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 (Fe(2+) and O2 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 (Fox) sites. Fe(2+) and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 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 (Kcat and kcat/Km), 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.

Keywords: BioIron; antioxidant; ferrihydrite; iron traffic; oxidoreductase enzyme activity.

Fig. 1.

Ion channels in soluble ferritin protein nanocages transport Fe²⁺ to multiple catalytic centers (F_ox or oxidoreductase sites). (A) The 24-subunit ferritin protein cage. Red helices, Fe²⁺ channels. (B) A ferritin Fe²⁺ ion channel (side view). Green spheres, metal ions. Conserved residues E57 and E136 are putative carboxylate links in Fe²⁺ transit between ferritin ion channel carboxylate (E130 and D127) and ferritin enzyme (F_ox) sites based on phylogenetic structural location and conformational flexibility in protein crystals (16, 18). (C) A single ferritin protein cage subunit (side view). Tan, channel helix segments. (D) Ion channel (viewed from inside the protein cage). Green spheres, metal ions. Prepared from PDB 3KA3 using PyMol.

Fig. 2.

Residues E57 and E136 are required for rapid access of Fe²⁺ substrate to ferritin catalytic sites for DFP formation as well as biomineralization. (A) Progress curves: diferric peroxo (DFP) catalytic intermediate, 48 Fe²⁺/cage (2 Fe²⁺/subunit) (A_{650 nm}). Ferritins E130A and D127A characterized earlier (14, 15) are analyzed here for comparison with E57A and E136A. (B) Catalytic efficiency (k_cat/K_m) and Fe²⁺ saturation of ferritin enzyme sites that oxidize 2 Fe²⁺(DFP formation). (C) Stabililzation of DFP at high iron:protein ratios (480 Fe²⁺/cage) in E57A, E136A, and E57A/E136A variant ferritins. (D) E57A and E136A inhibition of [Fe³⁺O]_x (biomineral) growth (A_{350 nm}) compared with WT. D127, E130, ion channel residues; E136, E57, flexible cage transfer residues. Concentrations of ferritin in solution (0.1 M Mops⋅Na, 0.1 M NaCl, pH 7.0) were (A and C) 50 µM, subunits (2.08 µM, protein cages); (B and D) 25 µM, subunits (1.04 µM, protein cages). Protein solutions, rapidly mixed (<5 ms) with fresh, acidic solutions of FeSO₄, were analyzed by UV-vis spectroscopy at 350 and 650 nm, as previously described. The data are averages of three to four independent experiments, using two different independent protein preparations for each protein; the error is the SD. Note the large differences in time scales for A (0–1 s, A_{650 nm}) and D (0–100 s, A_{350 nm}); in C the stabilization of DFP compared with WT and ion channel variants E130D and D127A, and in D, caged biomineral growth was faster in WT and ion channel ferritin variants E130D and D127A than in ferritin cage transfer residue variants E136A and E57A.

Fig. 3.

Selective effects of Co²⁺, Cu²⁺, and Zn²⁺ on ferritin activity. Solutions of metal cation inhibitors Co²⁺, Cu²⁺, or Zn²⁺ (6–96 metal ions/protein cage) were added to ferritin protein cage solutions in 0.2 M Mops⋅Na, 0.2 M NaCl, pH 7.0, for 1 h, before rapidly mixing (<5 ms) with acidic solutions of FeSO₄ (48 Fe²⁺/cage) at 25 °C; final protein concentrations were 50 µM, subunits (2.04 µM, protein cages). (A and B) Effects of Co²⁺, Cu²⁺, and Zn²⁺ on 2Fe²⁺/O₂ catalysis (diferric peroxo, A_{650 nm}, formation): (A) WT and (B) E130D. (C) Comparisons of metal cation inhibition in WT and E130D ferritin at 48 Fe/cage (2 Fe/enzyme site). Data for ferritin E130D with Cu²⁺ and Zn²⁺ were significantly different from WT: *P < 0.01; **P < 0.001. The inhibition of enzyme activity by metal cations in A of ferritins E136A and E57A/E136A was so great that reliable measurements were not possible. (D) Differences in UV-vis spectrum of Cu²⁺-WT ferritin and Cu²⁺-E130D ferritin. The spectrum of Cu²⁺-E130D ferritin is of a Cu–Cys charge transfer complex. The data in A–C are averages (±SD). All data are from two to four independent experiments, using two to three different protein preparations of each protein.

Fig. 4.

The Fe²⁺ bucket brigade that moves Fe²⁺ into ferritin protein cages. Three enzyme sites for the reaction of 2 Fe²⁺ with O₂ (rust) are shown in the three subunits that form one of the ion channels into a ferritin protein cage; in a 24-subunit eukaryotic ferritin, as studied here, there are eight ion channels and 24 enzyme sites (1/subunit). Green arrows show the path taken by entering Fe²⁺ through an ion channel to three enzyme sites in a ferritin protein cage, based on the results here. [The enzyme sites are also called ferroxidase (F_ox) sites or oxidoreductase sites.] F_ox site residues are E23, E58, H61, E103, Q137, and D140, described in ref. ; D140 can also be S140 or A140 in different eukaryotic ferritins, but in eukaryotic ferritins, residue 140 is never H (29), as it would be in a diiron cofactor. Q137 is a specific and conserved characteristic of eukaryotic ferritin enzyme sites (28). The multiple conformations of E136 and E57 observed in ferritin protein crystals (16, 18) are shown only for E136 for simplicity: 136_a, 136_b, and 136_c. The multiple conformations of E57, which are omitted, point either toward one of the E136 conformations or toward active site E58. Each of the three cage subunits contributing to the ion channel walls are drawn in different shades of blue. Residues in the Fe²⁺ bucket brigade are blue; O atoms are red. Colored spheres: green, multiple metal ions aligned down the middle of the ion channel, clustered at the D127 ring and en route to the catalytic sites; rust, Fe²⁺ at the ferritin di-Fe²⁺ enzyme centers.