The ferroxidase center is essential for ferritin iron loading in the presence of phosphate and minimizes side reactions that form Fe(III)-phosphate colloids

Ferritin iron loading was studied in the presence of physiological serum phosphate concentrations (1 mM), elevated serum concentrations (2–5 mM), and intracellular phosphate concentrations (10 mM). Experiments compared iron loading into homopolymers of H and L ferritin with horse spleen ferritin. Prior to studying the reactions with ferritin, a series of control reactions were performed to study the solution chemistry of Fe²⁺ and phosphate. In the absence of ferritin, phosphate catalyzed Fe²⁺ oxidation and formed soluble polymeric Fe(III)-phosphate complexes. The Fe(III)-phosphate complexes were characterized by electron microscopy and atomic force microscopy, which revealed spherical nanoparticles with diameters of 10–20 nm. The soluble Fe(III)-phosphate complexes also formed as competing reactions during iron loading into ferritin. Elemental analysis on ferritin samples separated from the Fe(III)-phosphate complexes showed that as the phosphate concentration increased, the iron loading into horse ferritin decreased. The composition of the mineral that does form inside horse ferritin has a higher iron/phosphate ratio (~1:1) than ferritin purified from tissue (~10:1). Phosphate significantly inhibited iron loading into L ferritin, due to the lack of the ferroxidase center in this homopolymer. Spectrophotometric assays of iron loading into H ferritin showed identical iron loading curves in the presence of phosphate, indicating that the ferroxidase center of H ferritin efficiently competes with phosphate for the binding and oxidation of Fe²⁺. Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe²⁺ and facilitate the transfer of the Fe³⁺ into apo transferrin in the presence of phosphate.     

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.     

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C 4 cage axes

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe₂O₃·H₂O minerals from Fe²⁺ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe²⁺ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe²⁺/O₂ catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe²⁺ + O₂ protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe₂O₃·H₂O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.     

Paragonimus westermani: molecular cloning,expression, and characterization of a recombinant yolk ferritin

Ferritin is an intracellular protein involved in iron metabolism. A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates. Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center. PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus. Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity. Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture. Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P. westermani lysate. PwYF-1 protein was localized to vitelline follicles and the eggs of P. westermani. Collectively, PwYF-1 protein was identified as a P. westermani yolk ferritin.     

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.     

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.     

The role of cysteine residues in the oxidation of ferritin

We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999-1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates.     

Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin

Fe(III) storage by ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a di-iron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site was obliterated. However, metal ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions toward the ferroxidase center directly through the protein shell and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted.     

Cloning and characterization of two ferritin subunit genes from bay scallop, <Emphasis Type="Italic">Argopecten irradians</Emphasis> (Lamarck 1819)

We have cloned two full-length cDNAs from two ferritin genes (Aifer1 and Aifer2) of the bay scallop, Argopecten irradians (Lamarck 1819). The cDNAs are 1,019 and 827 bp in length and encode proteins of 171 and 173 amino acids, respectively. The 5′ UTR of each contains a conserved iron response element (IRE) motif. Sequence analyses reveal that both proteins belong to the H-ferritin family with seven conserved amino acids in the ferroxidase center. Highest expression of Aifer1 is found in the mantle and adductor muscle, while that of Aifer2 is only in the latter tissue. These Aifer genes are differentially expressed following bacterial challenge of the scallop. The expression level of Aifer1 was acutely up-regulated (over 10 fold) at 6 h post-bacteria injection, whereas Aifer2 expression was not significantly changed by bacterial challenge. Both genes were effectively expressed in E. coli BL21 (DE3), producing proteins of similar molecular weight, approximately 23 kDa. Purified Aifer1 and Aifer2 proteins exhibited iron-chelating activity of 33.1% and 30.4%, respectively, at a concentration of 5 mg/ml. Cations, Mg²⁺, Zn²⁺ and Ca²⁺, depressed iron-chelating activity of both proteins. Additionally, the E. coli cells expressing recombinant Aifer1 and Aifer2 showed tolerance to H₂O₂, providing a direct evidence of the antioxidation function of ferritin. The results presented in this study suggest important roles of Aifer1 and Aifer2 in the regulation of iron homeostasis, immune response, and antioxidative stress in A. irradians.     

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.     

Model system oxidations supporting the crystal growth model of ferritin iron uptake

Fe²⁺ is oxidized and taken up by ferritin or ápoferritin in the presence of dioxygen. Iodate causes Fe²⁺ oxidation and uptake by ferritin, but not by apoferritin. Synthetic iron polymer facilitates Fe²⁺ oxidation by either dioxygen or iodate. Nitrilotriacetic acid or iminodiacetic acid facilitate oxidation of Fe²⁺ by oxygen but not by iodate. These results support the crystal growth model of ferritin iron uptake, with iron polymer serving as a model for the ferritin core and aminocarboxylic acids mimicking the metal-binding sites of apoferritin.     

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.     

Structural and Mechanistic Studies of a Stabilized Subunit Dimer Variant of Escherichia coli Bacterioferritin Identify Residues Required for Core Formation

Bacterioferritin (BFR) is a bacterial member of the ferritin family that functions in iron metabolism and protects against oxidative stress. BFR differs from the mammalian protein in that it is comprised of 24 identical subunits and is able to bind 12 equivalents of heme at sites located between adjacent pairs of subunits. The mechanism by which iron enters the protein to form the dinuclear (ferroxidase) catalytic site present in every subunit and the mineralized iron core housed within the 24-mer is not well understood. To address this issue, the properties of a catalytically functional assembly variant (E128R/E135R) of Escherichia coli BFR are characterized by a combination of crystallography, site-directed mutagenesis, and kinetics. The three-dimensional structure of the protein (1.8 Å resolution) includes two ethylene glycol molecules located on either side of the dinuclear iron site. One of these ethylene glycol molecules is integrated into the surface of the protein that would normally be exposed to solvent, and the other is integrated into the surface of the protein that would normally face the iron core where it is surrounded by the anionic residues Glu⁴⁷, Asp⁵⁰, and Asp¹²⁶. We propose that the sites occupied by these ethylene glycol molecules define regions where iron interacts with the protein, and, in keeping with this proposal, ferroxidase activity decreases significantly when they are replaced with the corresponding amides.Bacterioferritin (BFR)⁴ is a prokaryotic form of ferritin that has been identified in a number of bacteria (1–3). Despite low sequence similarity with eukaryotic ferritins, the three-dimensional structures and functional properties of BFRs from Escherichia coli, Rhodobacter capsulatus, Desulfovibrio desulfuricans, and Azotobacter vinelandii (4–10) are remarkably reminiscent of those reported for mammalian ferritins. For example, BFRs are oligomeric proteins comprised of 24 subunits (∼18 kDa each) that catalyze oxidation of Fe²⁺ by dioxygen (ferroxidase activity) to promote formation of a mineralized iron core that can contain as many as 2700 iron atoms/ 24-meric molecule (11). On the other hand, those BFRs that have been characterized differ from the mammalian proteins in that the 24 subunits are identical, and each possesses a catalytic dinuclear iron center that is referred to as the ferroxidase site (in mammalian ferritins, only the H-chains possess such catalytic sites). The pairwise arrangement of BFR monomers within the 24-mer creates 12 binding sites for heme, commonly protoheme IX but iron-coproporphyrin III in D. desulfuricans BFR, in which a methionyl residue on the surface of adjacent BFR monomers provides an axial ligand to create a b-type heme-binding site with bismethionine axial coordination (12, 13). Although a functional role for the heme of BFR has not been identified, the functional role of