Article pubs.acs.org/accounts
Solving Biology’s Iron Chemistry Problem with Ferritin Protein Nanocages Elizabeth C. Theil,* Takehiko Tosha,† and Rabindra K. Behera‡ Children’s Hospital Oakland Research Institute, Oakland, California 94609, United States Department of Structural and Molecular Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7313, United States CONSPECTUS: Ferritins reversibly synthesize iron-oxy(ferrihydrite) biominerals inside large, hollow protein nanocages (10−12 nm, ∼480 000 g/mol); the iron biominerals are metabolic iron concentrates for iron protein biosyntheses. Protein cages of 12- or 24-folded ferritin subunits (4-α-helix polypeptide bundles) self-assemble, experimentally. Ferritin biomineral structures differ among animals and plants or bacteria. The basic ferritin mineral structure is ferrihydrite (Fe2O3·H2O) with either low phosphate in the highly ordered animal ferritin biominerals, Fe/PO4 ∼ 8:1, or Fe/PO4 ∼ 1:1 in the more amorphous ferritin biominerals of plants and bacteria. While different ferritin environments, plant bacterial-like plastid organelles and animal cytoplasm, might explain ferritin biomineral differences, investigation is required. Currently, the physiological significance of plant-specific and animal-specific ferritin iron minerals is unknown. The iron content of ferritin in living tissues ranges from zero in “apoferritin” to as high as ∼4500 iron atoms. Ferritin biomineralization begins with the reaction of Fe2+ with O2 at ferritin enzyme (Fe2+/O oxidoreductase) sites. The product of ferritin enzyme activity, diferric oxy complexes, is also the precursor of ferritin biomineral. Concentrations of Fe3+ equivalent to 2.0 × 10−1 M are maintained in ferritin solutions, contrasting with the Fe3+ Ks ∼ 10−18 M. Iron ions move into, through, and out of ferritin protein cages in structural subdomains containing conserved amino acids. Cage subdomains include (1) ion channels for Fe2+ entry/exit, (2) enzyme (oxidoreductase) site for coupling Fe2+ and O yielding diferric oxy biomineral precursors, and (3) ferric oxy nucleation channels, where diferric oxy products from up to three enzyme sites interact while moving toward the central, biomineral growth cavity (12 nm diameter) where ferric oxy species, now 48-mers, grow in ferric oxy biomineral. High ferritin protein cage symmetry (3-fold and 4-fold axes) and amino acid conservation coincide with function, shown by amino acid substitution effects. 3-Fold symmetry axes control Fe2+ entry (enzyme catalysis of Fe2+/O2 oxidoreduction) and Fe2+ exit (reductive ferritin mineral dissolution); 3-fold symmetry axes influence Fe2+exit from dissolved mineral; bacterial ferritins diverge slightly in Fe/O2 reaction mechanisms and intracage paths of iron-oxy complexes. Biosynthesis rates of ferritin protein change with Fe2+ and O2 concentrations, dependent on DNA-binding, and heme binding protein, Bach 1. Increased cellular O2 indirectly stabilizes ferritin DNA/Bach 1 interactions. Heme, Fe-protoporphyrin IX, decreases ferritin DNA-Bach 1 binding, causing increased ferritin mRNA biosynthesis (transcription). Direct Fe2+ binding to ferritin mRNA decreases binding of an inhibitory protein, IRP, causing increased ferritin mRNA translation (protein biosynthesis). Newly synthesized ferritin protein consumes Fe2+ in biomineral, decreasing Fe2+ and creating a regulatory feedback loop. Ferritin without iron is “apoferritin”. Iron removal from ferritin, experimentally, uses biological reductants, for example, NADH + FMN, or chemical reductants, for example, thioglycolic acid, with Fe2+ chelators; physiological mechanism(s) are murky. Clear, however, is the necessity of ferritin for terrestrial life by conferring oxidant protection (plants, animals, and bacteria), virulence (bacteria), and embryonic survival (mammals). Future studies of ferritin structure/function and Fe2+/O2 chemistry will lead to new ferritin uses in medicine, nutrition, and nanochemistry. (Dps proteins) use Fe2+ and H2O2 for (Fe2O3·H2O).1,2 Mainly intracellular, glycosylated serum ferritin protects kidneys.3 Ferritin protein cages assemble spontaneously in solution from 24 folded, ferritin polypeptides (subunits). The cages have two natural sizes. Twelve subunit, bacterial mini-ferritins (Dps proteins),
1. INTRODUCTION Iron, a problem (“rusting”) for terrestrial life (Figure 1), is needed for bioenergetics, cell division and transporting O2. Ferritins, “anti-rust” proteins, are soluble, hollow, protein nanocages, ∼12 nm diameter, and ∼480 000 Da. They achieve iron concentrations equivalent to 2 × 10−1 M in neutral solutions,1,2 reversibly forming soluble, protein-encased ferrihydrite (Fe2O3·H2O) from Fe2+ and O2; 12 subunit mini-ferritins © 2016 American Chemical Society
Received: December 31, 2014 Published: May 2, 2016 784
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Figure 1. Ferritin ion channels in the protein cage. Left: View down a 3-fold symmetry axis. Middle: Cross section of a ferritin protein cage; 4-fold cage symmetry axis. Right: Single ion channel, cartoon; 24 subunit ferritin cages have 8 ion channels.
