Ferritins, highly symmetrical protein nanocages, are reactors for Fe2+ and dioxygen or hydrogen peroxide that are found in all kingdoms of life and in many different cells of multicellular organisms. They synthesize iron concentrates required for cells to make cofactors of iron proteins (heme, FeS, mono and diiron). The caged ferritin biominerals, Fe2O3•H2O are also antioxidants, acting as sinks for iron and oxidants scavenged from damaged proteins; genetic regulation of ferritin biosynthesis is sensitive to both iron and oxidants. Here, the emphasis here is ferritin oxidoreductase chemistry, ferritin ion channels for Fe 2+ transit into and out of the protein cage and Fe 3+ O mineral nucleation, and uses of ferritin cages in nanocatalysis and nanomaterial synthesis. The Fe2+ and O ferritin protein reactors, likely critical in the transition from anaerobic to aerobic life on earth, play central, contemporary roles that balance iron and oxygen chemistry in biology and have emerging roles in nanotechnology.

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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 Fe(2+) with O2 at ferritin enzyme (Fe(2+)/O oxidoreductase) sites. The product of ferritin enzyme activity, diferric oxy complexes, is also the precursor of ferritin biomineral. Concentrations of Fe(3+) equivalent to 2.0 × 10(-1) M are maintained in ferritin solutions, contrasting with the Fe(3+) 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 Fe(2+) entry/exit, (2) enzyme (oxidoreductase) site for coupling Fe(2+) 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 Fe(2+) entry (enzyme catalysis of Fe(2+)/O2 oxidoreduction) and Fe(2+) exit (reductive ferritin mineral dissolution); 3-fold symmetry axes influence Fe(2+)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 Fe(2+) 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 Fe(2+) binding to ferritin mRNA decreases binding of an inhibitory protein, IRP, causing increased ferritin mRNA translation (protein biosynthesis). Newly synthesized ferritin protein consumes Fe(2+) in biomineral, decreasing Fe(2)(+) and creating a regulatory feedback loo...

Mycobacterium tuberculosis (Mtb) expresses heme binding protein nanocages, bacterioferritin A (BfrA), along with nonheme bacterioferritin B (BfrB). BfrA is unique to bacteria and, like BfrB, carries out ferroxidase activity to synthesize iron oxide biominerals. The expression of BfrA, in the presence of BfrB, indicates that Mtb may utilize it for some additional purpose apart from its natural iron storage activity. However, the mechanism of ferroxidase activity (iron biomineralization) in Mtb BfrA still remains unexplored. H2O2 is secreted by the host during host–pathogen interaction. In some bacteria, heme containing Bfr and/or Dps (DNA binding protein during starvation) detoxify H2O2 by utilizing it during their ferroxidase activity. Interestingly, Mtb lacks the gene for Dps which protects DNA from H2O2-induced oxidative cleavage. Therefore, the current work investigates the kinetics of O2/H2O2-dependent ferroxidase activity, DNA protection, and catalase-like activity of recombinant Mtb BfrA. Ferroxidase activity by Mtb BfrA was found to proceed via the formation of a transient intermediate and its initial rate exhibited sigmoidal behavior, with increasing Fe2+ concentration. Moreover, Mtb BfrA exhibited catalase-like activity by evolving O2 upon reaction with H2O2, which gets inhibited in the presence of catalase inhibitors (NaN3 and NaCN). In addition, Mtb BfrA protected plasmid DNA from Fenton reagents (Fe2+ and H2O2), similar to Dps, by forming BfrA-DNA complexes. Thereby, Mtb BfrA executes multiple functions (ferroxidase, catalase, and Dps-like activities) in order to cope with the host generated oxidative stress and to promote pathogenesis.

Background: Ferritins, cytoplasmic protein nanocages, with internal and cytoplasmic pores terminating trans-cage ion channels, reversibly concentrate iron and scavenge oxidants. Results: Changing ferritin conserved channel residues altered Fe 2ϩ exit, channel flexibility, protein-crowding sensitivity, ion binding, and N-terminal folding. Conclusion: Eukaryotic ferritin N termini form cytoplasmic gates stabilized by hydrogen bonds and ionic bonds. Significance: Shared structure and function of ferritin with membrane ion channels includes cytoplasmic, N-terminal gates.

