From microorganisms to mankind, ferritin plays a central role in the biological management of iron. The ferritins function as iron storage and detoxification proteins by depositing iron as a hydrous ferric oxide mineral within their shell-like structures. This iron can be subsequently mobilized for the synthesis of heme. While ferritins from various organisms share many common structural features, being either 12 or 24 subunit proteins, they differ markedly in their chemistries of iron deposition. All known ferritins contain ferroxidase sites that catalyze iron(II) oxidation by either molecular oxygen or hydrogen peroxide; however they do so in significantly different ways. This proposal focuses on the mechanisms of iron deposition in a variety of recombinant 24mer ferritins that include human H- and L-chain ferritins, a newly discovered human mitochondrial ferritin, the heme-containing E. coli bacterioferritin (EcBFR) and the E. coil northeme bacterial ferritin (EcFtnA). Studies will also be conducted with the l2mer proteins: Listeria innocua ferritin and the DNA binding protein, Dps from E. coli. Important questions relating to dioxygen binding, ferritin-ferritin association during iron oxidation, transient radical and iron intermediates and the stoichiometric equations for iron oxidation and hydrolysis using dioxygen and hydrogen peroxide as oxidants will be addressed for the different proteins. Features of the mechanisms of iron deposition of the various ferritins will be elucidated through a combination of site-directed mutagenesis in conjunction with x-ray structure data, isothermal titration calorimetry, UV-visible stopped-flow kinetics, rapid-freeze quench Mossbauer and EPR spectroscopies, spin trapping, light scattering, oximetry and pH stat. The extensive studies proposed should lead to a detailed understanding of how various ferritins function as reversible iron storage proteins, assisting the cell cope with oxidative stress, and further our knowledge of the chemistry and biochemistry of iron biomineralization processes in general.
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