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

<jats:title>Abstract</jats:title><jats:p>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‐Fe<jats:sup>3+</jats:sup> site of Escherichia coli bacterioferritin (EcBFR) is stable and therefore does not function as a conduit for the transfer of Fe<jats:sup>3+</jats:sup> into the storage cavity, but instead acts as a true catalytic cofactor that cycles its oxidation state while driving Fe<jats:sup>2+</jats:sup> 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 Fe<jats:sup>2+</jats:sup> oxidation in the BFR cavity, to the di‐ferric catalytic site for safe reduction of O<jats:sub>2</jats:sub>.</jats:p>