Ferritin Iron Mineralisation: Route of Fe³⁺ Transfer from the Ferroxidase Centre to the Inner Cavity of Human H-chain ferritin

Ferritin-catalysed Fe2+ oxidation by reaction with O2 occurs at an intra-subunit diiron site known as the ferroxidase centre (FoC). Currently, how Fe3+, 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, Fe2+-bound human cytosolic H-chain ferritin (HuHF) to O2 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 Fe2+ oxidation and iron core mineralisation in wild-type HuHF and its E61A variant showed that rapid Fe2+ oxidation was unaffected by loss of Glu61, ruling out an important role for these sites in either guiding Fe2+ to the FoC, or in the mechanism of FoC-catalysed Fe2+ oxidation. Conversely, the transfer of Fe3+ out of the FoC and core mineralisation were both severely affected in the E61A variant. A mechanism for Fe3+ transfer from the FoC to the inner protein surface is proposed.