ReviewIron mineralization and core dissociation in mammalian homopolymeric H-ferritin: Current understanding and future perspectives
Graphical abstract
Iron Oxidation Mechanism in Human H Ferritin. From top left and counterclockwise, (1,2) diferrous and oxygen-bound diferrous-protein complex, (3, 4) deprotonated and protonated peroxo diFe(III)-protein complexes, (5) μ-oxo-diFe(III) protein complex.
Introduction
Iron is a critical nutrient required for virtually all forms of life. Iron redox-cycling ability and wide range of physiological oxidation-reduction potentials underline its essential involvement in many biological processes, but also its potential toxicity to aerobically respiring cells [[1], [2], [3], [4], [5], [6], [7]]. Because of the very low solubility of free ferric cations (~ 10−18 M at neutral pH), bioavailability, and propensity to participate in harmful free radical reactions, living organisms have developed sophisticated molecular machinery to safely control iron trafficking. One key component of this complex system is ferritin, a ubiquitous iron storage and detoxification molecule, and a key player in iron homeostasis [[2], [3], [4], [5], [6], [7]]. Although in cell-free systems (i.e. in-vitro) the mechanisms of iron(II) oxidation and mineral core formation in ferritin have been extensively studied, it is still unclear how iron is loaded into ferritin in-vivo, and virtually no mechanistic details are known about iron mobilization. Nonetheless, there is a widespread belief that the major pathway for iron mobilization from ferritin involves a lysosomal proteolytic degradation of ferritin, followed by the dissolution of the iron mineral core [1,8,9]. The generally accepted mechanism for ferritin mineral core formation involves the rapid diffusion of ferrous cations across the ~12 nm diameter protein nanocage, through eight hydrophilic 3-fold channels. Following the rapid oxidation of Fe2+ at the ferroxidase centers of H-subunits, the oxidized iron ions migrate to the protein cavity, and bind at nucleation sites on the L-subunits to form the iron mineral [2]. In an earlier review [10], we discussed the mechanism and physiological implication of iron reductive mobilization from ferritin in the presence of physiological reducing agents, and hypothesized that an auxiliary iron mobilization mechanism, in addition to ferritin proteolytic degradation, may exist. Other endogenous compounds, including oxidoreductases have also been proposed as potential ferritin-reducing agents. Here, we summarize our current knowledge of the mechanisms of iron oxidation, iron core mineralization and iron dissolution from ferritin, and highlight the need for additional studies to further our understanding of iron transport and storage in cells.
Section snippets
Labile iron pool and iron storage proteins
The majority of cellular iron is tightly bound to iron proteins, and does not interact with external chelate ligands. However, a minor fraction (less than 5%) of iron cations is loosely bound to a heterogeneous population of ligands, including polypeptides, and metal complexing groups such as carboxylates, phosphates, amines, thiolates, and hydroxylates [11,12]. This exchangeable pool of iron, predominantly in the form of iron(II) cations, because of cellular reductases and glutathione [13], is
Ferritin channels and iron transport
Iron(II) cations are highly labile [32] and relatively “soft” metals, that preferably form metal complexes with nitrogen and sulfur ligands. In the cytosol, iron(II) cations can be bound by histidine, cysteine [33], and glutathione [13,34], and some proteins [[35], [36], [37]] into highly kinetically labile complexes. In human ferritin, iron(II) cations enter the protein interior via eight hydrophilic three-fold channels lined with glutamate and aspartate residues. The binding of Fe2+ cations
Accessibility to ferritin interior by other molecules
Despite easy accessibility to a variety of metal cations and small molecules, the ferritin inner cavity appears to be well insulated to the diffusion of large organic molecules, as demonstrated by the ferritin disassembly-reassembly process at acidic pHs. The partial disassembly of the ferritin nanocage at pH ≤ 2 in the presence of different organic molecules resulted in the encapsulation of these molecules inside the ferritin cavity, and the full reassembly of the ferritin nanocage at neutral
Composition of the ferritin inorganic iron core
Whereas mineralized Fe3+ cations inside ferritin are in the form of insoluble hydrated iron(III) hydroxide, natural ferritins with differences in H- to L-subunit ratios correlate with different rates of ferritin biomineral formation and, likely, with biomineral order or degree of crystallinity and iron turnover [3]. For instance, disordered mineral cores in animal ferritins are mostly observed in L-rich heteropolymers having a large number of catalytically inactive L-subunits, as those found in
Reduction of the ferritin inorganic iron core