BFR is believed to be in iron storage and detoxification (14), thereby protecting against oxidative stress (15).The subunits of BFR are arranged to form eight 3-fold channels and six 4-fold channels. These channels have been proposed as possible entry and exit routes for iron incorporation into or release from the central iron core. For human ferritin, the 3-fold channel plays a significant role in the transport of iron into the iron core (16), but a similar role for this channel in BFR has not been demonstrated.The dinuclear ferroxidase site located within each subunit binds two iron atoms. Coordination of these iron atoms involves Glu⁵¹ and Glu¹²⁷ as bridging ligands for both irons, Glu¹⁸ and His⁵⁴ as ligands for FE1, and Glu⁹⁴ and His¹³⁰ as ligands for FE2. Previous studies of E. coli BFR have demonstrated that the ferroxidase center is essential for core formation and that core formation involves at least three kinetically distinguishable phases (11, 17). Phase 1 involves the very rapid reversible binding of two Fe²⁺ ions to each of the 24 dinuclear ferroxidase centers and can be studied by monitoring small changes in the spectrum of the bound heme. Phase 2 occurs in the presence of dioxygen (or an alternative oxidant such as hydrogen peroxide) and involves the rapid oxidation of each di-Fe²⁺ center to form an intermediate that is probably an oxo- or hydroxo-bridged di-Fe³⁺ center. In the presence of Fe²⁺ exceeding the amount required to saturate the ferroxidase centers, a slower reaction, Phase 3, is observed in which a large ferric oxyhydroxo mineral is synthesized within the protein cavity. The change in absorbance at 340 nm that is observed during aerobic addition of Fe²⁺ to apo-BFR results from Phases 2 and 3 but is influenced by the kinetics of Phase 1. Although Phases 1 and 2 are well characterized, less is known about Phase 3. This phase probably involves the interaction of Fe²⁺ (or Fe³⁺) with amino acid residues on the inner surface of the ferritin oligomer, as part of a complex and poorly defined process known as nucleation. Further information on this phase of core formation is now required.An assembly variant of E. coli BFR (E128R/E135R) has been shown previously to form stable subunit dimers that bind one equivalent of protoheme IX and not to form higher order oligomers (18). Each monomer in this minimal functional unit can form a dinuclear iron center that catalyzes the formation of a minimal iron core comprised of four to six iron atoms before precipitating (18, 19). The overall kinetics of Fe²⁺ oxidation observed on addition of Fe²⁺ to this variant are similar to those observed for wild-type BFR but have not been reported in detail. Nevertheless, the properties of this variant are of interest because the minimal structural unit that it forms constitutes a potentially important experimental model for evaluating detailed mechanistic features of BFR function. Simplification of the oligomeric structure of the protein as represented by this variant form of BFR makes the inner surface of the protein as accessible to bulk solvent as the outer surface, thereby removing any kinetic influences of the channels present in the 24-mer protein.The present paper reports detailed kinetic and structural studies that validate this dimeric variant of BFR as a model of the minimal functional unit of wild-type BFR. In addition, the crystallographic structural data suggest a likely functional role of acidic inner surface residues in iron core formation that led to construction of a family of variants of the stable subunit dimer involving replacement of Glu⁴⁷, Asp⁵⁰, and Asp¹²⁶ by site-directed mutagenesis. Kinetic studies of these additional variants confirm a functional role for these residues and lead to the proposal of a model of BFR action.     

