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Full ferritin heteropolymer H/L shell and schematic representation of the three neuroferritinopathy pathogenic variants showing the various frameshift mutations on the E-helix C-terminus.

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Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

Ferritins are highly conserved supramolecular protein nanostructures that play a key role in iron homeostasis. Thousands of iron atoms can be stored inside their hollow cavity as a hydrated ferric oxyhydroxide mineral. Although phosphate associates with the ferritin iron nanoparticles, the effect of physiological concentrations on the kinetics, structure, and reactivity of ferritin iron cores has not yet been explored. Here, the iron loading and mobilization kinetics were studied in the presence of 1–10 mM phosphate using homopolymer and heteropolymer ferritins having different H to L subunit ratios. In the absence of ferritin, phosphate enhances the rate of ferrous ion oxidation and forms large and soluble polymeric Fe­(III)–phosphate species. In the presence of phosphate, Fe­(II) oxidation and core formation in ferritin is significantly accelerated with oxidation rates several-fold higher than with phosphate alone. High-angle annular dark-field scanning transmission electron microscopy measurements revealed a strong phosphate effect on both the size and morphology of the iron mineral in H-rich (but not L-rich) ferritins. While iron nanoparticles in L-rich ferritins have spherical shape in the absence and presence of phosphate, iron nanoparticles in H-rich ferritins change from irregular shapes in the absence of phosphate to spherical particles in the presence of phosphate with larger size distribution and smaller particle size. In the presence of phosphate, the kinetics of iron-reductive mobilization from ferritin releases twice as much iron than in its absence. Altogether, our results demonstrate an important role for phosphate, and the ferritin H and L subunit composition toward the kinetics of iron oxidation and removal from ferritin, as well as the structure and reactivity of the iron mineral, and may have an important implication on ferritin iron management in vivo.