Abstact

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.