Structural and Mechanistic Studies of Prokaryotic Ferritins

Abstract

Prokaryotic ferritins are specialised proteins used by cells to uptake, oxidize and store iron. These proteins are composed by identical subunits forming a hollow spherical structure. Ftns and Bfrs are composed by 24 subunits that can store up to 4500 iron atoms. Dps (DNA-binding protein from starved cells) proteins are composed by 12 subunits and can store up to 500 iron atoms per shell. Ferritins contribute to iron homeostasis through three main processes: ferroxidation reaction, using H2O2 or O2; mineralization; and iron release in the ferrous form, essential for various cellular processes. In this Thesis, Bfr from D. vulgaris and Dps from M. hydrocarbonoclasticus, were characterized structurally and mechanistically. In general, Dps proteins use H2O2 to oxidize Fe2+ ions, while Bfr proteins use O2. We have proved that D. vulgaris Bfr is more catalytically efficient when uses H2O2 for the ferroxidation reaction, storing up to 600 Fe2+ ions per protein, and has the ability to bind DNA, a feature common to some Dps proteins but unknown in Ftns and Bfrs. DNA-binding activity was also investigated for Dps protein, which contains a flexible positively charged N-terminus. EMSA results showed that Dps binds to DNA with a !d of ~6 μM, that this interaction is dependent on the N-terminus, and it is inhibited when the protein contains the mineral core. Dps protein uses preferentially H2O2 to oxidize Fe2+ ions and can store 500 Fe2+ ions. The oxidation of 24 Fe2+/protein results in the formation of a small core, as demonstrated by Mössbauer spectroscopy. Additionally, using this technique and a mixture of 56Fe2+/57Fe2+, it was possible to observe a characteristic never reported for this ferritin proteins. In the absence of oxidant, the mineral core can oxidize Fe2+ ions and promote its incorporation.