Ferritin is a ubiquitous iron storage and detoxification protein found highly conserved in species from bacteria to plants to humans. In mammals, ferritin is composed of two functionally and genetically distinct subunit types, H (heavy, ~ 21,000 Da) and L (light, ~ 19,000 Da) subunits.  These two subunits co-assemble in various ratios with a tissue specific distribution to form a shell-like protein of 4/3/2 octahedral symmetry.  The H-subunit has a dinuclear iron center consisting of A & B binding sites where the fast conversion of Fe(II) to Fe(III) by dioxygen (or hydrogen peroxide) occurs, whereas the L-subunit is thought to contribute to the nucleation of the iron core and thus stores iron at a lower rate compared to the H-subunit.  In the present work, we investigated the iron oxidation and deposition mechanism in two recombinant heteropolymers ferritin samples of ~ 20H:4L (termed H/L) and ~ 22L:2H (termed L/H) ratios. The data indicate that iron oxidation occurs mainly on the H-subunit with a stoichiometry of 2Fe(II):1O2, suggesting the formation of H2O2.  However, unlike in the human H-chain homopolymer HuHF where most of the H2O2 produced at the ferroxidase center subsequently reacts with further Fe(II), most of the H2O2 produced in H/L remains in solution.  The H/L but not the L/H sample completely regenerates its ferroxidase activity within a short period of time (less than an hour) suggesting rapid movement of Fe(III) from the ferroxidase center to the cavity to form the mineral core, consistent with the role of L-chain in facilitating iron turn-over at the ferroxidase center of the H-subunit.  In the L/H heteropolymer, Fe(II) oxidation and mineralization appears to occur by two simultaneous pathways at all levels of iron additions: a ferroxidation pathway with a 2Fe(II)/1O2 ratio and a mineralization pathway with a 4Fe(II)/1O2 resulting in an average net stoichiometry of ~ 3Fe(II)/1O2. This result suggests the involvement of the two H-subunits in iron oxidation and mineral core formation in the L/H sample. A mechanism consistent with these observations is proposed.   * This work is supported by Grants Fondazione CARIPLO-2007 (SL), R01 GM20194 (NDC), a Faculty-Student Summer Undergraduate Research Program from SUNY Potsdam and the Cottrell College Science Award (ID # 7892) from Research Corporation (FBA).

Characterization of iron oxidation and mineral core formation in recombinant heteropolymer ferritins

240th National Meeting (2010)

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