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Moving Fe²⁺ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates
- Behera, Rabindra K., Theil, Elizabeth C.
- Proceedings of the National Academy of Sciences of the United States of America 2014 v.111 no.22 pp. 7925-7930
- cations, cobalt, copper, enzyme activity, ferric oxide, ferritin, ion channels, iron, manganese, oxidants, zinc
- Ferritin biominerals are protein-caged metabolic iron concentrates used for iron–protein cofactors and oxidant protection (Fe ²⁺ and O ₂ sequestration). Fe ²⁺ passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe ²⁺ substrate movement to ferritin enzyme (F ₒₓ) sites. Fe ²⁺ and O ₂ substrates are coupled via a diferric peroxo (DFP) intermediate, λ ₘₐₓ 650 nm, which decays to [Fe ³⁺–O–Fe ³⁺] 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 k cₐₜ/K ₘ), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe ²⁺ concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe ²⁺ access), Mn ²⁺ << Co ²⁺ < Cu ²⁺ < Zn ²⁺, reflecting metal ion–protein binding stabilities. Fe ²⁺–Cys126 binding in ferritin ion channels, observed as Cu ²⁺–S–Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu ²⁺ inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe ²⁺ movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe ²⁺ into ferritin enzymatic sites. The results clarify Fe ²⁺ transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations.