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

Abstract

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O]_n multimers within the protein cage, en route to the cavity, at sites distributed over ∼50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A_{650 nm}) and on mineral growth (Fe³⁺O-A_{350 nm}), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O]_n transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

Introduction

Ferritins are an ancient superfamily of protein nanocages that synthesize, reversibly, iron concentrates for cellular use, heme, FeS cluster, and Fe-protein synthesis and provide oxidant protection by consumption of dioxygen or hydrogen peroxide and ferrous iron during stress; the protein cavities containing the minerals are ∼60% of the cage volume (1–4). Ferritins differ in cage size, location, and mechanism of catalytic sites, mineral size, and mineral crystallinity; the Fe²⁺/O₂ oxidoreductase sites are also called ferroxidase sites or FC (ferroxidase centers) sites (1–5). In eukaryotic H ferritins, Fe²⁺ and dioxygen are substrates for oxidoreductase sites and are catalytically coupled at multiple protein sites to synthesize diferric oxo mineral precursors (Fig. 1). Consumption of both iron and oxygen by ferritins accounts for the antioxidant response and iron-controlled gene regulation of ferritins in eukaryotes (6). Many catalytic proteins use iron and oxygen to produce a variety of organic products. Such products include unsaturated fatty acids such as oleate stearoyl-CoA desaturase-1 (7), deoxyribose (ribonucleotide reductase), and prolyhydroxyl modification of oxygen-sensing DNA transcription factors, e.g. hypoxia-inducible factor-α (8). Only in ferritins are both substrates inorganic. The exclusive use of inorganic substrates may relate to the ancient origins of the ferritins, which are distributed in all kingdoms and in both anaerobes and aerobes; ferritin gene deletion is lethal early in mammalian embryogenesis (9).

FIGURE 1.

Ferritin nanocage structure. A, frog-M ferritin nanocage structure, viewed from the outside with the 3-fold axis near the center, shown with one subunit highlighted in blue (Protein Data Bank ID code 1MFR). Eight Fe²⁺ ion channels (10), each formed by residues from three subunits around the 3-fold axes, lead through the protein cage from the outside of the cage to the large (8-nm diameter) central cavity, near several residues of the active sites; B, a single subunit of the frog-M ferritin showing all of the residues studied by site-directed mutagenesis; C, steps in ferritin mineral synthesis.

There are two types of protein channels that move iron into and through the 24 subunit cages during synthesis of [Fe³⁺O]_n in eukaryotic ferritins. The two functional types of ferritin channels are: (i) ion entry channels around the 3-fold axes, for Fe²⁺ substrate access to oxidoreductase sites (Asp¹²⁷, Glu¹³⁰) and (ii) Fe³⁺O nucleation channels, distal to the active sites on the long axes of each subunit bundle (Ala²⁶, Val⁴², Thr¹⁴⁹), where diferric oxo mineral precursors produced by oxidoreduction fuse to tetramers and larger multimers before exiting from the protein cage for mineral growth.

Recent high resolution structural studies show that ferritin is a soluble ion channel protein with lines of multiple metal ions (10), much like K⁺ and other membrane ion channel proteins. The eight Fe²⁺ entry channels (Fig. 1) suggest roles beyond just electrostatics for the conserved carboxylates, such as ion selectivity at the channel constriction or directing Fe²⁺ substrate ions to active sites in three subunits that also form the entry channels (10). The Fe³⁺O nucleation channels were recently identified using ¹³C-¹³C solution NMR spectroscopy in the presence and absence of Fe³⁺, by the disappearance of resonances within 5 Å of Fe³⁺O moving away from the active sites (11). Nucleation channel exits into the cavity are clustered around the 4-fold axes of the cage, which facilitates ordered mineral growth. Functional studies of carboxylate residues in the Fe²⁺ ion entry channels have been mostly limited to later stages after diferric peroxo (DFP)⁴ decay (12–14) except (15), and none has examined the function of residues in the Fe³⁺O nucleation channel. Several recent reviews addressing the general topic of ferroxidation and catalysis in ferritin proteins include Refs. 1–4.

