Functionality of the Three-Site Ferroxidase Center of <i>Escherichia coli</i> Bacterial Ferritin (EcFtnA)

At least three ferritins are found in the bacterium Escherichia coli: the heme-containing bacterioferritin (EcBFR) and two nonheme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A and B sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA was further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV–vis, fluorescence, and EPR spectroscopic measurements on both the wild-type protein and site-directed variants of the A, B, and C sites. The data reveal that although H₂O₂ is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe²⁺/O₂ ∼ 3:1 most of the H₂O₂ produced is consumed in subsequent reactions with a 2:1 Fe²⁺/H₂O₂ stoichiometry, thus suppressing hydroxyl-radical formation. Although the A and B sites are essential for rapid iron oxidation, the C site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation, and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O₂ and H₂O₂ as oxidants. Furthermore, our data do not support a universal mechanism for iron oxidation in all ferritins whereby the C site acts as transit site, as has been recently proposed