NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

Abstract

Ferritin is a multimeric nanocage protein that directs the reversible biomineralization of iron. At the catalytic ferroxidase site two iron(II) ions react with dioxygen to form diferric species. In order to study the pathway of iron(III) from the ferroxidase site to the central cavity a new NMR strategy was developed to manage the investigation of a system composed of 24 monomers of 20 kDa each. The strategy is based on (13)C-(13)C solution NOESY experiments combined with solid-state proton-driven (13)C-(13)C spin diffusion and 3D coherence transfer experiments. In this way, 75% of amino acids were recognized and 35% sequence-specific assigned. Paramagnetic broadening, induced by iron(III) species in solution (13)C-(13)C NOESY spectra, localized the iron within each subunit and traced the progression to the central cavity. Eight iron ions fill the 20-A-long iron channel from the ferrous/dioxygen oxidoreductase site to the exit into the cavity, inside the four-helix bundle of each subunit, contrasting with short paths in models. Magnetic susceptibility data support the formation of ferric multimers in the iron channels. Multiple iron channel exits are near enough to facilitate high concentration of iron that can mineralize in the ferritin cavity, illustrating advantages of the multisubunit cage structure.

Fig. 1.

Sequential assignment of ferritin by solid-state NMR. (A) 3D structure of recombinant frog (R. catesbeiana) M ferritin (24 subunits, PDB 1MFR; PyMOL 0.99rc6). The four-helix-bundle subunits are displayed as gray helices and red loops. The green subunit illustrates the orientation of the monomeric units along the surface of the hollow sphere. (B) Representation of the four-helix-bundle subunit NMR-assigned residues (blue spheres) and ferroxidase residues (red spheres). (C) Primary and secondary structures of frog M ferritin-ferroxidase site residues (red font), NMR sequence-specific assigned residues (blue font), α-helices (bars), and loops (broken lines).

Fig. 2.

Combined use of solid-state and solution NMR. (A) Superimposition of the aliphatic region of the ferritin ¹³C-¹³C NOESY spectrum acquired in solution (blue trace) and in the solid-state¹³C-¹³C DARR spectrum (red trace) permits the transfer of the solid-state assignments to solution data and vice versa. The two 2D maps were overlaid by looking for the best superimposition of the aliphatic part. (B) Superimposition of the aliphatic region of the ferritin ¹³C-¹³C NOESY spectra acquired in solution before (blue) and after (green) the addition of 1 equivalent of iron (II) (2 iron(II)/active site; 48 iron(II)/nanocage); resonances disappearing upon formation of iron (III) products are labeled.

Fig. 3.

Tracing the iron channel in ferritin by paramagnetic effects. (A) NMR resonances that disappear in ¹³C-¹³C NOESY solution spectra, as the iron:protein ratio increases, are mapped onto the ribbon structure of a ferritin subunit as colored spheres (1 equivalent of iron = 2 iron(II)/active site = 48 iron(II)/nanocage). 1 equivalent: light blue, 2 equivalents: blue, 4 equivalents: dark blue. (B) View of the internal surface of the ferritin protein cage showing the relative spatial relationship near channel exits into the cavity; iron(III) products emerging from the channel of the red subunit have paramagnetic effects on residue V42 in the blue subunit and on R72 and G74 in the green subunit, after 4 equivalents of iron are added. Paramagnetically broadened residues are shown as spheres. (C) Ferritin channel exits in four adjacent subunits surround the four fold axes of the ferritin protein cage: V42 (blue spheres); I148, T149, L154, and L156 (red spheres). (D) Effective magnetic moments per subunit (μ_eff, red bars) and average magnetic moment per iron atom (μ_eff/iron atom, blue bars) obtained by Evans measurements at increasing concentrations of iron.