Iron core mineralisation in prokaryotic ferritins

Background

To satisfy their requirement for iron while at the same time countering the toxicity of this highly reactive metal ion, prokaryotes have evolved proteins belonging to two distinct sub-families of the ferritin family: the bacterioferritins (BFRs) and the bacterial ferritins (Ftns). Recently, Ftn homologues have also been identified and characterised in archaeon species. All of these prokaryotic ferritins function by solubilising and storing large amounts of iron in the form of a safe but bio-available mineral.

Scope of review

The mechanism(s) by which the iron mineral is formed by these proteins is the subject of much current interest. Here we review the available information on these proteins, with particular emphasis on significant advances resulting from recent structural, spectroscopic and kinetic studies.

Major conclusions

Current understanding indicates that at least two distinct mechanisms are in operation in prokaryotic ferritins. In one, the ferroxidase centre acts as a true catalytic centre in driving Fe²⁺ oxidation in the cavity; in the other, the centre acts as a gated iron pore by oxidising Fe²⁺ and transferring the resulting Fe³⁺ into the central cavity.

General significance

The prokaryotic ferritins exhibit a wide variation in mechanisms of iron core mineralisation. The basis of these differences lies, at least in part, in structural differences at and around the catalytic centre. However, it appears that more subtle differences must also be important in controlling the iron chemistry of these remarkable proteins.     

The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Protein Association and Dissociation Regulated by Ferric Ion: A NOVEL PATHWAY FOR OXIDATIVE DEPOSITION OF IRON IN PEA SEED FERRITIN*

