Ferritins: furnishing proteins with iron

Ferritins are a superfamily of iron oxidation, storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. The majority of ferritins consist of 24 subunits that individually fold into 4-α-helix bundles and assemble in a highly symmetric manner to form an approximately spherical protein coat around a central cavity into which an iron-containing mineral can be formed. Channels through the coat at inter-subunit contact points facilitate passage of iron ions to and from the central cavity, and intrasubunit catalytic sites, called ferroxidase centers, drive Fe2+ oxidation and O2 reduction. Though the different members of the superfamily share a common structure, there is often little amino acid sequence identity between them. Even where there is a high degree of sequence identity between two ferritins there can be major differences in how the proteins handle iron. In this review we describe some of the important structural features of ferritins and their mineralized iron cores and examine in detail how three selected ferritins oxidise Fe2+ in order to explore the mechanistic variations that exist amongst ferritins. We suggest that the mechanistic differences reflect differing evolutionary pressures on amino acid sequences, and that these differing pressures are a consequence of different primary functions for different ferritins

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin

Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV–vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein