Bacterioferritin (BFR) fromEscherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe 3؉ mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe 2؉ chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.

Ferritins solubilize and detoxify the essential metal iron through formation of a ferric mineral within the protein's central cavity. Key to this activity is an intrasubunit catalytic dinuclear iron center called the ferroxidase center. Here we show that the fluorescence intensity of Escherichia coli bacterioferritin (BFR), due to the presence of two tryptophan residues (Trp35 and Trp133) in each of the 24 subunits, is highly sensitive to the iron status of the ferroxidase center and is quenched to different extents by Fe2+ and Fe3+. Recovery of the quench following oxidation of Fe2+ to Fe3+ at the ferroxidase center was not observed, indicating that the di-Fe3+ form of the center is stable. Studies of the single-tryptophan variants W35F and W133F showed that Trp133, which lies approximately 10 A from the ferroxidase center, is primarily responsible for the observed fluorescence sensitivity to iron, while studies of a stable E. coli BFR subunit dimer demonstrated that the observed quench properties are principally derived from the interaction of iron with tryptophan residues within the subunit dimer. A double-tryptophan variant (W35F/W133F) was found to exhibit fluorescence from the seven tyrosine residues present in each subunit, which was also sensitive to the iron status of the ferroxidase center. Finally, we demonstrate using Zn2+, a potent competitive inhibitor of Fe2+ binding and oxidation, that the fluorescence response can be used to monitor the loss of iron from the ferroxidase center.