We now report the functional effects of amino acid substitutions on the initial Fe²⁺/O₂ reaction intermediate, diferric peroxo (Fe³⁺O-O-Fe³⁺), and mineral precursors (Fe³⁺O)_n (Fig. 1C), during multiple catalytic turnovers, for residues in the Fe²⁺ entry channels, identified by Co²⁺,Mg²⁺ protein co-crystallography (10) and earlier studies (16, 17), and on residues in the Fe³⁺ postoxidation/nucleation channel, identified by NMR spectroscopy (11). Our results show that conserved carboxylates in the Fe²⁺ ion entry channels, connecting the external cage surface to the central, mineralization cavity (Fig. 1, A and B), have two different functions, i.e. Fe²⁺ entry and Fe²⁺ distribution among multiple active sites. In addition, we show that amino acid substitutions in the Fe³⁺ postoxidation/nucleation channels affect oxidation and decay of the diferric peroxo intermediate. Concepts of ferritin function have usually considered that movement of iron through the protein cage depended on residues in localized regions within each 4-helix bundle subunit. The results of this study, by contrast, reveal functional residues spanning the length of each 4-helix bundle subunit, from Ala²⁶ to Thr¹⁴⁹, in the assembled ferritin protein cage.

EXPERIMENTAL PROCEDURES

Mutagenesis

Frog-M ferritin was used as the template because Fe-protein interactions have been studied extensively by Mössbauer, resonance Raman, EXAFS, UV-visible spectroscopies and x-ray crystallography as reviewed in Ref. 5, and more recently by active site amino acid substitutions (18), MCD/CD (19) and very high resolution x-ray crystallography (10). Site-directed amino acid substitutions in frog-M ferritin protein cages were generated by PCR on the expression plasmid pET-3a frog-M, using the QuikChange® II site-directed mutagenesis kit (Stratagene). Coding regions of all protein expression vectors were analyzed for DNA sequence confirmation (UC Berkeley DNA sequencing facility). The forward primers that were used are listed in the supplemental text, with bold nucleotides representing the mutated codon; reverse primers were complementary to the forward primers.

Protein Expression

pET-3a constructs encoding wild-type frog-M ferritin and mutants were transformed into Escherichia coli BL21(DE3)pLysS cells and subsequently cultured in LB medium containing ampicillin (0.1 mg/ml). Cells were grown at 37 °C, until A_{600 nm} reached 0.6–0.8. Induction, at 30 °C with isopropyl 1-thio-β-d-galactopyranoside (0.5 mm final concentration), was 4 h, when cells were harvested (14, 20). Recombinant ferritins were purified as described previously (14, 20–22). In short, cells were broken by sonication, and the cell free extract obtained after centrifugation (2 h, 18,000 rpm, 4 °C) was incubated for 15 min at 65 °C as first purification step. After removal of the aggregated proteins (30 min, 18,000 rpm, 4 °C), ferritin was precipitated with 65% ammonium sulfate, resolubilized in 25 mm bis-Tris-propane, pH 7.5, and dialyzed against the same buffer. Next, the sample was loaded onto a Q-Sepharose column (Vc = 200 ml) and eluted with a linear NaCl gradient of 0–1 m in bis-tris-propane, pH 7.5. Fractions containing ferritin (monitored by SDS-PAGE) were combined, precipitated with ammonium sulfate (65%), resolubilized in volumes of 100 mm MOPS, pH 7.0, containing 100 mm NaCl, and dialyzed against the same buffer. Protein concentration was determined with a Bradford assay, and iron content was analyzed after boiling in 1 n HCl, as the Fe²⁺-1,10-phenanthroline complex (23, 24).

Fe²⁺/O₂ Catalysis and Fe³⁺O Mineralization

Single turnover iron oxidation (uptake of 48 Fe²⁺/ferritin cage, 2 Fe²⁺/subunit), in frog-M ferritin, wild type or with amino acid substitutions, was monitored as the change in A_{650 nm} (diferric peroxo or DFP) (12, 25) or A_{350 nm} (Fe³⁺O) after rapidly mixing (<5 ms) equal volumes of 100 μm protein subunits (4.16 μm protein cages) in 200 mm MOPS, pH 7.0, containing 200 mm NaCl with a freshly prepared 200 μm ferrous sulfate in 1 mm HCl in a UV/visible, stopped-flow spectrophotometer (18). Generally, 2000 data points were collected during 10 s. The incubation time between subsequent additions of Fe²⁺ aliquots (48 Fe²⁺/nanocage) was 4–16 h (12, 25). The progress of oxidation (DFP + diferric oxo + Fe³⁺O tetramers + mineral nuclei + mineral) was monitored using the nonspecific absorbance between 300 and 450 nm, at ΔA_{350 nm}. Absorbance curves for Fe³⁺O between 300 and 450 nm were also collected for solutions of 240 μm protein subunits (10 μm ferritin cages) with an iron concentration of 480 μm (2 Fe²⁺/subunit; 48 Fe²⁺/protein cage).

