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Ferritin is an iron storage protein made of 24 subunits. Previous mutational analyses showed that ferritin C-terminal region has a major role in protein stability and assembly but is only marginally involved in the mechanism of iron incorporation. However, it has recently been shown that patients who carry alterations of ferritin C-terminal sequence caused by nucleotide insertions show neurological disorders possibly related to altered protein functionality and cellular iron deregulation. To re-evaluate the role of this region, five mutants of mouse H-ferritin were produced by 2-nucleotide insertions that modified the last 6–29 residues and extended the sequence of 14 amino acids. The mutants were expressed in Escherichia coli and analysed for solubility, stability and capacity to incorporate iron. The alteration of the last 6-residue non-helical extension had no evident effect on the properties of ferritin, while solubility and capacity to assemble in ferritin shells decreased progressively with the extension of the modified region. The results also showed that the modification of even a part of the terminal E-helix abolished the capacity of ferritin to incorporate iron during expression in the cells, probably caused by conformational modification of the hydrophobic channels. The data support the hypothesis that the pathogenic mutations alter cellular iron homeostasis.