Ferritins are multi-subunit iron storage and detoxification proteins that play a central role in the biological management of iron. In vertebrates, ferritins consist of 24 subunits of two types, H and L that co-assemble in various ratios with a tissue specific distribution. The homopolymer H- chain contains ferroxidase centers where the rapid oxidation of Fe(II) to Fe(III) occurs by either molecular oxygen or hydrogen peroxide. While ferritins from different organisms share many common structural features, the chemistries of iron uptake, oxidation, deposition and mobilization differ markedly. Extensive studies have been performed, separately, with either recombinant homopolymer H-chain or recombinant L-chain ferritin, but not with the heteropolymer H/L ferritin. [Surprisingly, and despite the widespread occurrence of heteropolymer ferritins of different H to L subunit ratio (isoferritins) in tissues of vertebrates], very little is known about these proteins and the complementary roles that H and L subunits play during iron uptake and mineralization. The goals of this research proposal are to investigate the structure-function relationships of iron uptake and deposition in recombinant heteropolymer ferritins, [which mimic naturally occurring ferritins in-vivo], and to characterize the stability and functionality of two pathogenic L-ferriti variants responsible for a hereditary ferritinopathy disorder. Specifically, we plan to study (a) te complementary roles of H and L subunits in iron oxidation and mineralization and identify iron-protein intermediates during this process, (b) the effect of L-chain mutations and iron content on the protein thermostability and (c) the magnetism and crystallinity of the iron core formed inside the ferritin cavity. To achieve this, a combination of site-directed mutagenesis, pH stat/oximetry, stopped-flow rapid kinetics techniques, UV- visible and fluorescence spectroscopy, differential scanning calorimetry and Mssbauer spectroscopy will be employed. The experiments proposed here should lead to a detailed understanding of the chemistry of iron deposition in heteropolymer ferritins and how different proportions of H and L subunits affect the biochemistry and functional properties of isoferritins. It will also provide insights into the biochemical processes responsible for the hereditary neuroferritinopathy disorder. Additionally, the proposed Mssbauer measurements of ferritin iron core should provide important insights into the structure, design, and development of controlled size nanoparticles within the ferritin shell for a broad range of applications ranging from electronics to biomedicine.
Ferritin plays a crucial role in iron homeostasis and human health as body levels and forms of iron must be appropriately maintained. The data that this proposal seeks to generate will be essential for (a) the rational development of new treatments for iron overload diseases and other defects in iron metabolism, (b) understanding the biochemical processes responsible for the hereditary neuroferritinopathy disorder and (c) the development of controlled size nanoparticles for biomedical applications.
