Ferritin Iron Mineralisation: Route of Fe3+ Transfer From the Ferroxidase Centre to the Inner Cavity of Human H-Chain Ferritin

Abstract

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.

Keywords: ferritin; iron; oxidation; storage; transport.