Ferrous ion binding to recombinant human H-chain ferritin. An isothermal titration calorimetry study

Iron deposition within the iron storage protein ferritin involves a complex series of events consisting of Fe2+ binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrous ferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamic properties of Fe2+ binding to recombinant human H-chain apoferritin (HuHF) by isothermal titration calorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the protein and the principal pathways by which the Fe2+ travels to the dinuclear ferroxidase center prior to its oxidation to Fe3+. Calorimetric titrations show that the ferroxidase center is the principal locus for Fe2+ binding with weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely the His65 containing A-site, preferentially binds Fe2+. That only one site of the ferroxidase center is occupied by Fe2+ implies that Fe2+ oxidation to form diFe(III) species might occur in a stepwise fashion. In dilute anaerobic protein solution (3-5 muM), only 12 Fe2+/protein bind at pH 6.51 increasing to 24 Fe2+/protein at pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding of Fe2+ to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe2+ binding. The total Fe2+ binding capacity of the protein is reduced in the 3-fold hydrophilic channel variant S14 (D1311+E134F), indicating that the primary avenue by which Fe2+ gains access to the interior of ferritin is through these eight channels. The binding, stoichiometry of the channel variant is one-third that of the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe2+ binding are similar for the two with an average values (DeltaHdegrees = 7.82 kJ/mol, binding constant K = 1.48 x 10(5) M-1 at pH 7.04). Since channel mutations do not completely prevent Fe2+ binding to the ferroxidase center, iron gains access to the center in approximately one-third of the channel variant molecules by other pathways.