Ferritin ion channel disorder inhibits Fe(II)/O2 reactivity at distant sites

Abstract

Ferritins, a complex, mineralized, protein nanocage family essential for life, provide iron concentrates and oxidant protection. Protein-based ion channels and Fe(II)/O(2) catalysis initiate conversion of thousands of Fe atoms to caged, ferritin Fe(2)O(3)·H(2)O minerals. The ion channels consist of six helical segments, contributed by 3 of 12 or 24 polypeptide subunits, around the 3-fold cage axes. The channel structure guides entering Fe(II) ions toward multiple, catalytic, diiron sites buried inside ferritin protein helices, ~20 Å away from channel internal exits. The catalytic product, Fe(III)-O(H)-Fe(III), is a mineral precursor; mineral nucleation begins inside the protein cage with mineral growth in the central protein cavity (5-8 nm diameter). Amino acid substitutions that changed ionic or hydrophobic channel interactions R72D, D122R, and L134P increased ion channel structural disorder (protein crystallographic analyses) and increased Fe(II) exit [chelated Fe(II) after ferric mineral reduction/dissolution]. Since substitutions of some channel carboxylate residues diminished ferritin catalysis with no effect on Fe(II) exit, such as E130A and D127A, we investigated catalysis in ferritins with altered Fe(II) exit, R72D, D122R and L134P. The results indicate that simply changing the ionic properties of the channels, as in the R72D variant, need not change the forward catalytic rate. However, both D122R and L134P, which had dramatic effects on ferritin catalysis, also caused larger effects on channel structure and order, contrasting with R72D. All three amino acid substitutions, however, decreased the stability of the catalytic intermediate, diferric peroxo, even though overall ferritin cage structure is very stable, resisting 80 °C and 6 M urea. The localized structural changes in ferritin subdomains that affect ferritin function over long distances illustrate new properties of the protein cage in natural ferritin function and for applied ferritin uses.

Figure 1. Ferritin (eukaryotic) protein cage structure

Ferritin protein cages with eight Fe(II) entry channels, ~ 15 Å long assemble from identical helix segments contributed by three polypeptide subunits, transport Fe(II) from the cage exterior to interior, exiting near the catalytic sites in each subunit. A second type of channel, for ferric oxo nucleation, ~ 20 Å long, connects catalytic sites to the central mineral growth cavity, moving the Fe(II)/O₂ catalytic product, Fe(III)-O(H)-Fe(III). A. Ferritin protein cage, outside view with a three-fold cage axis in the center; blue – helix segments from subunits forming an Fe(II) ion channel. B. Side view of a polypeptide subunit; blue-helix - segments that form an Fe(II) ion channel. C. A cutaway version of the ferritin protein cage with an empty mineral growth cavity viewed toward a threefold axis. Hazy blue helices in the center - an ion channel on the inner cage surface opposite the cut away section. D. View of a ferritin ion channel from outside the cage. A section of the N-terminal peptide extending from the helix bundle, through a network of bonds to the helices, slows (“gates’) the Fe(II) exit during mineral dissolution. Green - Mg(II) ions; red –ordered H₂O. E. Side view of the 16 Å long, ferritin ion channel; Green - Mg(II) ions. Pore diameters: Outer (top)-8.5; Inner (bottom)- 7.1 Å; mid-channel diameter is 5.4 Å. Figures from PDB 3KA3 and 3SH6 used PyMol, or Discovery Studio (visualizer), 2.0, for the cage cross section.

Figure 2. Progress curves of diferric peroxo formation in ferritin M

Fe(II) was added to solutions of frog M ferritin (wild type or with amino acid substitutions at pore residues) in air 25°C. The ratio of Fe(II): diiron oxidoreductase site = 2 (Total Fe = 48/cage). The absorbance was monitored at 650 _nm during ferritin catalysis, which is the λ_max for ferritin Fe(III)-O-O-Fe(III) (DFP). DFP forms in msec and decays ~ 1 sec. at A350 _nm is most informative for later stages of mineralization; some studies monitor the absorbance at 310 nm are monitored instead, since the transition is broad. Absorbance in the 300–400 nm range is due to a mixture of spectroscopically unresolved species that includes ferric oxo/hydroxo dimers, other multimers and caged Fe(III)(OH)₆ mineral. A. Progress curve for DFP (inset shows data from the early time domain); B. Ferric oxo/hydroxo formation.

Figure 3. Effect of changes in ferritin ion channel structure on metal ion binding

Figures were drawn with PyMol software, using PDB files WT-3KA3 (A, B); (C, D) D122R-3SH6. A. WT Fe(II) channel viewed from the top; B. WT Fe(II) channel viewed form the side. C. D122R channel viewed from the top; D. D122R viewed from the side. Green: Mg(II); red (H₂O).