Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a nonheme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly antiferromagnetically coupled, consistent with a mu-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure--including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin--coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites.

Synopsis and pictogram: Gated pores in the ferritin family of protein nanocages, illustrated in the pictogram, control transfer of ferrous iron into and out of the cages by regulating contact between hydrated ferric oxide mineral inside the protein cage, and reductants such as FMNH 2 on the outside. The structural and functional homology between the gated ion channel proteins in inaccessible membranes and gated ferritin pores in the stable, water soluble nanoprotein, make studies of ferritin pores models for gated pores in many ion channel proteins.Properties of ferritin gated pores, which control rates of FMNH 2 reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, are discussed in terms of the conserved pore gate residues (arginine 72-apspartate 122 and leucine 110-leucine 134), of pore sensitivity to heat at temperatures 30 °C below that of the nanocage itself, and of pore sensitivity to physiological changes in urea (1-10 mM). Conditions which alter ferritin pore structure/function in solution, coupled with the high evolutionary conservation of the pore gates, suggest the presence of molecular regulators in vivo that recognize the pore gates and hold them either closed or open, depending on biological iron need. The apparent homology between ferrous ion transport through gated pores in the ferritin nanocage and ion transport through gated pores in ion channel proteins embedded in cell membranes, make studies of water soluble ferritin and the pore gating folding/unfolding a useful model for other gated pores.

Pores regulate access between ferric-oxy biomineral inside and reductants/chelators outside the ferritin protein nanocage to control iron demineralization rates. The pore helix/loop/ helix motifs that are contributed by three subunits unfold independently of the protein cage, as observed by crystallography, Fe removal rates, and CD spectroscopy. Pore unfolding is induced in wild type ferritin by increased temperature or urea (1-10 mM), a physiological urea range, 0.1 mM guanidine, or mutation of conserved pore amino acids. A peptide selected for ferritin pore binding from a combinatorial, heptapeptide library increased the rate of Fe demineralization 3-fold (p < 0.001), similarly to a mutation that unfolded the pores. Conjugating the peptide to Desferal (desferrioxamine B mesylate), a chelator in therapeutic use, increased the rates to 8-fold (p < 0.001). A second pore binding peptide had the opposite effect and decreased the rate of Fe demineralization 60% (p < 0.001). The peptides could have pharmacological uses and may model regulators of ferritin demineralization rates in vivo or peptide regulators of gated pores in membranes. The results emphasize that small peptides can exploit the structural plasticity of protein pores to modulate function.Pores in ferritin protein cages are an example of pores in molecular or ionic barriers; other examples are pores in membranes. In ferritin, pores control reactions between reductants outside the protein and the ferric mineral inside. As with many gated pores, ferritin pores are formed by ␣-helices in multiple subunits that surround the ion path and are modulated by unfolding (1, 2) (see Fig. 1). Ferritin pores are arranged symmetrically around the protein cage, eight for 24 subunit maxi-ferritins and four for 12 subunit maxi-ferritins, also called Dps proteins (2, 3).Ferritins concentrate Fe for biological use and also detoxify Fe/O 2 or H 2 O 2 in the ferric/oxy biomineral inside the proteins of Archaea, bacteria, and Eukaryota, including higher plants and animals. Fe ions destined to enter the mineral appear to reach the ferroxidase coupling site through the pores for the first step in biomineralization (2, 4 -7). The critical roles of ferritins are illustrated by lethality of deletions in mice, neurological effects of mutations in humans, and pathogen responses to host-released oxidants (3,8,9). In addition, dual genetic regulatory systems with DNA (antioxidant-response elements) enhancers linking ferritin regulation to antioxidant response proteins (10, 11) and mRNA "promoters" (iron-response element) linking ferritin regulation to Fe trafficking proteins emphasize the central role of ferritin in Fe and oxygen metabolism. Finally, the ferritin Fe reactions with O 2 or H 2 O 2 (2, 4 -7) and the presence of ferritin in anaerobic archaea (12) suggest an ancient role for ferritins in the transition to aerobic metabolism.The rates of Fe transport from the ferritin mineral through the nanocage channels and pores to chelators on the outside of the nanocage are initiated by...

DNA Protection during Starvation (Dps) proteins are mini-ferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O 2 /H 2 O 2 , which accumulates in the large central cavity; for Dps, H 2 O 2 , is the more common Fe(II) oxidant contrasting with eukaryotic maxi-ferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two B. anthracis mini-ferritins; one in which two Fe(II) react with O 2 exclusively (Dps1) and a second in which both O 2 or H 2 O 2 can react with two Fe(II) (Dps2). Both result in the formation of iron oxy-biomineral. The data show: a single 5 or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30 × more stable than Dps1; and the lower limit of K d for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O 2 . The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.DNA Protection during Starvation (Dps) proteins, also known as mini-ferritins, are 12-monomer spherical proteins capable of storing iron mineral and thus are part of the ferritin super-family (1-4). Unlike maxi (24-monomer) ferritins, Dps proteins have been shown to bind and protect DNA from oxidation (2-4). The paired Dps proteins in Bacillus anthracis * To whom correspondence should be addressed: Elizabeth C. Theil etheil@chori.org; and Edward I. Solomon, edward.solomon@stanford.edu;. Supporting Information Available CD spectra showing effect of temperature on Dps1 transitions; MCD spectra of Dps1 overlaid with Fe(II) control spectrum, and MCD spectrum with control subtracted; Apparent initial rates of Fe 2+ oxidation in Dps1 & 2 scaled to overlay with the calculated concentration of binuclear Fe(II) active sites occupied at a given Fe 2+ /Dps2 ratio using fixed K D1 and varied K D2 values. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 December 14. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (Dps1 and Dps2), which share ~60% sequence homology(3), confer greatest protection when both are present. Dps proteins in pathogens, such as those from B. anthracis, are of particular interest as better knowledge of how these proteins p...

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