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.     

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.     

Could a dysfunction of ferritin be a determinant factor in the aetiology of some neurodegenerative diseases?

Background

The concentration of iron in the brain increases with aging. Furthermore, it has also been observed that patients suffering from neurological diseases (e.g. Parkinson, Alzheimer…) accumulate iron in the brain regions affected by the disease. Nevertheless, it is still not clear whether this accumulation is the initial cause or a secondary consequence of the disease. Free iron excess may be an oxidative stress source causing cell damage if it is not correctly stored in ferritin cores as a ferric iron oxide redox-inert form.

Scope

Both, the composition of ferritin cores and their location at subcellular level have been studied using analytical transmission electron microscopy in brain tissues from progressive supranuclear palsy (PSP) and Alzheimer disease (AD) patients.

Major conclusions

Ferritin has been mainly found in oligodendrocytes and in dystrophic myelinated axons from the neuropili in AD. In relation to the biomineralization of iron inside the ferritin shell, several different crystalline structures have been observed in the study of physiological and pathological ferritin. Two cubic mixed ferric–ferrous iron oxides are the major components of pathological ferritins whereas ferrihydrite, a hexagonal ferric iron oxide, is the major component of physiological ferritin. We hypothesize a dysfunction of ferritin in its ferroxidase activity.

General significance

The different mineralization of iron inside ferritin may be related to oxidative stress in olygodendrocites, which could affect myelination processes with the consequent perturbation of information transference.     

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.     

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 unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

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.     

Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin

Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV–vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein.     

Multiple pathways for mineral core formation in mammalian apoferritin. The role of hydrogen peroxide

Human ferritins sequester and store iron as a stable FeOOH((s)) mineral core within a protein shell assembled from 24 subunits of two types, H and L. Core mineralization in recombinant H- and L-subunit homopolymer and heteropolymer ferritins and several site-directed H-subunit variants was investigated to determine the iron oxidation/hydrolysis chemistry as a function of iron flux into the protein. Stopped-flow absorption spectrometry, UV spectrometry, and electrode oximetry revealed that the mineral core forms by at least three pathways, not two as previously thought. They correspond to the ferroxidase, mineral surface, and the Fe(II) + H2O2 detoxification reactions, respectively: [see reactions]. The H-subunit catalyzed ferroxidase reaction 1 occurs at all levels of iron loading of the protein but decreases with increasing iron added (48-800 Fe(II)/protein). Reaction 2 is the dominant reaction at 800 Fe(II)/protein, whereas reaction 3 occurs largely at intermediate iron loadings of 100-500 Fe(II)/protein. Some of the H2O2 produced in reaction 1 is consumed in the detoxification reaction 3; the 2/1 Fe(II)/H2O2 stoichiometry of reaction 3 minimizes hydroxyl radical production during mineralization. Human L-chain ferritin and H-chain variants lacking functional nucleation and/or ferroxidase sites deposit their iron largely through the mineral surface reaction 2. H2O2 is shown to be an intermediate product of dioxygen reduction in L-chain as well as in H-chain and H-chain variant ferritins.     

Unique iron binding and oxidation properties of human mitochondrial ferritin: a comparative analysis with Human H-chain ferritin

Ferritins are ubiquitous iron mineralizing and storage proteins that play an important role in iron homeostasis. Although excess iron is stored in the cytoplasm, most of the metabolically active iron is processed in the mitochondria of the cell. Little is known about how these organelles regulate iron homeostasis and toxicity. The recently discovered human mitochondrial ferritin (MtF), unlike other mammalian ferritins, is a homopolymer of 24 subunits that has a high degree of sequence homology with human H-chain ferritin (HuHF). Parallel experiments with MtF and HuHF reported here reveal striking differences in their iron oxidation and hydrolysis chemistry despite their similar diFe ferroxidase centers. In contrast to HuHF, MtF does not regenerate its ferroxidase activity after oxidation of its initial complement of Fe(II) and generally has considerably slower ferroxidation and mineralization activities as well. MtF exhibits sigmoidal kinetics of mineralization more characteristic of an L-chain than an H-chain ferritin. Site-directed mutagenesis reveals that serine 144, a residue situated near the ferroxidase center in MtF but absent from HuHF, is one player in this impairment of activity. Additionally only one-half of the 24 ferroxidase centers of MtF are functional, further contributing to its lower activity. Stopped-flow absorption spectrometry of Fe(II) oxidation by O(2) in MtF shows the formation of a transient diiron(III) mu-peroxo species (lambda(max) = 650 nm) as observed in HuHF. Also, as for HuHF, minimal hydroxyl radical is produced during the oxidative deposition of iron in MtF using O(2) as the oxidant. However, the 2Fe(II) + H(2)O(2) detoxification reaction found in HuHF does not occur in MtF. The structural differences and the physiological implications of the unique iron oxidation properties of MtF are discussed in light of these results.     

