Ferritin Contains Less Iron (59Fe) in Cells When the Protein Pores Are Unfolded by Mutation

Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mM) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t(1/2) decreased 17-fold), and (iii) decreased alpha-helix (8%). Overexpression (>4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell Fe-59 but increased chelatable iron, 16%, p < 0.01 (Fe-59 in the deferoxamine-containing medium), and decreased Fe-59 in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased Fe-59 in open pore ferritin with increased chelatable Fe-59 in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (Fe-59) in ferritin in vivo.