A third metal site near the diiron catalytic centers has been observed in bacterial
maxi-ferritins co-crystallized with various metal ions, and name site C or Fe 3
(Stillman et al. 2003; Crow et al. 2009); soaking in iron of co-crystallization with
ferrous anaerobically indicates Fe 1, Fe 2, and Fe 3 (Crow et al. 2009). To date, no
specific kinetic intermediates have been characterized in prokaryotic maxi-ferritins,
contrasting with the well-described differic peroxo intermediate in animal ferritins
(Pereira et al. 1998; Moe ¨nne-Loccoz et al. 1999; Bou-Abdallah et al. 2002); although
changes in heme absorption have been observed during ferrous oxidation of hemecontaining BFR (Le Brun et al. 1993). Most likely, Fe 3 represents an alternate ironbinding site related to ferrous ions traveling through the protein cage to the catalytic
sites, especially since mutation of the ligands slows down oxidation (Treffry et al.
1998). Alternated conformations of ferritin protein cage side chains have been
observed in high-resolution structures of ferritin, dependent on metals present during
crystallization (e.g., Trikha et al. 1995; Toussaint et al. 2007) indicate the flexibility
of the ligands at and near the active sites needed to move iron through the cage.
The ferritin catalytic sites are diiron sites related to the diiron cofactors sites in
dioxygenases by simple, DNA codon differences in two amino acids of the Fe 2 site
(Liu and Theil 2005). However, in eukaryotic ferritins, ferrous iron is substrates
rather than cofactors, as in the oxygenases. Iron leaves the active sites, after
catalytic coupling to dioxygen, as differic oxo products that are mineral precursors
(Liu and Theil 2005). In prokaryotes, where primary structure varies as much as
80%, the ferritin protein cages are studded with catalytic sequences within each
subunit or at the cavity surface between subunits that bind two iron atoms as
cofactors, or are mono or diiron substrates sites (Chiancone and Ceci 2010;
Le Brun et al. 2010). Since many of the amino acids at the ferritin catalytic sites
are highly conserved, in both eukaryotes and prokaryotes (Table 2.3), the variable
amino acids at and around the active sites provide significant tissue and species
selectivity (e.g., (Tosha et al. 2008).
At the ferritin catalytic sites, the ligating residues for Fe 1(A) are highly
conserved in prokaryotes and eukaryotes (91.3–100%). By contrast, Fe 2 (B)
ligands vary considerably (46.2–93.3%) (Table 2.4). In general, iron site 2 has
two, nonvariant residues and several variable residues that have specific kinetic
effects and are genetically regulated so the abundance differ (Tosha et al. 2008).
There appears to be some type of cooperativity among the catalytic sites of multiple
ferritin subunits. Since each ferrous ion binds independently at the diiron sites in
each ferritin subunit, and the other substrate, dioxygen, is absent, the coooperativity
is likely protein: protein (Schwartz et al. 2008).
The diiron sites in maxi-ferritins of both prokaryotes and eukaryotes, Fe 1(A)
and Fe 2 (B), are highly conserved (all above 90.9%). Conservation of site Fe 2 of
mini-ferritins (46.2%) is low, unless the second most abundant site Fe2 motif is
included a consensus sequence; thus mutation of aspartate to the glutamate, in the
E. coli template, increases the conservation to 92.7%. A similar approach for
bacterioferritin increases for Fe 2 ligands from 76.3% to 88.5%. However, in
eukaryotic ferritins of organisms with multiple H subunit (catalytically active)
genes, Fe 2 site differences as small as changing alanine to serine occur, and in
36
L.E. Bevers and E.C. Theil
maxi-ferritins co-crystallized with various metal ions, and name site C or Fe 3
(Stillman et al. 2003; Crow et al. 2009); soaking in iron of co-crystallization with
ferrous anaerobically indicates Fe 1, Fe 2, and Fe 3 (Crow et al. 2009). To date, no
specific kinetic intermediates have been characterized in prokaryotic maxi-ferritins,
contrasting with the well-described differic peroxo intermediate in animal ferritins
(Pereira et al. 1998; Moe ¨nne-Loccoz et al. 1999; Bou-Abdallah et al. 2002); although
changes in heme absorption have been observed during ferrous oxidation of hemecontaining BFR (Le Brun et al. 1993). Most likely, Fe 3 represents an alternate ironbinding site related to ferrous ions traveling through the protein cage to the catalytic
sites, especially since mutation of the ligands slows down oxidation (Treffry et al.
1998). Alternated conformations of ferritin protein cage side chains have been
observed in high-resolution structures of ferritin, dependent on metals present during
crystallization (e.g., Trikha et al. 1995; Toussaint et al. 2007) indicate the flexibility
of the ligands at and near the active sites needed to move iron through the cage.
The ferritin catalytic sites are diiron sites related to the diiron cofactors sites in
dioxygenases by simple, DNA codon differences in two amino acids of the Fe 2 site
(Liu and Theil 2005). However, in eukaryotic ferritins, ferrous iron is substrates
rather than cofactors, as in the oxygenases. Iron leaves the active sites, after
catalytic coupling to dioxygen, as differic oxo products that are mineral precursors
(Liu and Theil 2005). In prokaryotes, where primary structure varies as much as
80%, the ferritin protein cages are studded with catalytic sequences within each
subunit or at the cavity surface between subunits that bind two iron atoms as
cofactors, or are mono or diiron substrates sites (Chiancone and Ceci 2010;
Le Brun et al. 2010). Since many of the amino acids at the ferritin catalytic sites
are highly conserved, in both eukaryotes and prokaryotes (Table 2.3), the variable
amino acids at and around the active sites provide significant tissue and species
selectivity (e.g., (Tosha et al. 2008).
At the ferritin catalytic sites, the ligating residues for Fe 1(A) are highly
conserved in prokaryotes and eukaryotes (91.3–100%). By contrast, Fe 2 (B)
ligands vary considerably (46.2–93.3%) (Table 2.4). In general, iron site 2 has
two, nonvariant residues and several variable residues that have specific kinetic
effects and are genetically regulated so the abundance differ (Tosha et al. 2008).
There appears to be some type of cooperativity among the catalytic sites of multiple
ferritin subunits. Since each ferrous ion binds independently at the diiron sites in
each ferritin subunit, and the other substrate, dioxygen, is absent, the coooperativity
is likely protein: protein (Schwartz et al. 2008).
The diiron sites in maxi-ferritins of both prokaryotes and eukaryotes, Fe 1(A)
and Fe 2 (B), are highly conserved (all above 90.9%). Conservation of site Fe 2 of
mini-ferritins (46.2%) is low, unless the second most abundant site Fe2 motif is
included a consensus sequence; thus mutation of aspartate to the glutamate, in the
E. coli template, increases the conservation to 92.7%. A similar approach for
bacterioferritin increases for Fe 2 ligands from 76.3% to 88.5%. However, in
eukaryotic ferritins of organisms with multiple H subunit (catalytically active)
genes, Fe 2 site differences as small as changing alanine to serine occur, and in
36
L.E. Bevers and E.C. Theil