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Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

The Encapsulated ferritin-like proteins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. The encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question due to differences in organisation of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a putative metal binding site on the inner surface of Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase centre. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase centre and suggest a dual role for the secondary site as an electrostatic trap guiding ferrous ions toward the ferroxidase centre and, at the same time, acting as a barrier protecting the ferroxidase site against non-cognate inhibiting species. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ferritin ability to undergo catalysis, does not influence the specific function of the secondary site.