"Opening" the ferritin pore for iron release by mutation of conserved amino acids at interhelix and loop sites

Ferritin concentrates, stores, and detoxifies iron in most organisms. The i ron is a solid, ferric oxide mineral (less than or equal to 4500 Fe) inside the protein shell. Eight pores are formed by subunit trimers of the 24 sub unit protein. A role for the protein in controlling reduction and dissoluti on of the iron mineral was suggested in preliminary experiments [Takagi et al. (1998) J. Biol. Chem. 273, 18685-18688] with a proline/leucine substitu tion near the pore. Localized pore disorder in frog L134P crystals coincide d with enhanced iron exit, triggered by reduction. In this report, nine add itional substitutions of conserved amino acids near L134 were studied for e ffects on iron release. Alterations of a conserved hydrophobic pair, a cons erved ion pair, and a loop at the ferritin pores all increased iron exit (3 -30-fold). Protein assembly was unchanged, except for a slight decrease in volume (measured by gel filtration); ferroxidase activity was still in the millisecond range, but a small decrease indicates slight alteration of the channel from the pore to the oxidation site. The sensitivity of reductive i ron exit rates to changes in conserved residues near the ferritin pores, as sociated with localized unfolding, suggests that the structure around the f erritin pores is a target for regulated protein unfolding and iron release.