Initial rates of DFP and [Fe³⁺O]_n species formation were determined from the linear fitting of the initial phases of 650 nm and 350 nm trace (0.01–0.03 s). The rate of decay of DFP complex was determined by exponential fitting of the decreasing part of the progress curve at 650 nm (0.1–1.5 s). The data presented are averages from a minimum of 4–6 experiments each using a minimum of two or more independent protein preparations, and the error is presented as the S.D.

Fe³⁺O Mineral Dissolution/Chelation

Empty ferritin protein cages were mineralized with ferrous sulfate (480 Fe²⁺/cage), in 100 mm MOPS, pH 7.0, containing 100 mm NaCl, as described previously (20). Mineral dissolution was monitored as formation of Fe²⁺-bipyridyl, at A_{522 nm}, outside of the ferritin nanocavity after reduction of Fe³⁺ as described previously (20, 21, 26). A mixture of reductant (NADH and FMN) was added to a solution of 0.5 μm mineralized ferritin (0.25 mm Fe) containing 2.5 mm bipyridyl at 25 °C; final concentrations of NADH and FMN were 2.5 mm. Initial rates of iron release were calculated from the linear part of the Fe²⁺-bipyridyl formation (522 nm trace). The data were averaged from a minimum of four experiments using a minimum of two independent protein preparations.

RESULTS

Fe²⁺ Enters Ferritin Ion Channels Controlled by Glu¹³⁰ and Is Directed to Active Sites by Asp¹²⁷

Negatively charged ferritin residues Glu¹³⁰ and Asp¹²⁷ were originally identified by sequence conservation and location in 3-fold pores (16, 17). The three Glu¹³⁰ residues are in the middle of the Fe²⁺ ion channels, one from each of the three subunits that form the channels, at a constriction (Fig. 1B); the 8 channels are formed by the juxtaposition of the helix₃-loop helix₄ regions of three subunits around the 3-fold axes of the protein cage. Three Asp¹²⁷ residues are at the end of the channels at the entries to the large central cavity (8-nm diameter) where mineral forms. The influence of Glu¹³⁰ and Asp¹²⁷ on catalysis at the Fe²⁺/O₂ oxidoreductase sites, i.e. on diferric peroxo formation, has never been studied, although substitutions at Glu¹³⁰ and Asp¹²⁷ are known to inhibit formation of the multiple Fe³⁺O species measured in the range A_{305–450 nm} (27, 28), where Asp¹²⁷ and Glu¹³⁰ are equivalent to Asp¹³¹ and Glu¹³⁴ in the human ferritin studied. The three Glu¹³⁰ residues and the three Asp¹²⁷ residues from each of the three subunits that form the eight Fe²⁺ entry channels in the 24-subunit cage structure, all participate in protein-metal ion interactions in the channels, based on crystal structures (10, 29–31). We predict that E130A and D127A will have different effects during multiple catalytic turnovers.