often associated with virulence, and 24 subunit animal, plant, and bacterial ferritins, central to iron metabolism, are more extensively studied and emphasized here.
Figure 2. Close-up: long (L) loops between two ferritin protein cage subunits. The L loops, in each of two ferritin polypeptide subunits, connect subunit helices 2 and 3, likely “sealing” the cage. Ferritin polypeptide subunit folded in the common 4-α-helix bundle motif.7,8
2. FERRITIN IN BIOLOGY Living cells need ferritin proteins to survive; they make and maintain protein-coated iron concentrates. The ferritin ironconcentrating reaction is 2n[Fe2+·H2O] + nO2 → nFe2O3(S) + 4nH+. Clear, neutral, amber-colored ferritin solutions maintain iron concentrations, in air, equivalent to 2 × 10−1 M, contrasting with a Ks ∼ 10−18 M for Fe3+, at pH 7 (Figure 1). A stable, protein cage, ferritin resists 80 °C for 10−20 min in neutral, buffered solutions. Amounts of ferritin iron vary in Nature, to ∼4500 iron atoms/ cage; sucrose gradient centrifugation (∼100 000g) separates ferritin molecules based on iron content. Biosynthesis of ferritin “anticipates” iron need, for example, ferritin accumulates in immature red blood cells before globin and heme biosynthesis begin. Later, iron from old hemoglobin is concentrated in ferritin of macrophage cells for “recycling” to new, immature red blood cells.4 Reduction/chelation remove ferritin iron, experimentally, producing “apoferritin”; mercaptoacetic acid or FMNH2 are effective reductants. In vivo reductants are unidentified. Ferritin enzymology, ion traffic, biomineralization, and regulated biosynthesis are all active study areas. Solving ferritin “puzzles” (section 8) is crucial for new ferritin applications in medicine and nanotechnology.
The combinations of 24 different H-type (enzymatically active) subunits in plant and bacterial ferritins, contrast with animal ferritin cages, combinations of H subunits with enzymatically inactive L subunits; “H:L ratios”7 are tissue specific. Bacterial mini-ferritins (Dps proteins), associated with virulence, have different active site ligands than larger ferritins.1,2 3.2. Iron/Oxy Nanomineral Composition/Structure
Fe2O3·H2O (ferrihydrite), a metastable mineral,11 is similar to animal ferritin biominerals, which also contain phosphate, Fe:PO4 ∼ 8:1, and are relatively ordered.12 Plant and bacterial ferritin biominerals, hydrated ferric phosphate, Fe:P ∼ 1:1, are relatively disordered, and less studied. The physiological significance of different ferritin biominerals is unknown. Ferritin protein nanocages (Figure 1) contain 24 subunits (folded polypeptides); mini-ferritin (Dps proteins) of bacteria have 12 subunits. Subunits are 4-α-helix bundles. Cage assembly mechanism(s) in vivo are little studied.
