Ferritin reactions: direct identification of the site for the diferric peroxide reaction intermediate

Abstract

Ferritins managing iron-oxygen biochemistry in animals, plants, and microorganisms belong to the diiron carboxylate protein family and concentrate iron as ferric oxide approximately 10(14) times above the ferric K(s). Ferritin iron (up to 4,500 atoms), used for iron cofactors and heme, or to trap DNA-damaging oxidants in microorganisms, is concentrated in the protein nanocage cavity (5-8 nm) formed during assembly of polypeptide subunits, 24 in maxiferritins and 12 in miniferritins/DNA protection during starvation proteins. Direct identification of ferritin ferroxidase (F(ox)) sites, complicated by multiple types of iron-ferritin interactions, is now achieved with chimeric proteins where putative F(ox) site residues were introduced singly and cumulatively into an inactive host, an L maxiferritin. A dimagnesium ferritin cocrystal model guided site design and the diferric peroxo F(ox) intermediates (A at 650 nm) monitored activity. Diferric peroxo formation in chimeric and WT proteins had similar K(app) values and Hill coefficients. Catalytic activity required cooperative ferrous substrate binding to two sites A (E, EXXH) and B (E, QXXD). The weaker B sites in ferritin contrast with stronger B sites (E, EXXH) in diiron carboxylate oxygenases, explaining diferric oxo/hydroxo product release in ferritin vs. diiron cofactor retention in oxygenases. Codons for Q/H and D/E differ by single nucleotides, suggesting simple DNA mutations relate site B diiron substrate sites and diiron cofactor sites in proteins. The smaller k(cat) values in chimeras indicate the absence of second-shell residues important for ferritin substrate-product channeling that, when identified, will outline the entire iron path from ferritin pores through the F(ox) site to the mineral cavity.

Fig. 1.

Ferrous oxidation by natural or chimeric ferritin F_ox sites. Progress curves for the conversion of ferrous to ferric oxo/hydroxo species (A at 350 nm), after rapid mixing solutions of ferrous sulfate (2 mM in 1 mM HCl) and recombinant ferritin protein nanocages (4.16 μM, or 100 μM subunits, in 200 mM Mops, pH 7.0). Guest diFe (A, B) F_ox site design used sequence conservation (Table 1) and Mg²⁺ or Ca²⁺ ligands in ferritin crystals (11, 12). ♦, H-WT A site: E23, E58, and H61; B site: E103, Q137, and S140. ▵, L+3 A site: E23, E58, and H61; B site: Q103, Q137, and D140. ▪, M(H′)-WT A site: E23, E58, and H61; B site: E103, Q137, and D140. ▴, L+4 A site: E23, E58, and H61; B site: E103, Q137, and D140. □, L+2 A site: E23, E58, and H61; B site: Q103, Q137, and T140. L+1: ○, L-K23E; +, L-S137Q; –, Q103E; ○, L-T140D; and ×, S137Q/T140D. •, L-WT (host).

Fig. 2.

Formation of the DFP intermediate by natural or guest F_ox sites. Curves show progress of DFP formation (A at 650 nm) (28) when using proteins and mixing conditions described for Fig. 1. (A) pH = 7, 0–0.5 s: □, M(H′)-WT A site: E23, E58, and H61; B site: E103, Q137, and D140. ▵, H-WT A site: E23, E58, and H61; B site: E103, Q137, and S140. ♦, L+3 A site: E23, E58, and H61; B site: Q103, Q137, and D140. ⋄, L+4 A site: E23, E58, and H61; B site: E103, Q137, and D140. (B) pH = 7, 0–10 s: □, M(H′)-WT; ▵, H-WT; ⋄, L+4; ♦, L+3. (C) pH = 8, 0–0.5 s: □, M(H′)-WT; ⋄, L+4. (D) pH = 8, 0–10 s: □, M(H′)-WT; ⋄, L+4.

Fig. 3.

Effect of pH on F_ox site kinetics in chimeric and WT ferritins. The ratios of the initial rates of ferrous oxidation (A at 350 nm) and DFP formation (A at 650 nm) at different values of pH were computed from progress curves for solutions of ferritin (4.16 μM = 100 μM in subunits) in Mops buffers at different values of pH after rapid mixing with solutions of ferrous sulfate in 1 mM HCl (480 iron atoms per assembled protein molecule). No DFP was detected in L+4 at pH 6 and the value for pH 7/6 was computed by using as the value for the minimum detectable rate, 0.27 ΔA₆₅₀·s^–1 per mg of protein.

Fig. 4.

Model of the ferritin F_ox site amino acids required to form the DFP intermediate in ferrous substrate oxidation and diferric oxo/hydroxo product (biomineral precursor) in eukaryotic ferritins. Required amino acids were identified by sequential introduction of residues into guest F_ox sites of a F_ox inactive ferritin and kinetic analyses of the DFP intermediates (A at 650 nm) (28) in recombinant ferritins. The figure is developed from data in Table 2, Figs. 1,2,3, and references and models in refs. – and .