Three Aromatic Residues are Required for Electron Transfer during Iron Mineralization in Bacterioferritin

Ferritins are iron storage proteins that overcome the problems of toxicity and poor bioavailability of iron by catalyzing iron oxidation and mineralization through the activity of a diiron ferroxidase site. Unlike in other ferritins, the oxidized di-Fe3+ site of Escherichia coli bacterioferritin (EcBFR) is stable and therefore does not function as a conduit for the transfer of Fe3+ into the storage cavity, but instead acts as a true catalytic cofactor that cycles its oxidation state while driving Fe2+ oxidation in the cavity. Herein, we demonstrate that EcBFR mineralization depends on three aromatic residues near the diiron site, Tyr25, Tyr58, and Trp133, and that a transient radical is formed on Tyr25. The data indicate that the aromatic residues, together with a previously identified inner surface iron site, promote mineralization by ensuring the simultaneous delivery of two electrons, derived from Fe2+ oxidation in the BFR cavity, to the di-ferric catalytic site for safe reduction of O2. A radical view of bacterioferritin: Three aromatic residues located close to the catalytic ferroxidase site are shown to be important for iron mineralization. A transient radical is associated with Tyr25, consistent with a mechanism where electrons from Fe2+ oxidation are transferred to the oxidized, di-Fe3+ ferroxidase site, ensuring that two electrons arrive at the site simultaneously and avoid formation of toxic reactive oxygen species.