| Abstract
| - Bacterioferritin from Escherichia coli is able to accumulate large quantities of iron in theform of an inorganic iron(III) mineral core. Core formation in the wild-type protein and a number offerroxidase center variants was studied to determine key features of the core formation process and, inparticular, the role played by the ferroxidase center. Core formation rates were found to be iron(II)-dependent and also depended on the amount of iron already present in the core, indicating the importanceof the core surface in the mineralization reaction. Core formation was also found to be pH-dependent interms of both rate and iron-loading characteristics, occurring with maximum efficiency at pH 6.5. Evenat this optimum pH, however, the effective iron capacity was ∼2700 per molecule, i.e., well below thetheoretical limit of ∼4500, suggesting that competing oxidation/precipitation processes have a majorinfluence on the amount of iron accumulated. Disruption of the ferroxidase center, by site-directedmutagenesis or by chemical inhibition with zinc(II), had a profound effect on core formation. Effectiveiron capacities were found to be linked to iron(II) oxidation rates, and in zinc(II)-inhibited wild-type andE18A bacterioferritins core formation was severely restricted. Zinc(II) was also able, even at lowstoichiometries (12−60 ions/protein), to significantly inhibit further core formation in protein alreadycontaining a substantial core, indicating the importance of the ferroxidase center throughout the coreformation process. A mechanism is proposed that incorporates essential roles for the core surface and theferroxidase center. A central feature of this mechanism is that dioxygen cannot readily gain access to thecore, perhaps because the channels through the bacterioferritin coat are hydrophilic and dioxygen isnonpolar.
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