Moving Fe2+from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

Abstract

Ferritin biominerals are protein-caged metabolic iron concentrates used for iron-protein cofactors and oxidant protection (Fe2+ and O 2 sequestration). Fe2+ passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe2+ substrate movement to ferritin enzyme (Fox) sites. Fe2+ and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 650 nm, which decays to [Fe3+-O-Fe3+] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (K cat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe2+ concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe2+ access), Mn2+ ≪ Co2+ < Cu2+ < Zn2+, reflecting metal ion-protein binding stabilities. Fe2+-Cys126 binding in ferritin ion channels, observed as Cu2+-S-Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu2+ inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe2+ movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade thatmoves external Fe2+ into ferritin enzymatic sites. The results clarify Fe2+ transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations.