Ferritin Iron Mineralisation: Route of Fe ³⁺ Transfer From the Ferroxidase Centre to the Inner Cavity of Human H‐Chain Ferritin

ABSTRACT Ferritin‐catalysed Fe 2+ oxidation by reaction with O 2 occurs at an intra‐subunit diiron site known as the ferroxidase centre (FoC). Currently, how Fe 3+ , the key substrate for iron core nucleation/mineralisation, transfers from the FoC to the inner protein surface/central cavity where the mineral is laid down is unknown. Iron‐binding sites that become occupied following exposure of anaerobic, Fe 2+ ‐bound human cytosolic H‐chain ferritin (HuHF) to O 2 were identified by time‐resolved x‐ray crystallography. In addition to the two FoC iron sites, three further sites were identified, each involving Glu61 as a coordinating residue. Substitution by a non‐coordinating residue (variant E61A) eliminated binding at these additional iron sites. Solution kinetic studies of Fe 2+ oxidation and iron core mineralisation in wild‐type HuHF and its E61A variant showed that rapid Fe 2+ oxidation was unaffected by loss of Glu61, ruling out an important role for these sites in either guiding Fe 2+ to the FoC, or in the mechanism of FoC‐catalysed Fe 2+ oxidation. Conversely, the transfer of Fe 3+ out of the FoC and core mineralisation were both severely affected in the E61A variant. A mechanism for Fe 3+ transfer from the FoC to the inner protein surface is proposed.