Iron stored in phytoferritin plays an important role in the germination and early growth of seedlings. The protein is located in the amyloplast where it stores large amounts of iron as a hydrated ferric oxide mineral core within its shell-like structure. The present work was undertaken to study alternate mechanisms of core formation in pea seed ferritin (PSF). The data reveal a new mechanism for mineral core formation in PSF involving the binding and oxidation of iron at the extension peptide (EP) located on the outer surface of the protein shell. This binding induces aggregation of the protein into large assemblies of ∼400 monomers. The bound iron is gradually translocated to the mineral core during which time the protein dissociates back into its monomeric state. Either the oxidative addition of Fe²⁺ to the apoprotein to form Fe³⁺ or the direct addition of Fe³⁺ to apoPSF causes protein aggregation once the binding capacity of the 24 ferroxidase centers (48 Fe³⁺/shell) is exceeded. When the EP is enzymatically deleted from PSF, aggregation is not observed, and the rate of iron oxidation is significantly reduced, demonstrating that the EP is a critical structural component for iron binding, oxidation, and protein aggregation. These data point to a functional role for the extension peptide as an iron binding and ferroxidase center that contributes to mineralization of the iron core. As the iron core grows larger, the new pathway becomes less important, and Fe²⁺ oxidation and deposition occurs directly on the surface of the iron core.The chemistry of iron and oxygen in a number of non-heme di-iron proteins has been a subject of intense interest because of their varied roles in oxygen activation and catalysis, substrate hydrolysis, oxygen transport, redox reactions, H₂O₂, and iron detoxification and iron storage. Di-iron centers have similar structural motifs consisting of a combination of carboxylate and histidine ligands that either bind or bridge the two metal ions of the di-nuclear active site; di-iron proteins containing these centers include methane monooxygenase, ribonucleotide reductase, rubrerythrin, stearoyl desaturase, purple acid phosphatase, hemerythrin, the Dps proteins, and ferritins (1–6). Despite their similar di-nuclear centers, each of these proteins fulfills a distinct biological role that seems to be mediated by the nature of the first and second coordination sphere of the di-iron center.Ferritins are a class of intracellular iron storage and detoxification proteins that facilitate the oxidation of iron by molecular oxygen or hydrogen peroxide to form a hydrous ferric oxide mineral core within their interiors (1–3). Rapid Fe²⁺ oxidation occurs at the di-iron ferroxidase center located on the H-subunit of the mammalian protein. Unlike the H-chain, the more acidic L-subunit lacks the ferroxidase center but contains a putative nucleation site responsible for slower iron oxidation and mineralization (7). The shapes of both the H- and L-subunit are nearly cylindrical and composed of a four-α-helix bundle containing two antiparallel helix pairs (A, B and C, D) connected by a long non-helical stretch, the BC-loop, between B and C helices. A fifth short helix (E helix) lies at one end of the bundle at 60° to its axis (1, 2).From an evolutionary point of view, plant and animal ferritins arose from a common ancestor, but plant ferritins exhibit various specific features as compared with animal ferritins. Plant ferritins are observed in plastids (chloroplasts in leaves, amyloplasts in tubers and seeds, etc.) where iron is incorporated into the ferritin shell to form the mineral core, whereas animal ferritins are largely found in the cytoplasm of cell (1, 8). Ferritins from dried soybean and pea seed consist of two subunits of 26.5 and 28.0 kDa, which are designated H-1 and H-2, respectively, share ∼80% amino acid sequence identity (9, 10), and contain ferroxidase centers. The two subunits are synthesized from a 32-kDa precursor with a unique two-domain N-terminal sequence containing a transit peptide (TP)² and an extension peptide (EP). The TP is responsible for precursor targeting to plastids (11). Upon transport to plastids, the TP is cleaved from the subunit precursor, resulting in the formation of the mature subunit which assembles into a 24-mer apoferritin within the plastids (11, 12). The EP domain is kept at the N-terminal extremity of the mature plant ferritins, but its function remains unknown. It is absent in animal ferritins. For soybean ferritin, further processing of the 26.5-kDa subunit occurs through excising a short C-terminal amino acid sequence (16 amino acid residues) that corresponds to the E helix of the mammalian ferritin subunit (9). Compared with their own precursors, H-1 is devoid of both the N-terminal TP domain and the C-terminal E helix, whereas H-2 lacks the N-terminal TP domain only (13). However, the amino acids constituting the ferroxidase center are strictly conserved in all the plant ferritins except for pea seed ferritin where His-62 replaces Glu-62 (14, 15).Iron oxidation/mineralization in human ferritin occurs by at least three reaction pathways (16, 17). After Fe²⁺ binding at the ferroxidase site, the protein-catalyzed oxidation of Fe²⁺ occurs, H₂O₂ is the product of O₂ reduction, and a mineral core of Fe³⁺ is produced, written for simplicity as Fe(O)OH_(core) (Equation 1) (18, 19). Some of the H₂O₂ generated in Equation 1 reacts with additional Fe²⁺ in the Fe²⁺ + H₂O₂ detoxification reaction (Equation 2) to produce H₂O (16, 20). Once a mineral core of sufficient size has developed, Fe²⁺ autoxidation becomes significant, and iron oxidation and hydrolysis occurs primarily on the growing surface of the mineral through an autocatalytic process where O₂ is reduced completely to H₂O (Equation 3) (1, 7, 17). Based on the above reactions and on identification of intermediates by resonance Raman spectroscopy, Mössbauer spectroscopy, and EXAFS (extended x-ray absorption fine structure), the mechanism of mineral core formation in mammalian ferritins has been reasonably well established (18, 21–23). Initially two Fe²⁺ are oxidized by O₂ at the ferroxidase center to form a transient μ-1,2-peroxodi-Fe³⁺ intermediate, which rapidly decays to form a μ-oxo di-Fe³⁺ complex(es), releasing H₂O₂ to the solution. The μ-oxo(hydroxo) bridged di-Fe³⁺ dimer(s) then translocates from the ferroxidase center to the inner cavity to form an incipient core, which ultimately leads to the formation of the mineral core itself. However, this mechanism of oxidative deposition of iron is only applicable to ferritins such as horse spleen ferritin and human H-chain ferritin, which regenerate activity at their ferroxidase centers. In contrast, the ferroxidase centers of human mitochondrial ferritin (24), Escherichia coli bacterioferritin (25), E. coli bacterial ferritin A (26), and pea seed ferritin (PSF) (27) lack significant regeneration activity, and Fe³⁺ produced at their centers migrates with difficulty from the center to the cavity to form the initial core. These observations raise the question as to whether alternate pathways exist for core formation in these proteins, in particular in phytoferritins, the focus of this work.In the present study we have looked for alternate mechanisms of iron deposition in PSF and demonstrate that ferritin-ferritin aggregation occurs during the oxidative deposition of iron in PSF when more than 48 Fe²⁺/shell are added to the apoprotein (apoPSF), an amount exceeding the 24 ferroxidase center binding capacity for Fe^2+/3+. Such aggregation was also induced by the addition of Fe³⁺ directly to PSF. The data indicate that binding occurs on the outer surface of the ferritin shell at sites involving the extension peptide. Upon standing, the iron induced aggregate reversibly dissociates to its original undissociated form, whereas the iron migrates to the mineral core. This finding represents an alternate pathway for iron deposition in phytoferritin.     