4. FERRITIN STRUCTURE RELATED TO FUNCTION Ferritins in plants and animals, the 24 subunit maxi-ferritins, are the more extensively studied. Bacterial ferritins have either 24 subunits or 12 subunits (called Dps proteins). Ferritins with 24 subunits are the focus here. Each polypeptide subunit of ferritin protein cages contains a distinctively long loop, L, connecting helices 2 and 3 of the 4-α-helix bundles (Figure 2); the short polypeptide loops connecting subunit helices 1−2 and 3−4 are more typical of other proteins. Ferritin subunit L loops are anchored by conserved, intersubunit ion pairs, aspartate 80 and lysine 82; the loops fill intersubunit spaces between pairs of subunits;7,8 it is likely that L loops contribute to the high stability of ferritin at neutral pH (60 °C for 10 min or 6 M urea, 25 °C). Ferritin cage stability, important in selective tissue ferritin purification, can be exploited in ferritin-based nanotechnology and nanomedicine.14,16 Animal ferritins differ from plant and bacterial ferritin because they are mixtures of polypeptide subunits: H (heavy or higher), M/H′ (middle) and L (lower). [Ferritin subunit nomenclature reflects electrophoresis mobility but often diverges from molecular mass.]
3. FERRITIN PROTEIN STRUCTURE The very complex ferritin structure5,6 is incompletely understood. Ferritin “unity”, prematurely proposed recently,15 obscures mechanistic complexities, for example, different di-iron site ligands and enzyme rates, impeding understanding. Moreover, ferritin variants, newly discovered or engineered, require structure/function study. 3.1. Nanocage
Ferritin cages assemble from polypeptides (subunits of ∼170−180 amino acids, folded in 4 α-helix bundles). A long (L), intrasubunit loop contributes to ferritin cage assembly and function (Figure 2).7,8 Ferritin protein cage symmetry and temperature stability are unusually high.1 Enzyme sites (Fe2+/O oxidoreductase sites), within ferritin protein cage subunits, initiate Fe2O3·H2O nanomineral formation.9,10 While quaternary (nanocage) and secondary structure (4-α-helix bundles) are conserved in ferritins, amino acid sequences are conserved as much as 80%.1,13 Moreover, enzyme site amino acids vary among different ferritins. 785
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Figure 3. Iron ion traffic within ferritin protein cages. Subunit interfaces at 2-fold, 3-fold, and 4-fold cage symmetry axes of ferritin protein cages contribute to ferritin function. (A) Eight Fe2+ entry and exit channels at protein cage 3-fold symmetry axes. (B) Side view of an Fe2+ channel, viewed from inside a ferritin protein cage. Negatively charged carboxylate side chains of conserved amino acids, E130 and D127, project into the Fe2+ entry/exit channel from three cage subunits (red, cyan, or tan).17,30,31 Fox site, Fe2+/O2 oxidoreductase enzyme site; arrows path of entering Fe2+. (C) Single ferritin cage subunit. Side view: green spheres are metal ions. Fe2+ distribution among multiple ferritin enzyme sites is likely random, but unknown. (D) A single ferritin ion channel, cage interior view; arrows and dotted lines are apparent Fe2+ ion trajectory between cage entrance and Fox enzyme site, ∼27 Å. Data from the authors’ work.30,31 RCSB PDB 3KA3.
