Literature summary for 1.16.3.1 extracted from

Bernacchioni, C.; Pozzi, C.; Di Pisa, F.; Mangani, S.; Turano, P.
Ferroxidase activity in eukaryotic ferritin is controlled by accessory-iron-binding sites in the catalytic cavity (2016), Chemistry, 22, 16213-16219 .

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant iron-free E57A/E136A/D140A ferritin mutant variant, hanging drop vapor diffusion technique, mixing of 0.002 ml of 7 mg/ml protein in 20 mm Tris, pH 7.5, with 0.002 ml of reservoir solution composed of 1.6-2.0m MgCl2 and 0.1m bicine, pH 8.0, and equilibration against 0.6 ml reservoir solution, 8°C, 36 days to 10 days, for Fe2+-bound enzyme mutant, 0.1m bis-tris propane buffer at pH 6.5 is used for precipitation, X-ray diffraction structure determination and analysis at 1.50 A resolution, modeling	Lithobates catesbeianus

Crystallization (Comment)

Organism

purified recombinant iron-free E57A/E136A/D140A ferritin mutant variant, hanging drop vapor diffusion technique, mixing of 0.002 ml of 7 mg/ml protein in 20 mm Tris, pH 7.5, with 0.002 ml of reservoir solution composed of 1.6-2.0m MgCl2 and 0.1m bicine, pH 8.0, and equilibration against 0.6 ml reservoir solution, 8°C, 36 days to 10 days, for Fe2+-bound enzyme mutant, 0.1m bis-tris propane buffer at pH 6.5 is used for precipitation, X-ray diffraction structure determination and analysis at 1.50 A resolution, modeling

Lithobates catesbeianus

Protein Variants

Protein Variants	Comment	Organism
E130A	site-directed mutagenesis, inactive mutant	Lithobates catesbeianus
E136A	site-directed mutagenesis, the mutant enzyme activity is reduced compared to wild-type due to a reduced ability of the variant nanocages to populate the ferroxidase sites Fe1 and Fe2, reduced catalytic activity compared to wild-type	Lithobates catesbeianus
E57A	site-directed mutagenesis, the mutant enzyme activity is reduced compared to wild-type due to a reduced ability of the variant nanocages to populate the ferroxidase sites Fe1 and Fe2, reduced catalytic activity compared to wild-type	Lithobates catesbeianus
E57A/E136A	site-directed mutagenesis, the mutant enzyme activity is reduced compared to wild-type due to a reduced ability of the variant nanocages to populate the ferroxidase sites Fe1 and Fe2, reduced catalytic activity compared to wild-type	Lithobates catesbeianus
E57A/E136A/D140A	site-directed mutagenesis, inactive mutant, structure comparison to the wild-type enzyme. In the triple variant, only one Mg2+ ion is bound at the Fe1 site, and the ability of the variant cage to process Fe2+ ions is altered. The mutant shows reduced biomineralization efficiency	Lithobates catesbeianus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	stopped-flow kinetics and single-turnover oxidoreductase kinetics, measuring diferric-peroxo intermediate and Fe3+	Lithobates catesbeianus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Cu2+	required	Lithobates catesbeianus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
480000	-	about, gel filtration, wild-type enzyme and mutant E57A/E136A/D140A	Lithobates catesbeianus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
4 Fe(II) + 4 H+ + O2	Lithobates catesbeianus	-	4 Fe(III) + 2 H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Lithobates catesbeianus	-	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4 Fe(II) + 4 H+ + O2	-	Lithobates catesbeianus	4 Fe(III) + 2 H2O	-	?
4 Fe(II) + 4 H+ + O2	two Fe2+ ions occupy sites Fe1 and Fe2 in the ferroxidase cavity, structure overview	Lithobates catesbeianus	4 Fe(III) + 2 H2O	-	?
additional information	formation of the diferric-peroxo (DFP) intermediate and of the ferric-oxo products of the ferroxidase reactions	Lithobates catesbeianus	?	-	?

Synonyms

Synonyms	Comment	Organism
H' ferritin	-	Lithobates catesbeianus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	9	assay at	Lithobates catesbeianus

General Information

General Information	Comment	Organism
additional information	residue Asp140 and previously identified residues Glu57 andGlu136 are essential residues to promote the iron oxidation at the ferroxidase site, but the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe2+ substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild-type cages	Lithobates catesbeianus
physiological function	ferritins are iron-storage nanocage proteins that catalyze the oxidation of Fe2+ to Fe3+ at ferroxidase sites. Ferroxidase activity in eukaryotic ferritin is controlled by accessory-iron-binding sites in the catalytic cavity, a ferroxidase-active cage, overview	Lithobates catesbeianus