residues L-96-138 and H-66-96; the major DNP-dipeptide was Lys-DNP-Lys. Using the structure of an all L-subunit ferritin as a model, the most likely site for the H-L DNP crosslink is L-Lys 104 (C helix) and H-Lys 67 (B helix). The B helix forms the internal subunit dimer interface, a putative site of iron core nucleation. Alteration by crosslinks of the B helix could, therefore, explain the effect of crosslinks on ferritin iron uptake, release, and iron content.     

Fe2+ and phosphate interactions in bacterial ferritin from Azotobacter vinelandii.

Fe2+ binding to both apo- and holo- bacterial ferritin from Azotobacter vinelandii (AVBF) was measured as a function of pH under carefully controlled anaerobic conditions. Fe2+ binding to apo-AVBF is strongly pH dependent with 25 Fe2+ ions/apo-AVBF binding tightly at pH 5.5 and over 150 Fe2+/apo-AVBF at pH 9.0. Holo-AVBF gave a similar pH-dependent binding profile with over 400 Fe2+/AVBF binding at pH of 9.0. Proton release per Fe2+ bound to either AVBF protein increases with increasing pH until a total of about two protons are released at pH 9.0. These binding results are both qualitatively and quantitatively different from corresponding measurements (Jacobs et al., 1989) on apo- and holo- mammalian ferritin (MF) where less Fe2+ binds in both cases. The high level of Fe2+ binding to holo-AVBF relative to that of mammalian ferritin is a consequence of the higher phosphate content in the core of AVBF. Reduction of AVBF by either dithionite or methyl viologen in the absence of chelating agents demonstrated that phosphate, but not Fe2+, is released from the AVBF core in amounts commensurate with the degree of iron reduction, although even at 100% reduction considerable phosphate remains associated with the reduced mineral core. Fe2+ binding to holo-AVBF made deficient in phosphate was lower than that of native AVBF, while the addition of phosphate to native holo-AVBF increased the Fe2+ binding capacity. These results clearly support the role of phosphate as the site of interaction of Fe2+ with the AVBF mineral core.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena)

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.     

On the limited ability of superoxide to release iron from ferritin

Reductive release of iron from ferritin may catalyze cytotoxic radical reactions like the Haber-Weiss reaction. The ability of .O2- to mobilize Fe(II) from ferritin was studied by using the xanthine/xanthine oxidase reaction, with and without superoxide dismutase, and with bathophenanthroline sulphonate as the chelator. Not more than one or two Fe(II)/ferritin molecules could be released by an .O2(-)-dependent mechanism, even after repeated exposures of ferritin to bursts of .O2-. The amount of releaseable iron depended on the size and the age of the iron core, but not on the iron content of the protein shell of ferritin which was manipulated by chelators and addition of FeCl3. The kinetic characteristics of the .O2(-)-mediated iron release indicated the presence of a small pool of readily available iron at the surface of the core. The very limited .O2(-)-dependent release of iron from ferritin is compatible with a protective role of ferritin against toxic iron-catalyzed reactions.     

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.     

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.     

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).     

Redox reactions associated with iron release from mammalian ferritin

The early redox events involved in iron reduction and mobilization in mammalian ferritin have been investigated by several techniques. Sedimentation velocity measurements of ferritin samples with altered core sizes, prepared by partial reduction and Fe2+ chelation, suggest two different events occur during iron loss from the ferritin core. Reductive optical titrations confirm this biphasic behavior by showing that the first 20-30% of core reduction has different optical properties than the latter 70-80%. Proton uptake during initial core reduction is near zero, but as the percent core reduction increases, the proton uptake (H+/e) values increase to 2 H+/e (2 H+/Fe3+ reduced) as core reduction approaches 1 e/Fe3+. Coulometric reduction of ferritin by mediators of different redox potential and different cross-sectional areas show a two-phase sigmoidal reaction pattern in which initial core reduction occurs at a slower rate than later core reduction. The above experiments were all conducted in the absence of iron chelators so that the observed results were all attributed to core reduction rather than the combined effects of core reduction accompanied by Fe2+ chelation. The coulometric reduction of ferritin by various mediators shows a correlation more with reduction potential than with molecular cross-sectional area. The role of the ferritin channels in core reduction is considered in terms of the reported results.     

Ferroxidase kinetics of horse spleen apoferritin.

Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.     

铁核结构对马脾铁蛋白释放铁动力学的影响

建立Ｈ^% 参与马脾铁蛋白释放铁的动力方程,Ｈ^ 以１／２级反应方式参与铁蛋白释放铁核表层的铁。在酸性介质（ＰＨ６．５）中,铁蛋白释放铁的总平均速率（３３２Ｆe^3 /HSF.min)比在碱性介质（Ｐ8Ｈ８．０）中放铁的总平均速率（７３Ｆe^3 /HSF.min)高４．６倍,铁蛋白的铁核结构和外加的磷酸盐均能影响该蛋白释放的速率,但并不改变其反应级数。     

Rate of iron transfer through the horse spleen ferritin shell determined by the rate of formation of Prussian Blue and Fe-desferrioxamine within the ferritin cavity

Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)6]3-, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe2+ penetrating into the ferritin interior by adding external Fe2+ to [Fe(CN)6]3- encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe2+ reacts with [Fe(CN)6]3- in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe2+ through the HoSF protein shell. The calculated diffusion coefficient, D approximately 5.8x10(-20) m2/s corresponds to the measured lag time of 10-20 s before PB forms within the HoSF interior. The activation energy for Fe2+ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 10 approximately 40 degrees C. The reaction of Fe3+ with encapsulated [Fe(CN)6]4- also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe2+ reaction. The rate for Fe3+ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell.     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献