Ferritins play a crucial role in iron homeostasis and detoxification in organisms from all kingdoms of life. They are composed of 24 α-helical subunits arranged around a hollow interior cavity in which an iron-containing mineral can be reversibly stored.1,2 Animal cells contain a cytosolic ferritin, which is composed of a tissue-dependent mixture of two different subunit types, H- and L-chains. H-chain contains a catalytic site, called the ferroxidase centre, which drives Fe2+ oxidation, while L-chain lacks the ferroxidase centre, but contains a nucleation site. Despite decades of research, high-resolution structural information on the mineral iron core generated by ferroxidase centre activity is lacking.
Animal cells with high metabolic activity express another ferritin, composed of H-chain-type subunits, that is targeted to mitochondria.3 Each subunit contains a ferroxidase centre highly related to that of the cytosolic ferritin,4 and a presumed, but undefined, nucleation site for mineral core formation. Here, by exploiting the presence of both iron oxidation and core nucleation sites within the mitochondrial ferritin subunit, together with a novel method for iron enrichment that solved the issue of metal ion competition, we report high-resolution time-resolved X-ray crystal structures that map out the mineralization process for mitochondrial ferritin. At extended O2-exposure time, a ferrihydrite-like hydrated iron-oxo cluster containing five iron ions, representative of the nascent native mineral core of ferritin, was observed. Kinetic data for wild-type mitochondrial ferritin and variants lacking potential coordinating ligands of the nucleation site demonstrated its functional importance in solution.