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.     

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)     

Chemiluminescence in the presence of ferritin

The ability of ferritins with differing iron contents to catalyze the oxidation of luminol by hydrogen peroxide was studied. The least efficient catalysts were iron-rich ferritins, and the most potent were those with iron to protein ratios of less than 0.1.     

Isolation and characterization of phytoferritin from pea (Pisum sativum) and Lentil (Lens esculenta).

Ferritin was isolated from the seeds of pea (Pisum sativum) and lentil (Lens esculenta). The homogeneity of the phytoferritins was established by polyacrylamide-gel electrophoresis. The subunit molecular weights were respectively 20 300 and 21 400 for hte pea and lentil proteins. A neutron low-angle scattering study established the molecular weight of the oligomer as 480 000 for pea apoferritin and 510 000 for lentil apoferritin. Although the quaternary structure of 24 polypeptide chains is preserved, the phytoferritins have a larger cavity in the interior than mammalian ferritins and can thus potentially store 1.2-1.4 times as much iron. The amino acid composition of the phytoferritins show some similarities to those of mammalian apoferritins; tryptic 'fingerprinting' reveals that there are many differences in the amino acid sequence of plant and mammalian apoferritins.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of an uncharacterized branch of the ferritin-like superfamily

Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins. In an attempt to identify new iron-storage systems, we have found and characterized a new member of the ferritin-like superfamily of proteins, which unlike the multimeric storage system of ferritin, bacterioferritin, and Dps is monomeric in the absence of iron. Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin. We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.     

The formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake

Ferritin has a high capacity as an iron store, incorporating some 4500 iron atoms as a microcrystalline ferric oxide hydrate. Starting from apoferritin, or ferritin of low iron content, Fe²⁺ and an oxidizing agent, the uptake of iron can be recorded spectrophotometrically. Progress curves were obtained and the reconstituted ferritin was shown by several physical methods to be similar to natural ferritin. The progress curves of iron uptake by apoferritin are sigmoidal; those for ferritins of low iron content are hyperbolic. The rate of iron uptake is dependent on the amount of iron already present in the molecule. The distribution of iron contents among reconstituted ferritin molecules is inhomogeneous. These findings are interpreted in terms of a crystal growth model. The surface area of the crystallites forming inside the protein increases until the molecule is half full, and then declines. This surface controls the rate at which new material is deposited. The experimental results can best be accounted for by a two-stage mechanism, an initial slow `nucleation' stage, which is apparently zero order with respect to [Fe<sup>2+</sup>], followed by a more rapid `growth' stage. The rate of Fe²⁺ oxidation is increased in the presence of apoferritin as compared with controls. Ferritin can therefore be regarded as an enzyme to which the product remains firmly attached. The protein appears to increase the rate of `nucleation'. The apparent zero order of this stage suggests the presence of binding sites on the protein, which are saturated with respect to Fe²⁺. These sites are presumed also to be oxidation sites. The oxidation and subsequent formation of the ferric oxide hydrate may proceed according to one of three alternative models.     

X-ray structures of ferritins and related proteins

Ferritins are members of a much larger superfamily of proteins, which are characterised by a structural motif consisting of a bundle of four parallel and anti-parallel α helices. The ferritin superfamily itself is widely distributed across all three living kingdoms, in both aerobic and anaerobic organisms, and a considerable number of X-ray structures are available, some at extremely high resolution. We describe first of all the subunit structure of mammalian H and L chain ferritins and then discuss intersubunit interactions in the 24-subunit quaternary structure of these ferritins. Bacteria contain two types of ferritins, FTNs, which like mammalian ferritins do not contain haem, and the haem-containing BFRs. The characteristic carboxylate-bridged di-iron ferroxidase sites of H chain ferritins, FTNs and BFRs are compared, as are the potential entry sites for iron and the ‘nucleation’ site of L chain ferritins. Finally we discuss the three-dimensional structures of the 12-subunit bacterial Dps (DNA-binding protein from starved cells) proteins as well as their intersubunit di-iron ferroxidase site.     