Ferritins, a complex, mineralized, protein nanocage family essential for life, provide iron concentrates and oxidant protection. Protein based ion channels and Fe(II)/O2 catalysis initiate conversion of thousands of Fe atoms to caged, ferritin Fe2O3•H2O minerals. The ion channels consist of six helical segments, contributed by 3 of 12 or 24 polypeptide subunits, around the three-fold cage axes. The channel structure guides entering Fe(II) ions toward multiple, catalytic, diiron sites buried inside ferritin protein helices, ~20 Å away from channel internal exits. The catalytic product, Fe(III)-O(H)-Fe(III) is a mineral precursor; mineral nucleation begins inside the protein cage with mineral growth in the central protein cavity (5–8 nm diameter). Amino acid substitutions that changed ionic or hydrophobic channel interactions R72D, D122R, and L134P, increased ion channel structural disorder (protein crystallographic analyses), increased Fe(II) exit [chelated Fe(II) after ferric mineral reduction/dissolution]. Since substitutions of some channel carboxylate residues diminished ferritin catalysis with no effect on Fe(II) exit, such as E130A and D127A, we investigated catalysis in ferritins with altered Fe(II) exit, R72D, D122R and L134P. The results indicate that simply changing the ionic properties of the channels, as in the R72D variant, need not change the forward catalytic rate. However both D122R and L134, which had dramatic effects on ferritin catalysis also caused larger effects on channel structure and order, contrasting with R72D. All three amino acid substitutions, however, decreased the stability of the catalytic intermediate, diferric peroxo, even though overall ferritin cage structure is very stable, resisting 80°C and 6 M urea. The localized structural changes in ferritin subdomains that affect ferritin function over long distances, illustrate new properties of the protein cage in natural ferritin function and for applied ferritin uses.

Ferritins are supramolecular nanocage proteins, which synthesize hydrated ferric oxyhydroxide mineral via protein mediated rapid Fe 2+ sequestration and ferroxidase reactions. Ferritin minerals are also associated with a significant amount of phosphate, which contribute toward their structure and reactivity. Like iron, phosphate also regulates the pathogenesis of Mycobacterium tuberculosis (Mtb), which expresses two types of ferritin: heme binding bacterioferritin A (BfrA) and nonheme binding bacterioferritin B (BfrB). Unlike Mtb BfrA, the rapid kinetics and mechanism of ferroxidase activity, and the mineral core formation/dissolution in Mtb BfrB are not well explored. Moreover, the effect of physiological levels of phosphate (0−10 mM) on the synthesis, structure, and reactivity of ferritin mineral core also require investigation in detail. Therefore, the stopped-flow rapid kinetics of ferroxidase activity (ΔA 650 /Δt) of Mtb BfrB was carried out, which detected a transient intermediate similar to diferric peroxo species as observed in frog and human ferritins. Increasing phosphate concentration increased the initial rate of iron mineralization (ΔA 350 /Δt) and dissolved O 2 consumption (both ∼1.5−2-fold).Phosphate not only decreased the amount of iron loading and size of the BfrB mineral core (both up to 2-fold) but also decreased its crystallinity, resembling the variations observed in the core morphology of different native ferritins. In addition, phosphate inhibited the kinetics of reductive iron mobilization (∼6−8-fold) indicating its influence on the stability of the iron mineral core. Hence, the current work provides the kinetic/mechanistic insight toward the ferroxidase activity in Mtb BfrB, apart from demonstrating the role of phosphate toward the structure/reactivity of its iron mineral.

Ferritins, the cellular iron repositories, are self-assembled, hollow spherical nanocage proteins composed of 24 subunits. The self-assembly process in ferritin generates the electrostatic gradient to rapidly sequester Fe­(II) ions, thereby minimizing its toxicity (Fenton reaction). Although the factors that drive self-assembly and control its kinetics are little investigated, its inherent reversibility has been utilized for cellular imaging and targeted drug delivery. The current work tracks the kinetics of ferritin self-assembly by laser light scattering and investigates the factors that influence the process. The formation of partially structured subunit-monomers/dimers, at pH ≤ 1.5, serves as the starting material for the self-assembly, which upon increasing the pH exhibits biphasic behavior (a rapid assembly process coupled with subunit folding followed by a slower reassembly/reorganization process) and completes within 10 min. The ferritin self-assembly accelerated with subunit concentration and ionic strength (t 1/2 decreases in both the cases) but slowed down with the pH of the medium from 5.5 to 7.5 (t 1/2 increases). These findings would help to regulate the ferritin self-assembly to enhance the loading/unloading of drugs/nanomaterials for exploiting it as a nanocarrier and nanoreactor.