mechanism of ferritin biomineral synthesis is ferric hydrolysis. Apparently, ferritin
protein cages contribute to a much larger fraction of the mineralization process than
previously thought, but the fraction of ferritin biominerals laid down without
passage nucleation in the protein cage remains unknown.
2.5 Ferritin Iron Biomineral Dissolution
Recovering iron from the ferritin mineral core is crucial for the biosynthesis of heme
and iron-sulfur cluster-containing proteins that are important for all forms of life.
However, the exit of iron from the ferritin cage, which requires the delivery of
electrons and protons to dissolve the mineral, is much less studied than entry/
oxidation/mineralization. The reduction and subsequent release of iron from the
ferritin cavity can be measured in an assay as first described by Jones et al. (1978),
and has been used to study maxi-ferritins and mini-ferritins from plants, animals,
bacteria, and cyanobacteria (Bellapadrona et al. 2009; Takagi et al. 1998; Richards
et al. 1996; Castruita et al. 2007). Usually, NADH/FMN, or sodium dithionite is the
source of electrons, added to mineralized ferritin protein in neutral buffer
(Bellapadrona et al. 2009), with a chelator such as bipyridyl, as the reporter for
iron exit the pink Fe(II)-chelate (A 522 nm); other chelators such as desferrioxamine
B (DFO) are also used. Curves for the formation of the Fe–chelator complexes are
multiphasic and complex due to multiple sequential and simultaneous reactions; iron
reduction, hydration of ferric oxo bridges in the mineral, transport of iron and
reductants through the protein cage in the case of Fe(III) chelators, the competition
between binding to the chelator or to the catalytic sites of the protein (Liu et al. 2007).
In both mini- and maxi-ferritins, iron leaves the mineral cage via pore structures
in the protein that connect the inner cavity with the outside of the protein (Liu and
Theil 2005; Bellapadrona et al. 2009). These funnel-shaped, hydrophilic pores are
localized at the threefold symmetry axes by junctions of three subunits (Takagi et al.
1998). There are eight of these hydrophilic pores in maxi-ferritin and four in miniferritin. Experiments with the H-frog maxi-ferritin, using mutagenesis, protein
crystallography, helix content (CD spectroscopy) have shown that the helix content
around ferritin pores is sensitive to slight changes in temperature, ionic strength, or
by the presence of low concentrations (1–10 mM) of chaotropes like urea or
guanidine (Liu et al. 2003). Partial unfolding of these pore structures was observed
simultaneously with an increase in rate of iron reduction and iron release from the
proteins inner core illustrating the regulatory role of ferritin cage pores in controlling
mineral/reductant access. Similar effects of threefold pore mutations were observed
Listeria innocua mini-ferritin where replacement of conserved, negatively charged
residues with neutral amino acids changed the initial rate of iron release from the
mineral core. Even though the amino acid sequences of mini-ferritin and maxiferritin subunits vary considerably (up to 80%), quaternary structures have pores, at
the junctions of matched helix turns in three subunits, which control reductant/
chelator access/Fe (II) exit during demineralization of both maxi-ferritins and mini42
L.E. Bevers and E.C. Theil
protein cages contribute to a much larger fraction of the mineralization process than
previously thought, but the fraction of ferritin biominerals laid down without
passage nucleation in the protein cage remains unknown.
2.5 Ferritin Iron Biomineral Dissolution
Recovering iron from the ferritin mineral core is crucial for the biosynthesis of heme
and iron-sulfur cluster-containing proteins that are important for all forms of life.
However, the exit of iron from the ferritin cage, which requires the delivery of
electrons and protons to dissolve the mineral, is much less studied than entry/
oxidation/mineralization. The reduction and subsequent release of iron from the
ferritin cavity can be measured in an assay as first described by Jones et al. (1978),
and has been used to study maxi-ferritins and mini-ferritins from plants, animals,
bacteria, and cyanobacteria (Bellapadrona et al. 2009; Takagi et al. 1998; Richards
et al. 1996; Castruita et al. 2007). Usually, NADH/FMN, or sodium dithionite is the
source of electrons, added to mineralized ferritin protein in neutral buffer
(Bellapadrona et al. 2009), with a chelator such as bipyridyl, as the reporter for
iron exit the pink Fe(II)-chelate (A 522 nm); other chelators such as desferrioxamine
B (DFO) are also used. Curves for the formation of the Fe–chelator complexes are
multiphasic and complex due to multiple sequential and simultaneous reactions; iron
reduction, hydration of ferric oxo bridges in the mineral, transport of iron and
reductants through the protein cage in the case of Fe(III) chelators, the competition
between binding to the chelator or to the catalytic sites of the protein (Liu et al. 2007).
In both mini- and maxi-ferritins, iron leaves the mineral cage via pore structures
in the protein that connect the inner cavity with the outside of the protein (Liu and
Theil 2005; Bellapadrona et al. 2009). These funnel-shaped, hydrophilic pores are
localized at the threefold symmetry axes by junctions of three subunits (Takagi et al.
1998). There are eight of these hydrophilic pores in maxi-ferritin and four in miniferritin. Experiments with the H-frog maxi-ferritin, using mutagenesis, protein
crystallography, helix content (CD spectroscopy) have shown that the helix content
around ferritin pores is sensitive to slight changes in temperature, ionic strength, or
by the presence of low concentrations (1–10 mM) of chaotropes like urea or
guanidine (Liu et al. 2003). Partial unfolding of these pore structures was observed
simultaneously with an increase in rate of iron reduction and iron release from the
proteins inner core illustrating the regulatory role of ferritin cage pores in controlling
mineral/reductant access. Similar effects of threefold pore mutations were observed
Listeria innocua mini-ferritin where replacement of conserved, negatively charged
residues with neutral amino acids changed the initial rate of iron release from the
mineral core. Even though the amino acid sequences of mini-ferritin and maxiferritin subunits vary considerably (up to 80%), quaternary structures have pores, at
the junctions of matched helix turns in three subunits, which control reductant/
chelator access/Fe (II) exit during demineralization of both maxi-ferritins and mini42
L.E. Bevers and E.C. Theil