Maintaining a constant concentration of labile iron cations in living cells requires a careful balance between the rate of iron mineralization and the rate of iron mobilization in ferritin, whether through proteolytic degradation [10], or possibly through non-proteolytic mobilization processes, including reduction and/or direct chelation [10]. No reliable evidence of allosteric modulation of either pathway has been provided, although the kinetics of these processes play a central role in
Ferritin ferroxidase centers
Following initial contact at the opening of the three-fold hydrophilic channels, Fe2+ cations follow a 15 to 20 Å pathway to the ferritin ferroxidase centers [77]. In addition to the three-fold channels, other acidic residues (i.e. glutamate and aspartate), and possibly histidines may, assist Fe2+ cations in their journey to the dinuclear centers (Fig. 2). Because of the rapid oxidation of Fe2+ in ferritin, the diffusion pathway has been studied predominantly using model redox inactive cations,
Iron dissociation from the dinuclear centers and iron mineralization
The dissociation constant for the strong Fe2+ binding site (Fe1) at the ferroxidase center is moderately strong (Kd ~ 5–10 μM [80] for human H-chain ferritin and Kd ~ 0.3 μM for the E. coli bacterial ferritin EcFtnA [85]) at neutral pH. There are conflicting reports in the literature about the value of Km,Fe2+, ranging between 380 μM for horse spleen ferritin [86], and 9 μM for bovine spleen ferritin [87]. Even large differences have been reported for ferritin from the same species and tissues [
Iron mineralization in homopolymer L-subunit ferritin
As discussed above, ferritin ferroxidase activity is due to a dinuclear ferroxidase center located on the H-subunit. Whereas weak ferroxidase activity has been reported for L-ferritin lacking a ferroxidase center, a recent study employing heteropolymer ferritin of 20H:4 L ratio revealed an improved ferroxidase and mineralization activity, and a dual role the L-subunit in facilitating iron turnover at the ferroxidase center, and in the mineralization of the iron core [3]. Serum ferritin, for
The inorganic iron core does not possess ferroxidase-like activity
The unusual kinetics of iron mineralization [79,100] in human H-chain ferritin suggest the existence of multiple pathways for iron mineralization that include the ferroxidase center, the iron core surface, and the hydrogen peroxide detoxification reactions [68,102]. However, a more recent investigation from our laboratory revisited on the prospect of iron oxidation on the surface of a growing mineral core [89]. Our data [89] demonstrate that for human homopolymer and heteropolymer ferritins,
Effect of oxygen on ferritin iron mineralization and mobilization
The dependence of the iron mineralization rate on the concentration of dissolved oxygen is another physiologically important parameter in ferritin function. In living cells, the concentration of oxygen is in the range of 1.3–2.5 kPa (18–34 μM) [106] and ~ 7 μM in mitochondria. The oxygen concentration can be even lower under strenuous exercises, hypoxia, and cardiovascular disorders [107]. If oxygen concentration drops significantly below the experimentally measured Km,O2 in ferritin (i.e. ~
Hydrogen peroxide production and ferritin peroxidase activity
One of the byproducts of Fe(II) oxidation by molecular oxygen in ferritin is hydrogen peroxide [42]. Whereas most of the produced hydrogen peroxide at the ferroxidase center of ferritin is rapidly reduced by incoming iron(II) cations, a small amount can diffuse out of the ferritin shell, as detected by the enzyme catalase [112]. Because there are more ferroxidase centers in a pure homopolymer H-chain ferritin (HuHF), the amount of hydrogen peroxide produced in HuHF is much higher than in L-rich
Role of autophagy in the demineralization of the ferritin iron core
As briefly discussed at the beginning of this review, the Nuclear Receptor Coactivator-4 (NCOA4) is involved in ferritin degradation by lysosomal autophagy [31], and that the NCOA4-mediated ferritinophagy is an essential mechanism for normal iron homeostasis, but also in several pathological conditions including neurodegenerative diseases, ischemia/reperfusion injury and cancer [117]. In mammalian cells, the divalent metal transporter 1 (DMT1) is a transmembrane iron transporter that is
Conclusions and future perspectives
The pathway of Fe2+ ions entry through the ferritin shell and our understanding of iron mineralization in ferritin has significantly improved in recent years. That ferritins from different sources oxidize iron differently illustrates the diversity of iron mineralization in these major iron storage proteins [65,129]. Depending on the ferritin type, the fate of the ferric-oxo complex formed at the ferroxidase center of ferritin varies from being a true and stable catalytic cofactor site that
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgements
This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM104879 (F. B. A.)
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