possibly reflecting physiological temperature differences: warmblooded humans vs cold-blooded frogs. The sequence similarity between ferritin enzyme sites, E, ExxH/E, QxxD, and di-iron cofactor sites, E, ExxH/E, and ExxH, which also catalyze Fe/O2 reactions, is tantalizing. However, replacing ferritin active site ligands with di-iron oxygenase ligands, using site-directed mutagenesis, inhibited the Fe2+/O2 reaction, illustrating the importance of protein “background” on enzyme activity.21 Di-iron enzyme sites in ferritin are more coordinately saturated than di-iron oxygenases.21 Fe2+ transit into or out of ferritin cages, and thus enzyme activity, depends on conserved, ion channel amino acids with carboxylate side chains.17,22,23 E130 and D127 carboxylates project into ferritin ion channels connecting the outside environment to ferritin enzyme sites, which are within the protein cage. Ion channel residue D127, near the internal channel exit, is close to carboxylates E136; E136 near E57 which is near enzyme site residues; the “carboxylate chain” is required for ferritin enzyme activity (blue diferric peroxo (λmax 650nm) formation from the enzymatic reactions of Fe2+ with O2); ferritin diferric peroxo complexes are the precursors of ferritin ferric oxy dimers and ferric oxy multimers, the ferritin biomineral precursors;5,19,24 and diferric peroxo formation/decay rates facilitated ferritin mechanistic understanding.25 Figure 3 illustrates Fe2+ movement from ferritin protein cage exterior to cage enzyme sites. “Unity”, proposed for all ferritin Fe2+/O2 oxidoreductase enzyme mechanisms15 is not possible with current knowledge. Known are the facts that enzyme site iron ligands, enzyme rates, and mineral order all vary, even within different tissue ferritins of
Ferritin H, L, or M/H′ subunits, encoded in different genes, are selectively synthesized by different animal tissues; only H or H′ ferritin subunits have active enzyme sites. In plant and bacterial ferritins, all subunits are enzymatically active, H-type. In animals, by contrast, ferritins are tissue-specific mixtures of enzymatically active (H-type) or inactive (L-type) subunits. The relationships among iron physiology, ferritin protein-encased iron biominerals, and ferritin protein cage subunits are, themselves, incompletely studied.
5. FERRITIN ENZYME ACTIVITY How Fe2+ is directed into ferritin ion channels embedded in the protein cages, through the ion channels and into the protein cage interior, binding at Fe2+/O2 enzyme sites, is only partly understood (Figure 3). The required ion channel and intracage transfer residues, as well as multiple, di-iron enzyme substrate sites and residues in interhelix subunit loops contributing to ferritin Fe2+/O2 oxidoreductase enzyme rates are relatively wellknown,5,7,8,17−19 but mechanistic information is lacking. Structure/function ferritin data show the flexible, ion channel carboxylate chain delivers Fe2+ to multiple enzyme sites.18 While enzyme sites (Fe/O oxidoreductases) occur in all ferritin cage subunits, of plants and bacteria in higher animals, including humans, the ferritins are tissue-specific mixtures of enzymatically active (H/H′/M) and inactive (L) subunits with different Fe2+ oxidation rates.4,19 Ferritin di-iron site A is invariant: E, ExxH; site B can be E, QxxA or E, QxxS or E, QxxD.19,20 Comparison of Fe2+ movement through ferritin protein cages in crystallo, for human site B (E, QxxA) and frog site B (E, QxxD)6 revealed differences, 786
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Figure 4. L-Ferritin subunits, naturally lacking enzyme sites and in animal ferritins, influence biomineral formation. Ferritin H, H′, or M protein cage subunits have enzyme sites (Fe2+/O2 oxidoreductase). “L” subunits, lacking enzyme activity, coassemble with H subunits in animal ferritins; identifying H ferritin enzyme sites used L ferritin insertional mutagenesis and protein activity measurements.19
Figure 5. Ferritin IRE-RNA bound to repressor protein, IRP. IRE-RNA, noncoding mRNA, binds regulatory protein, IRP in animal mRNAs of iron homeostasis.41 IRP/IRE-RNA blocks ribosome binding. Fe2+/IRE- RNA releases IRP, facilitating protein biosynthesis.35 Fe2+-IRE-RNA structure is unknown. IRE-RNA, aqua; IRE-RNA paired bases, red and yellow; IRP protein, purple; black lines, conserved, IRE-RNA/IRP contact sites.
7. FERRITIN PROTEIN CAGE STRUCTURAL SUBDOMAINS RELATED TO FUNCTION Ferritin biomineral formation is reversible, experimentally, with external reductants, for example, thioglycolic acid or FMNH2. Physiological ferritin mineral reduction “iron release” is poorly understood.
the same organism! Until the enzyme and biomineralization mechanisms of all ferritins25 are known, mechanistic “unity” is premature and discounts the thousands of years of natural selection leading to contemporary variations in ferritin enzyme mechanisms and biomineral properties (see subsection 3.2 and section 6). Diferric oxy enzyme products in ferritin protein cages move into “nucleation channels”;5 distinct from Fe2+ entry/exit channels; nucleation channels connect enzyme sites to the central mineral growth cavity (Figure 4). In 24 subunit ferritin protein cages, 3 nucleation channels exit near each other (Figure 4), at cage 4-fold symmetry axes.5 Reactions within ferritin nucleation channels yield ferric oxy multimers, averaging ∼48 Fe atoms5, indicating contributions of ferritin protein cages to early biomineral growth.