A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu-44, a site C ligand, and Glu-130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe³⁺ exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe²⁺ oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.     

Influence of site-directed modifications on the formation of iron cores in ferritin

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.     

Phytoferritin and its implications for human health and nutrition

Background

Plant and animal ferritins stem from a common ancestor, but plant ferritins exhibit various features that are different from those of animal ferritins. Phytoferritin is observed in plastids (e.g., chloroplasts in leaves, amyloplasts in tubers and seeds), whereas animal ferritin is largely found in the cytoplasm. The main difference in structure between plant and animal ferritins is the two specific domains (TP and EP) at the N-terminal sequence of phytoferritin, which endow phytoferritin with specific iron chemistry. As a member of the nonheme iron group of dietary iron sources, phytoferritin consists of 24 subunits that assemble into a spherical shell storing up to ∼ 2000 Fe^3 + in the form of an iron oxyhydroxide-phosphate mineral. This feature is distinct from small molecule nonheme iron existing in cereals, which has poor bioavailability.

Scope of review

This review focuses on the relationship between structure and function of phytoferritin and the recent progress in the use of phytoferritin as iron supplement.

Major conclusions

Phytoferritin, especially from legume seeds, represents a novel alternative dietary iron source.

General significance

An understanding of the chemistry and biology of phytoferritin, its interaction with iron, and its stability against gastric digestion is beneficial to design diets that will be used for treatment of global iron deficiency.     

Ferritins,iron uptake and storage from the bacterioferritin viewpoint

Ferritins constitute a broad superfamily of iron storage proteins, widespread in all domains of life, in aerobic or anaerobic organisms. Ferritins isolated from bacteria may be haem-free or contain a haem. In the latter case they are called bacterioferritins. The primary function of ferritins inside cells is to store iron in the ferric form. A secondary function may be detoxification of iron or protection against O(2) and its radical products. Indeed, for bacterioferritins this is likely to be their primary function. Ferritins and bacteroferritins have essentially the same architecture, assembling in a 24mer cluster to form a hollow, roughly spherical construction. In this review, special emphasis is given to the structure of the ferroxidase centres with native iron-containing sites, since oxidation of ferrous iron by molecular oxygen takes place in these sites. Although present in other ferritins, a specific entry route for iron, coupled with the ferroxidase reaction, has been proposed and described in some structural studies. Electrostatic calculations on a few selected proteins indicate further ion channels assumed to be an entry route in the later mineralization processes of core formation.     

Stability and iron oxidation properties of a novel homopolymeric plant ferritin from adzuki bean seeds: A comparative analysis with recombinant soybean seed H-1 chain ferritin

Background

All reported plant ferritins are heteropolymers comprising two different H-type subunits. Whether or not homopolymeric plant ferritin occurs in nature is an open question.

Methods

A homopolymeric phytoferritin from adzuki bean seeds (ASF) was obtained by various protein purification techniques for the first time, which shares the highest identity (89.6%) with soybean seed H-1 ferritin (rH-1). Therefore, we compared iron oxidation activity and protein stability of ASF with those of rH-1 by stopped-flow combined with light scattering or UV/Vis spectrophotography, SDS- and native- PAGE analyses. Additionally, a new rH-1 variant (S68E) was prepared by site-directed mutagenesis approach to elucidate their difference in protein stability.

Results

At high iron loading of protein, the extension peptide (EP) of plant ferritin was involved in iron oxidation, and the EP of ASF exhibited a much stronger iron oxidative activity than that of rH-1. Besides, ASF is more stable than rH-1 during storage, which is ascribed to one amino acid residue, Ser68.

Conclusions

ASF exhibits a different mechanism in iron oxidation from rH-1 at high iron loading of protein, and a higher stability than rH-1. These differences are mainly stemmed from their different EP sequences.

General significance

This work demonstrates that plant cells have evolved the EP of phytoferritin to control iron chemistry and protein stability by exerting a fine tuning of its amino acid sequence.