Review
Iron mineralization and core dissociation in mammalian homopolymeric H-ferritin: Current understanding and future perspectives

https://doi.org/10.1016/j.bbagen.2020.129700Get rights and content

Highlights

  • Ferrous cations enter the ferritin shell through hydrophilic channels.

  • Rapid Fe(II) oxidization occurs at the di‑iron ferroxidase centers.

  • H2O2 can react with Fe(II) at ferroxidase centers, at separate sites, or on surface of the iron core.

  • In-vitro ferritin iron mobilization can be achieved using a variety of reducing agents.

  • In-vivo iron retrieval includes proteolytic degradation and possible iron reductive mechanisms.

Abstract

Background

The mechanism of iron oxidation and core formation in homopolymeric H-type ferritins has been extensively studied in-vitro, so has the reductive mobilization of iron from the inorganic iron(III) core. However, neither process is well-understood in-vivo despite recent scientific advances.

Scope of review

Here, we provide a summary of our current understanding of iron mineralization and iron core dissolution in homopolymeric H-type ferritins and highlight areas of interest and further studies that could answer some of the outstanding questions of iron metabolism.

Major conclusions

The overall iron oxidation mechanism in homopolymeric H-type ferritins from vertebrates (i.e. human H and frog M ferritins) is similar, despite nuances in the individual oxidation steps due to differences in the iron ligand environments inside the three fold channels, and at the dinuclear ferroxidase centers. Ferrous cations enter the protein shell through hydrophilic channels, followed by their rapid oxidization at di‑iron centers. Hydrogen peroxide produced during iron oxidation can react with additional iron(II) at ferroxidase centers, or at separate sites, or possibly on the surface of the mineral core. In-vitro ferritin iron mobilization can be achieved using a variety of reducing agents, but in-vivo iron retrieval may occur through a variety of processes, including proteolytic degradation, auxiliary iron mobilization mechanisms involving physiological reducing agents, and/or oxidoreductases.

General significance

This review provides important insights into the mechanisms of iron oxidation and mobilization in homopolymeric H-type ferritins, and different strategies in maintaining iron homeostasis.

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.

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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.)

References (129)

  • C.C. Philpott et al.

    Cytosolic iron chaperones: proteins delivering iron cofactors in the cytosol of mammalian cells

    J. Biol. Chem.

    (2017)
  • S. Leidgens et al.

    Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits Iron chaperone activity toward ferritin

    J. Biol. Chem.

    (2013)
  • S. Haldar et al.

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

    J. Biol. Chem.

    (2011)
  • B. Chandramouli et al.

    Electrostatic and structural bases of Fe2+ translocation through ferritin channels

    J. Biol. Chem.

    (2016)
  • T. Zhang et al.

    Encapsulation of anthocyanin molecules within a ferritin nanocage increases their stability and cell uptake efficiency

    Food Res. Int.

    (2014)
  • L. Chen et al.

    Encapsulation of beta-carotene within ferritin nanocages greatly increases its water-solubility and thermal stability

    Food Chem.

    (2014)
  • L. Chen et al.

    Encapsulation of curcumin in recombinant human H-chain ferritin increases its water-solubility and stability

    Food Res. Int.

    (2014)
  • E.C. Theil

    Ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry

    Curr. Opin. Chem. Biol.

    (2011)
  • N.D. Chasteen et al.

    Mineralization in ferritin: an efficient means of iron storage

    J. Struct. Biol.

    (1999)
  • V.J. Wade et al.

    Structure and composition of ferritin cores from pea seed (Pisum sativum)

    Biochim. Biophys. Acta

    (1993)
  • S. Mann et al.

    Reconstituted and native iron-cores of bacterioferritin and ferritin

    J. Mol. Biol.

    (1987)
  • G. Zhao

    Phytoferritin and its implications for human health and nutrition

    Biochim. Biophys. Acta

    (2010)
  • W. Wang et al.

    Serum ferritin: past, present and future

    Biochim. Biophys. Acta Gen. Subj.

    (2010)
  • M. Mehlenbacher et al.

    Iron oxidation and core formation in recombinant heteropolymeric human ferritins

    Biochemistry

    (2017)
  • E.C. Theil

    Ferritin: the protein nanocage and iron biomineral in health and in disease

    Inorg. Chem.

    (2013)
  • E.C. Theil et al.

    Solving biology’s iron chemistry problem with ferritin protein nanocages

    Acc. Chem. Res.

    (2016)
  • M.C. Linder

    Mobilization of stored iron in mammals: a review

    Nutrients

    (2013)
  • A. La et al.

    Mobilization of iron from ferritin: new steps and details

    Metallomics

    (2018)
  • F. Bou-Abdallah et al.

    Reductive mobilization of iron from intact ferritin: mechanisms and physiological implication

    Pharmaceuticals

    (2018)
  • Z.I. Cabantchik

    Labile iron in cells and body fluids: physiology, pathology, and pharmacology

    Front. Pharmacol.

    (2014)
  • R.C. Hider et al.

    Glutathione: a key component of the cytoplasmic labile iron pool

    Biometals

    (2011)
  • T.W. Giessen et al.

    Large protein organelles form a new iron sequestration system with high storage capacity

    ELife

    (2019)
  • D. He et al.

    Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

    ELife

    (2016)
  • R. Lebo et al.

    Human ferritin light chain gene-sequences mapped to several sorted chromosomes

    Hum. Genet.

    (1985)
  • X. Liao et al.

    Structure, function, and nutrition of phytoferritin: a newly functional factor for iron supplement

    Crit. Rev. Food Sci. Nutr.

    (2014)
  • J.C. Sibille et al.

    Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein

    Hepatology

    (1988)
  • M.J. Leimberg et al.

    Macrophages function as a ferritin iron source for cultured human erythroid precursors

    J. Cell. Biochem.

    (2008)
  • G.A. Ramm et al.

    Identification and characterization of a receptor for tissue ferritin on activated rat lipocytes

    J. Clin. Invest.

    (1994)
  • L. Li et al.

    Binding and uptake of H-ferritin are mediated by human transferrin receptor-1

    Proc. Natl. Acad. Sci. U. S. A.

    (2010)
  • R.R. Crichton et al.

    Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH

    Biochem. J.

    (1973)
  • M. Kim et al.

    pH-dependent structures of ferritin and apoferritin in solution: disassembly and reassembly

    Biomacromolecules

    (2011)
  • K. Iwasaki et al.

    Hemin-mediated regulation of an antioxidant-responsive element of the human ferritin H gene and role of Ref-1 during erythroid differentiation of K562 cells

    Mol. Cell. Biol.

    (2006)
  • K.J. Hintze et al.

    DNA and mRNA elements with complementary responses to hemin, antioxidant inducers, and iron control ferritin-L expression

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • N. Wilkinson et al.

    The IRP/IRE system in vivo: insights from mouse models

    Front. Pharmacol.

    (2014)
  • J.D. Mancias et al.

    Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis

    Elife

    (2015)
  • A.E. Martell et al.

    Critical Stability Constants

    (1975)
  • R.C. Hider et al.

    Iron speciation in the cytosol: an overview

    Dalton Trans.

    (2013)
  • H. Shi et al.

    A cytosolic Iron chaperone that delivers Iron to ferritin

    Science

    (2008)
  • F. Bou-Abdallah et al.

    Facilitated diffusion of iron (II) and dioxygen substrates into human H-chain ferritin. A fluorescence and absorbance study employing the ferroxidase center substitution Y34W

    J. Am. Chem. Soc.

    (2008)
  • C. Pozzi et al.

    Time-lapse anomalous X-ray diffraction shows how Fe(2+) substrate ions move through ferritin protein nanocages to oxidoreductase sites

    Acta Crystallogr. D Biol. Crystallogr.

    (2015)
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