7.1. Fe2+ Entry and Exit
Fe2+ entry and exit in ferritin occur through the same intracage ion channels. Each ion channel contains segments of three helices contributed by each of three adjacent subunits around at the 3-fold symmetry axes of the protein cage. (Figure 3B). Fe2+ is transferred to ferritin enzyme sites, after traversing ferritin ion channels. Fe2+ appears to move between conserved aspartate and glutamate residues (a “carboxylate chain” or “bucket brigade”) that begins within the ion channels and continues through ferritin protein cages to enzyme sites buried within each cage subunit17(Figure 3B).
6. FERRITIN BIOMINERALS Maximum diameters of the hydrated ferric oxides (ferrihydrite biominerals) in ferritin are limited by the 8 nm inner diameter of the protein cage. The biominerals contain up to ∼4500 iron atoms. Differences in ferritin biomineral size reflect tissue iron metabolism; ferritin with low to zero iron is generally destined for destruction (turnover). Ferritin biomineral order (amorphous to microcrystalline) generally coincides with biomineral phosphate, varying from amorphous/disordered plant and microbial ferritins (Fe:P ∼ 1:1) to nanocrystalline/ordered animal ferritins (Fe:P ∼ 8:1). However, different ferritin subcellular environments, plant organelles (plastids) or animal cytoplasm, may also explain ferritin biomineral differences.
7.2. Ferritin Enzyme (Oxidoreductase) Sites
Ferritin enzyme sites in ferritin polypeptide subunits, catalyze the reaction between 2 Fe2+ and O2, yielding di-iron oxy mineral precursors. Enzyme activity is contained in H (H′/M)-type ferritin subunits; animals, contrasting with plants and bacteria, produce a cage subunit, L, lacking enzyme activity. L-type subunits, genetically engineered with increasing numbers of putative H-type enzyme site ligands, and monitoring diferric peroxo production, identified ferritin enzyme sites.19 In natural ferritins, differences in H:L subunit ratios coincide with different 787
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ferritins the two Fe2+ bind transiently as enzyme substrates and, after reacting with the second substrate, O2 (or H2O2 the smaller, Dps protein mini-ferritins), are rapidly released as diferric oxy product.
rates of ferritin biomineral formation and, likely, biomineral order and turnover (Figure 4). When ferritin di-iron oxy products emerge at the mineral growth cavity, they are diferric oxy multimers.5,6 The structure(s) of the diferric oxy complexes, the products of Fe2+/O2 enzyme activity, are currently unknown. In ferritin protein cages, specific, conserved amino acids are required to move Fe3+oxy complexes from enzyme sites through nucleation channels to the mineral growth cavity5,6,8,10,26 but nucleation channel properties remain largely unexplored. When diferric-oxy products of ferritin Fe2+/O2 enzyme activity traverse ferritin protein cages toward the central, mineral growth cavity, they move slowly enough to interact with each other, entering the central mineral growth cavity as ∼48-mers.5 Experimentally, proton release (∼2.5 H+/Fe) is detected during the process likely from reactions among hydrated ferric ions inside the protein cage. In vivo, ferritin mineral sizes vary depending on available metabolic iron, and reflecting biological signals. Fe2+, reductively dissolved from ferritin biominerals, leaves the protein cage through the same protein channels as entering Fe2+. However, the conserved ion channel amino acids required for Fe2+ exit, different from those for Fe2+ entry.18,22 Possibly, though unexplored, electron transfer to mineralized Fe3+ inside ferritin requires specific ion channels amino acids. FMNH2, effectively experimentally, could also function in vivo, but nothing is currently known about ferritin biomineral reductants, or e− or H+ transfers during ferritin mineral dissolution in vivo.