Three ferritin subunits involved in immune defense from bay scallop Argopecten irradians

Ferritin is a ubiquitous protein that plays an important role in iron storage and iron-withholding strategy of innate immunity. In this study, three genes encoding different ferritin subunits were cloned from bay scallop Argopecten irradians (AiFer1, AiFer2 and AiFer3) by rapid amplification of cDNA ends (RACE) approaches based on the known ESTs. The open reading frames of the three ferritins are of 516 bp, 522 bp and 519 bp, encoding 171,173 and 172 amino acids, respectively. All the AiFers contain a putative Iron Regulatory Element (IRE) in their 5′-untranslated regions. The deduced amino acid sequences of AiFers possess both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Gene structure study revealed two distinct structured genes encoding a ferritin subunit (AiFer3). Quantitative real-time PCR analysis indicated the significant up-regulation of AiFers in hemocytes after challenged with Listonella anguillarum, though the magnitudes of AiFer1 and AiFer2 were much higher than that of AiFer3. Taken together, these results suggest that AiFers are likely to play roles in both iron storage and innate immune defense against microbial infections.     

Ferritin protein nanocage ion channels: gating by N-terminal extensions

Ferritin protein nanocages, self-assembled from four-α-helix bundle subunits, use Fe²⁺ and oxygen to synthesize encapsulated, ferric oxide minerals. Ferritin minerals are iron concentrates stored for cell growth. Ferritins are also antioxidants, scavenging Fenton chemistry reactants. Channels for iron entry and exit consist of helical hairpin segments surrounding the 3-fold symmetry axes of the ferritin nanocages. We now report structural differences caused by amino acid substitutions in the Fe²⁺ ion entry and exit channels and at the cytoplasmic pores, from high resolution (1.3–1.8 Å) protein crystal structures of the eukaryotic model ferritin, frog M. Mutations that eliminate conserved ionic or hydrophobic interactions between Arg-72 and Asp-122 and between Leu-110 and Leu-134 increase flexibility in the ion channels, cytoplasmic pores, and/or the N-terminal extensions of the helix bundles. Decreased ion binding in the channels and changes in ordered water are also observed. Protein structural changes coincide with increased Fe²⁺ exit from dissolved, ferric minerals inside ferritin protein cages; Fe²⁺ exit from ferritin cages depends on a complex, surface-limited process to reduce and dissolve the ferric mineral. High concentrations of bovine serum albumin or lysozyme (protein crowders) to mimic the cytoplasm restored Fe²⁺ exit in the variants to wild type. The data suggest that fluctuations in pore structure control gating. The newly identified role of the ferritin subunit N-terminal extensions in gating Fe²⁺ exit from the cytoplasmic pores strengthens the structural and functional analogies between ferritin ion channels in the water-soluble protein assembly and membrane protein ion channels gated by cytoplasmic N-terminal peptides.     

Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus)

Ferritin is a conserved iron binding protein existing ubiquitously in prokaryotes and eukaryotes. In this study, the gene encoding a ferritin M subunit homologue (SoFer1) was cloned from red drum (Sciaenops ocellatus) and analyzed at expression and functional levels. The open reading frame of SoFer1 is 531 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE) preserved in many ferritins. The deduced amino acid sequence of SoFer1 possesses both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Expression of SoFer1 was tissue specific and responded positively to experimental challenges with Gram-positive and Gram-negative fish pathogens. Treatment of red drum liver cells with iron, copper, and oxidant significantly upregulated the expression of SoFer1 in time-dependent manners. To further examine the potential role of SoFer1 in antioxidation, red drum liver cells transfected transiently with SoFer1 were prepared. Compared to control cells, SoFer1 transfectants exhibited reduced production of reactive oxygen species following H₂O₂ challenge. Finally, to examine the iron binding potential of SoFer1, SoFer1 was expressed in and purified from Escherichia coli as a recombinant protein. Iron-chelating analysis showed that purified recombinant SoFer1 was capable of iron binding. Taken together, these results suggest that SoFer1 is likely to be a functional ferritin involved in iron sequestration, host immune defence against bacterial infection, and antioxidation.     

The C-Terminal Regions Have an Important Role in the Activity of the Ferroxidase Center and the Stability of Chlorobium tepidum Ferritin

The recombinant Chlorobium tepidum ferritin (rCtFtn) is able to oxidize iron using ferroxidase activity but its ferroxidase activity is intermediate between the H-chain human ferritin and the L-chain human ferritin. The rCtFtn has an unusual C-terminal region composed of 12 histidine residues, as well as aspartate and glutamate residues. These residues act as potential metal ion ligands, and the rCtFtn homology model predicts that this region projects inside the protein cage. The rCtFtn also lacks a conserved Tyr residue in position 19. In order to know if those differences are responsible for the altered ferroxidase properties of rCtFtn, we introduced by site-directed mutagenesis a stop codon at position 166 and a Tyr residue replaced Ala19 in the gene of rCtFtn (rCtFtn 166). The rCtFtn166 keeps the canonical sequence considered important for the activity of this family of proteins. Therefore, we expected that rCtFtn 166 would possess similar properties to those described for this protein family. The rCtFtn 166 is able to bind, oxidize and store iron; and its activity is inhibit by Zn(II) as was described for other ferritins. However, the rCtFtn 166 possesses a decrease ferroxidase activity and protein stability compared with the wild type rCtFtn. The analysis of the Ala19Tyr rCtFtn shows that this change does not affect the kinetic of iron oxidation. Therefore, these results indicate that the C-terminal regions have an important role in the activity of the ferroxidase center and the stability of rCtFtn.     

Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O]_n multimers within the protein cage, en route to the cavity, at sites distributed over ∼50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A_{650 nm}) and on mineral growth (Fe³⁺O-A_{350 nm}), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O]_n transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.     

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.