Structural characterization and biological implications of di-zinc binding in the ferroxidase center of Streptococcus pyogenes Dpr

Dps proteins contain a ferroxidase site that binds and oxidizes iron, thereby preventing hydroxyl radical formation by Fenton reaction. Although the involvement of a di-iron ferroxidase site has been suggested, X-ray crystal structures of various Dps members have shown either one or two iron cations with various occupancies despite the high structural conservation of the site. Similarly, structural studies with zinc, a redox-stable replacement for iron, have shown the binding of either one or two zinc ions. Here, the crystal structure of Streptococcus pyogenes Dpr in complex with zinc reveals the binding of two zinc cations in the ferroxidase center and an additional zinc-binding site at the surface of the protein. The results suggest a structural basis for the protection of Streptococcus pyogenes in zinc stress conditions and provide a clear evidence for a di-zinc and di-iron ferroxidase site in Streptococcus pyogenes Dpr protein.     

Mineralization in Ferritin: An Efficient Means of Iron Storage

Ferritins are a class of iron storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral "ferrihydrite." The eight hydrophilic channels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mammalian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O(2), producing H(2)O(2). The L subunit lacks this site but contains additional glutamate residues on the interior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxidase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a mu-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus minimizing the production of potentially harmful H(2)O(2).     

New Vistas on the Recruiting of Ferrous Iron and Dioxygen by Ferritins: A Case Study of the Escherichia coli 24‐mer Ferritin by All‐Atom Molecular Dynamics in Aqueous Medium

It is shown here that Fe²⁺ and O₂ ligands are displaced from the ferroxidase center of the C1 four‐helix bundle of E. coli 24‐mer ferritin under molecular dynamics (MD) aided by a randomly oriented external force applied to the ligand. Under these conditions, ligand egress toward the external aqueous medium occurs preferentially from the same four‐helix bundle, in the case of O₂, or other bundle, in the case of Fe². Viewing ligand egress from the protein as the microscopic reverse of ligand influx into the protein under unbiased MD, these findings challenge current views that preferential gates for recruitment of Fe²⁺ are 3‐fold channels with human ferritin, or the short path from the ferroxidase center to H93 with bacterial ferritins.     

Influence of site-directed mutagenesis on protein assembly and solubility of tadpole H-chain ferritin

In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferritin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced inEscherichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine. Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritins were capable of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further functional analysis was also made on the individual amino acid residues of ferroxidase center.     

Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Å resolution to R = 0.195 and R _free = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.     

mu-1,2-peroxo diferric complex formation in horse spleen ferritin. A mixed H/L-subunit heteropolymer

Previous kinetics studies with homopolymer ferritins (bullfrog M-chain, human H-chain and Escherichia coli bacterial ferritins) have established that a mu-1,2-peroxo diferric intermediate is formed during Fe(II) oxidation by O2 at the ferroxidase site of the protein. The present study was undertaken to determine whether such an intermediate is formed also during iron oxidation in horse spleen ferritin (HoSF), a naturally occurring heteropolymer ferritin of H and L-subunits (approximately 3.3 H-chains/HoSF), and to assess its role in the formation of the mineral core. Multi-wavelength stopped-flow spectrophotometry of the oxidative deposition of iron in HoSF demonstrated that a transient peroxo complex (lambda(max) approximately 650 nm) is produced in this protein as for other ferritins. The peroxo complex in HoSF is formed about fourfold slower than in human H-chain (HuHF) and decays more slowly (approximately threefold) as well, at an iron level of two Fe(II)/H-chain. However, as found for HuHF, a second intermediate is formed in HoSF as a decay product of the peroxo complex. Only one-third of the expected peroxo complex forms at the ferroxidase centers of HoSF when two Fe(II)/H-subunits are added to the protein, dropping to only approximately 14% when 20 Fe(II)/H-chain are added, indicating a declining role of the peroxo complex in iron deposition. In contrast to HuHF, HoSF does not enzymatically regenerate the observable peroxo complex. The kinetics of mineralization in HoSF are modeled satisfactorily by a mechanism in which the ferroxidase site rapidly produces an incipient core from a single turnover of iron, upon which subsequent Fe(II) is oxidized autocatalytically to build the Fe(O)OH(s) mineral core. This model supports a role for the L-chain in iron mineralization and helps to explain the widespread occurrence of heteropolymer ferritins in tissues of vertebrates.     

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.