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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-06, Vol.111 (22), p.7925-7930
• Main Authors: Behera, Rabindra K., Theil, Elizabeth C.
• Format: Article
• Language: English
• Subjects: Active sites; Animals; Antioxidants - chemistry; Antioxidants - metabolism; Anura; Biological Sciences; Biomineralogy; Carboxylates; Catalysis; Conserved Sequence; Enzyme substrates; Enzymes; Ferric Compounds - chemistry; Ferric Compounds - metabolism; Ferritins; Ferritins - chemistry; Ferritins - genetics; Ferritins - metabolism; Heme - chemistry; Heme - metabolism; Hydroxides - chemistry; Hydroxides - metabolism; Ion channels; Ion Channels - chemistry; Ion Channels - metabolism; Ions; Iron - chemistry; Iron - metabolism; Metal ions; Metals - chemistry; Metals - metabolism; Minerals; Minerals - chemistry; Minerals - metabolism; Models, Chemical; Mutagenesis, Site-Directed; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Physical Sciences; Protein Structure, Secondary; Protein Structure, Tertiary; Substrate Specificity; Sulfates - chemistry; Sulfates - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1318417111

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⁺