7.5. Fe3+ Oxy Nucleation Channels
In 24 subunit ferritins, diferric oxo complexes are synthesized at ferritin oxidoreductase enzyme sites and then move through internal cage channels (Figure 4).28 Experimentally, four additions of Fe2+, equivalent to 2 Fe2+/ferritin enzyme site, are required before any Fe3+ reaches the internal exits of the channels.5 The internal exits of four nucleation channels are symmetrically clustered around the 4-fold symmetry axes of the ferritin protein cage. As a result, ferritin biomineral nucleation is facilitated by the physical proximity of the ferric oxy complexes in the nucleation channels and the resulting interactions to form larger ferric oxy multimers as they move away from the enzyme coupling sites en route to the internal biomineral growth cavity.5,28 7.6. Summary of Distinctive Ferritin Protein Cage Sudomain Structure and Function
Ferritin polypeptides spontaneously fold into subunits and form hollow nanocages, at least in solution. The protein cages, 8−12 nm in diameter, have central internal cavity 5−8 nm in diameter. After enzymatic coupling at ferritin intracage enzyme sites, small, ferric oxy clusters with two to eight iron atoms emerge from the intracage channels into the mineral growth cavity (Figure 4). The complex of iron mineral inside a protein cage remains stable in solution or lyophilized to dryness for many years at 4 °C.
7.3. Fe2+ Ion Entry and Exit Channels.
Ferritin protein cage channels at the 3-fold cage symmetry axes are ion channels controlling both Fe2+ entry and exit. X-ray crystallography of ferritin cocrystallized with Co2+, modeling Fe2+, show multiple metal ions aligned within the ion channels, similarly to membrane ion channel proteins.18 Protein engineering and enzyme activity analyses demonstrated the dependence of Fe2+ entry on conserved, ferritin ion channel carboxylates D127 and E130 and the functional analogy of ferritin 3-fold pores and channels to those of ion channel proteins in cell membranes.18,22 The ferritin protein cage enzyme sites, where Fe2+/O2 react, are more than 10 Å from the inner exit of Fe2+ channels. Near the internal exits of the Fe2+ ion channels, on the inner surface of ferritin nanocages, conserved amino acids, with flexible carboxylate side chains, facilitate Fe2+ movement from the Fe2+ channel to the ferritin enzyme sites; carboxylate flexibility was observed as multiple conformations in protein crystals.8,20,22 Fe2+ oxidation is slower when ferritin ion channel carboxylates were changed by genetic engineering.20
7.7. Iron Biomineral Reduction, Dissolution, and Fe2+ Exit
The biominerals that form inside ferritin protein cages can, upon biological demand or chemical reduction experimentally, be dissolved to release Fe2+. Ferritin protein cage pores can be unfolded without affecting overall ferritin protein cage structure,29 but pore unfolding increases rates of Fe2+ exit whether by amino acid substitution, or changing solution condition.19,22,29 While ferritin is stable to 80−85 °C and 6 M urea at pH 7, localized unfolding of ferritin pores can be induced by solution changes such as heat (35−45 °C) or chaotropes such as 6 M urea at pH 7, the presence of reductants such conditions accelerated Fe2+ exit.18−20,22,29,30 Engineered ferritin proteins were used to demonstrate that that Fe2+, dissolved from ferritin biominerals, exits through the same ion channels as Fe2+ entering the protein cage.10,31 Although conserved carboxylates in ferritin Fe2+ channels guide Fe2+ entry, such residues are not essential for Fe2+ exit. Rather, based on crystallographic studies of ferritin variants, most often produced by protein engineering, Fe2+ exit rates depend on localized folding/unfolding of the protein cage around the 8 ferritin pores.18,29 Such ferritin pore unfolding activity could be contained in intracellular proteins or peptides, yet to be identified.
7.4. Fe2+/O2 Oxidoreduction Enzyme Sites
Ferritin enzyme activity sites, in the 24 subunit ferritins of plants and animals, are embedded in the 4-α-helix polypeptide bundles that comprise ferritin protein cages. The enzyme sites are di-iron sites with two sets of 3−4, highly conserved amino acids; histidine imidazole and glutamate carboxylate, for example, are always present. Ferritin enzyme sites bind two Fe2+ atoms in close proximity, which are released as a diferric oxo complex, after reacting with O2. Ferritin enzyme sites share properties with the other di-iron enzymes, including ribonucleotide reductase (DNA synthesis) and soluble methane monooxygenase (oxidation of methane to methanol); all form diferric peroxo intermediates, for example.20,27 However, the di-iron center in the other enzymes is stable (a protein-cofactor), whereas in
7.8. Distinctive Properties of Ferritin Protein cages
Structural specificity in ferritin protein cage pores, the external ion channel entry sites, is emphasized by the fact that only a very small number of peptides from a large, combinatorial peptide library could bind to native ferritin protein cages; a different peptide subset bound to ferritin with abnormally unfolded cage pores. One of the peptides that selectively bound to folded ferritin pores regulated both Fe2+ entry (enzyme activity) and Fe2+ exit after mineral reduction/dissolution.10 Physiological control of ferritin pore unfolding is regulated in vivo because a 788
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(transcription) of the encoded mRNA is inhibited, resulting is less biosynthesis of the encoded proteins such as ferritin or thioredoxin reductase; when heme binds to Bach 1, the DNA/Bach 1 complex dissociates, resulting in more encoded mRNA and, thus, more of the encoded protein. Because of the ARE-DNA/Bach 1 interaction, ferritin DNA transcription (mRNA biosynthesis) is sensitive to oxidants. Similarly, because of the IRE-RNA/IRP interaction, ferritin mRNA translation (protein biosynthesis) is sensitive to iron.38 The combination creates a regulatory feedback loop:38 ferritin substrates, O2 and Fe2+, stimulate ferritin DNA activity (transcription) and ferritin mRNA (translation), respectively, which increases ferritin protein concentrations. Ferritin protein makes ferric oxy biomineral, thereby consuming the genetic regulators, Fe2+ and O2, that increase ferritin DNA transcription and ferritin mRNA translation. The cumulative result of ferritin biomineral formation in extant ferritin molecules, is decreased ferritin protein biosynthesis.
range of iron distribution is observed among individual ferritin molecules from natural tissues; the average iron content of such ferritin preparations also varies from tissue to tissue.32 Ferritin is an unusually stable protein that survives, intact, at 80 °C or the presence of chaotropes such as 6 M urea,19,33 which inactivate most proteins. Reversible folding and unfolding of ferritin pores, which depends on highly conserved amino acids, changes the accessibility of ferritin minerals to external reductants. Experimentally, Fe2+, reduced and dissolved from ferritin iron biominerals, is trapped by a nonbiological chelator.31 It is likely that in vivo a specific intracellular protein and/or small molecule also binds and transports Fe2 reduced and dissolved from ferritin biominerals, to other intracellular sites; such biological carriers and mechanisms remain little studied.
8. REGULATION OF FERRITIN BIOSYNTHESIS (DNA AND mRNA) BY IRON AND OXYGEN Intracellular ferritin protein depends on environmental iron concentrations. Ferritin regulatory mechanisms in animals reflect local concentrations of both ferritin substrates, Fe and O2. O2 influences ferritin DNA activity;34 while iron, as Fe2+, controls ferritin protein biosynthesis.35 Fe2+, a substrate of the encoded ferritin protein, binds directly to ferritin mRNA, causing a ferritin mRNA conformational change that enhances ribosome binding and biosynthesis of the encoded protein.35 Currently, the control mechanism is novel in biology.
9. CONCLUSION Natural protein nanocages of ferritin concentrate metabolic iron as iron- oxy bionanominerals, minimizing free radicals from aqueous Fe2+/O2 chemistry. Understanding ferritin chemistry facilitates contemporary exploitation of evolutionary tweaking of ferritin for uses in nanochemistry, nanobiology, and nanomedicine. Ferritin biosynthesis, part of iron and oxygen regulatory networks, illustrates Nature’s crossroad at ferritin for Fe and O2 biochemistry. Ferritin proteins convert Fe and O2 to proteincoated, ferritin biominerals; ferritin DNA genes and mRNA use Fe and heme to regulate ferritin biosynthesis. The Fe-protoporphyrin IX complex (heme) activates ferritin DNA transcription (ferritin mRNA biosynthesis) by binding Bach 1, which increases ferritin mRNA biosynthesis (DNA transcription).34 Moreover, Fe2+ ion, activates ferritin mRNA translation (ferritin protein biosynthesis); when Fe2+ bind to an animal mRNA structure (the IRE riboregulator), IRP, an mRNA binding protein is released and ferritin biosynthesis increases.35,39 In each case, iron, either as the complex (Fe-protoporphyrin IX) or the ion (Fe2+), binds to a protein biosynthesis regulator: heme binds to ARE-DNA and Fe2+ binds to IRE-RNA. To date, such complementary (DNA and mRNA) genetic control of protein biosynthesis (ferritin) by the enzyme substrates, Fe2+ and O2, is unusual. Either substrate control of other enzyme biosyntheses is undiscovered, or distinctive properties of ferritin require such regulation, or both. The site(s) of Fe2+ binding to ferritin IRE-RNA is currently unknown.36 Why is ferritin needed? An example is the fate of iron in “wornout” red blood cells: in humans, they generate ∼0.54 mmol of iron/day. Without ferritin as an iron recycling site, we would need to drink ca. 1013 liters of water/day to prevent kidney “rusting”. How iron moves around within ferritin, in and out of the protein cage itself, to and from ferritin enzyme (Fe2+/O2 oxidoreductase) sites, into the mineral growth cavity17 are reasonably clear (see Figures 3 and 4). However, still incomplete are detailed mechanisms for coupling Fe2+/O2 (eukaryotic) or Fe2+/H2O2 (prokaryotic) in ferritin and microbial Dps proteins, identification of Fe2+ binding sites on IRE- mRNA, e−/ H+/H2O flow during dissolution of ferritin biomineral to Fe2+. Ferritin family members share (1) hollow protein nanocages, (2) internal, self-synthesized, ferric oxide minerals from Fe2+ and O, initiated at ferritin enzyme sites, and (3) biosynthesis
8.1. Ferritin DNA Transcription/mRNA Biosynthesis
Rates of ferritin gene transcription (ferritin mRNA biosynthesis) are controlled by a DNA sequence, called ARE (Antioxidant Response Element). ARE-DNA is also present in genes of proteins that repair oxidative damage such as thioredoxin reductase 1, heme oxygenase 1, and NADP(H) quinone (oxido) reductase 1. ARE-DNA binds a heme-sensitive, protein repressor, Bach 1. Thus, the “iron signal” for ferritin gene transcription is an iron complex, which contrasts with the iron signal for ferritin mRNA translation/protein biosynthesis, the Fe2+ ion itself.35 8.2. Ferritin mRNA Translation/Protein Biosynthesis
Ferritin mRNA translation, producing ferritin protein, is controlled by IRE (Iron Responsive/Regulatory Element)RNA, an mRNA loop that binds IRP, an inhibitor of protein biosynthesis (Figure 5). Fe2+ binds to IRE-RNA and prevents IRP binding. The result is increased ferritin mRNA activity and ferritin protein biosynthesis The IRE-RNA sequence, 17−19 bases, folds into a small, RNA A-helix; the IRE sequence occurs in mRNAs encoding a number of different proteins in iron metabolism. Six bases in the IRE-RNA terminal loop bases are highly conserved. Two of the bases, a C-G base pair, span the RNA loop, thereby exposing the intervening base triplet, AGU, to solvent. All five bases of the CAGUG loop are required for normal IRE-mRNA function and IRP (Iron Regulatory Protein) binding.36 Fe2+/IRE-RNA binding is the signal for ferritin mRNA translation (Figure 5). The IRE-RNA/IRP complex (Figure 5) prevents ribosome/ mRNA binding. Fe2+ binding to IRE-RNA releases IRP protein, allowing ribosome binding and protein biosynthesis.35,37 Effects of Fe2+ on mRNA translation (iron induction of protein biosynthesis)37 reflect Fe2+ binding to mRNA35 (Figure 5). An indirect effect of iron on ferritin protein biosynthesis is through the reversible binding of heme (iron-protoporphyrin IX complex) to the Bach 1 protein. When Bach 1 binds ferritin DNA (or the DNA of other antioxidant genes), the synthesis 789
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