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-Å-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.¹³C direct detection | high molecular weight NMR | iron channels | solid-state NMR | paramagnetic NMR F erritins are a family of protein nanocages that concentrate iron in biominerals for controlled release and use in enzyme iron cofactors. A large cavity that occupies about 30% of the total protein volume at the center of the cage is the mineralization site of ferritin ferrihydrite. The importance of ferritin is illustrated by embryonic lethality of gene deletion in mammals (1) and resistance to oxidants in animals, plants, bacteria, and archea that reflects consumption of iron(II) and dioxygen or hydrogen peroxide (2-5). Some classes of nanomaterials use ferritin nanocages as templates (6). The protein nanocages self-assemble from fourhelix-bundle subunits into cages of two sizes: 12-subunit, hydrogen peroxide-consuming miniferritins of bacteria and archea, and 24-subunit maxiferritins of archea, bacteria, and higher organisms (3, 4, 7). Miniferritins may be the more primitive and are alternatively named DNA-binding proteins because some of them coat DNA.The first step in the conversion of iron(II) and dioxygen to ferric oxide mineral, in the 24-subunit maxiferritins, occurs at catalytic oxidoreductase (ferroxidase) sites in the center of each of the four-helix bundles, which have long axes oriented almost parallel to the protein surface (Fig. 1A). Each active site has residues contributed by each of the four helices (Fig. 1B) that create a diiron(II) site similar to the diiron cofactor sites in ribonucleotide reductases and stearoyl desaturases (8). When dioxygen, the second substrate, binds to the active site after iron(II) binding, diferric oxo products form via a diferric peroxo intermediate. The active site has...

Moving Iron through Ferritin Protein Nanocages Depends on Residues throughout Each Four α-Helix Bundle Subunit

“…1) suggest roles beyond just electrostatics for the conserved carboxylates, such as ion selectivity at the channel constriction or directing Fe 2ϩ substrate ions to active sites in three subunits that also form the entry channels (10). The Fe 3ϩ O nucleation channels were recently identified using 13 (10) and earlier studies (16,17), and on residues in the Fe 3ϩ postoxidation/nucleation channel, identified by NMR spectroscopy (11). Our results show that conserved carboxylates in the Fe 2ϩ ion entry channels, connecting the external cage surface to the central, mineralization cavity (Fig.…”

“…1C). The role of the protein in mineral buildup, beyond the catalytic reactions at the catalytic (oxidoreductase or ferroxidase) centers, has only recently been suggested by structural studies (11), which show that nucleation occurs inside the long (ϳ20 Å) Fe 3ϩ O nucleation channels (Fig. 1, A-C).…”

“…The nucleation channels, also called post-oxidation channels, are distal to the active sites, on the long axis of each subunit bundle; they open around the 4-fold axes on the inner surface of the cage. The Fe 3ϩ paramagnetic effects indicated that the residues were ϳ5 Å away from Fe 2ϩ oxidized in situ (11). Note that the apparent absence of carboxylate residues in the Fe 3ϩ O channels relates in part to incomplete site-specific NMR assignments that underrepresented carboxylates due to large size and number of residues (480 kDa, 175 residues/subunit, 24 subunits), an unusually large number of carboxylate residues, the small 13 C chemical shifts for Glu and Asp, the narrow dispersion between Asn/Asp or Gln/Glu, and the effects of ␣-helix bundle structure (low chemical shift dispersion).…”

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ 3 -oxo)Tris[(μ 2 -peroxo)] triiron(III) cluster

“…Consistently, the biomineralization reaction in H-ferritin cages does not require a nucleation site (24,25), and His57 and Glu61 are involved in controlling the access of ferrous ions to the ferroxidase site (6); the reaction at the ferroxidase site is much faster than in L-subunits, with time scales for the iron oxidation in homopolymeric H-cages of <1 s. Even with a low number of H-subunits (1 to 2 per cage), the reaction rate is dominated by the catalytic oxidation of iron occurring at the ferroxidase sites. The formation of ferric clusters of high nuclearity in ferroxidase-active (H or H′) subunits has been inferred from Mössbauer data (26)(27)(28) and NMR data (29,30), but these clusters have never been detected by time-lapse anomalous crystallography approaches. This observation supports the occurrence of a different iron mineralization process in ferroxidase-active subunits, leading to spatially disordered clusters that escape detection by X-ray crystallography.…”

Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

“…For the solid‐state NMR investigation, ANSII (either free or its MenC conjugate) was freeze‐dried, packed into the magic‐angle spinning (MAS) rotors, then rehydrated to restore the resolution 30, 34, 35, 36. The spectra collected on the pelleted ANSII‐MenC conjugate (Figure 2) are largely superimposable to those of the native and PEGylated forms of the enzyme (see Figure S2 in the Supporting Information) making possible the assessment of the preservation of the protein fold after conjugation with the MenC polysaccharide 37, 38, 39, 40. The resonances of the protein residues were easily reassigned starting from the assignment of the native form of ANSII, by using a carbon‐detection approach (Figure S3) 41.…”