Ferritin protein nanocages with E130A or D127A substitutions in each subunit were analyzed for differences in diferric peroxo formation, compared with wild type; DFP has a λ_max at 650 nm (12, 33, 34). Fe²⁺ was added in multiple aliquots, each of which saturated the active sites, i.e. 48 Fe²⁺/ferritin nanocage (2 Fe²⁺/active site) to achieve distinguishable turnovers of the active site. Based on the NMR experiments (11), which determined where Fe³⁺O is, in the protein cage, four turnovers were required for [Fe³⁺O]_n complexes (see Fig. 1C) to complete the transit through the nucleation channels to the cavity. In addition to the DFP complex, we also measured absorbance changes at 350 nm. Many Fe³⁺O species contribute to the absorbance in 300–450 nm range, which monotonically decreases with increasing wavelength (27, 28, 35, 36). We monitored changes in A_{350 nm} because they can be observed for long time periods, during mineral growth, after DFP has decayed (Fig. 2B). During ferritin mineral formation, the many Fe³⁺O species absorbing in the 300–450 nm range (Figs. 1C and 2D) include DFP at the active sites (12, 13, 37), diferric oxo product (12, 25), and mineral nuclei that form as Fe³⁺O multimers moving through the channels to the cavity (11) (reaction scheme shown in Fig. 1C). Fig. 2D shows the absorbance versus wavelength spectrum of WT ferritin (48 Fe²⁺/cage) immediately after the addition of Fe²⁺ or 60 min later. Mineral growing in the protein cavity also contributes to the absorbance in this range. Assignment of specific spectral properties to each of the Fe³⁺O species that occur during iron oxidation and mineral formation is not possible without additional different spectroscopic information analyses related to kinetics of mineral formation; currently the mixed spectra of the Fe³⁺O species are unresolved. We use changes in A_{350 nm} to monitor mineral formation because it is far enough away from spectral contributions due to aromatic amino acids and still high enough to provide sensitive measurements.

FIGURE 2.

Negatively charged residues in the 3-fold pore region are required for iron entry and active site access. For single turnover iron oxidation (uptake of 48 Fe²⁺/ferritin cage) (A–C), monitored by the change in A_{650 nm} (DFP) or A_{350 nm} (all Fe³⁺O) rapid mixing (<5 ms) protein concentrations were 2.08 μm ferritin cages (50 μm ferritin subunits) 100 μm ferrous sulfate solution in 100 mm MOPS, pH 7.0, 100 mm NaCl. Subsequent additions Fe²⁺ were 48 Fe nanocage (2/diiron active site) (see “Experimental Procedures”). For the absorbance spectra of Fe³⁺O in ferritin (D), the final protein concentration was 10 μm ferritin cages and 480 μm Fe²⁺ in 100 mm MOPS, pH 7.0, 100 mm NaCl. A, initial rates of DFP formation (ΔA_{650 nm}/s) for the four catalytic cycles. Inset, DFP absorbance spectrum in WT frog-M ferritin, modified from Ref. 29. Progress curves at 650 nm, after each addition of iron, are illustrated in supplemental Fig. S1. B, Progress curves of A_{350 nm} for the fourth (192 Fe²⁺/cage) catalytic cycle: wild type, solid line; D127A, ▴; and E130A, *. C, initial rates of single turnover for the formation of the DFP (ΔA_{650 nm}/s) and iron oxidation (ΔA_{350 nm}/s) for the first (48 Fe²⁺/cage) and fourth (192 Fe²⁺/cage) catalytic cycles. The numbers are averages from at least four to six experiments, each performed on at least two independent preparations of protein. D, spectra of Fe³⁺O species between 300 and 450 nm measured immediately (manual mixing) after Fe²⁺ addition and after 60 min.

Glu¹³⁰ is absolutely required for formation of the DFP catalytic intermediate of ferritin (Fe²⁺/O₂ oxidoreductase activity) because insignificant amounts of DFP formed in E130A under any conditions tested (up to 8 Fe²⁺/active site or 192 Fe²⁺/cage) in stepwise Fe²⁺ addition experiments (Fig. 2A). In fact, the relative rates of Fe²⁺ oxidation are comparable with those for the animal-specific, L-type ferritin subunit that lacks a catalytic site (14) and where Fe²⁺ oxidation is thought to be facilitated by iron chelation at residues on the protein or on the caged, inorganic mineral (38). The initial rate of oxidation measured at 350 nm (ΔA_{350 nm}/s) was ∼1% wild type.

Asp¹²⁷ is required for DFP formation when the amount of Fe²⁺ added is low (Fig. 2, A and C); partial rescue occurs when six or more Fe²⁺ are added/active site (Fig. 2, A and C), likely due to saturation of competing, weaker Fe²⁺ sites that are inactive when Asp¹²⁷ is present. Rates of DFP formation in D127A ferritin are 45% of WT (Fig. 2C) when an equivalent of six or more Fe²⁺/active site were added. When changes in A_{350 nm} were analyzed at six or more Fe²⁺/active site, the initial rates were only 20% of the WT (Fig. 2, A and C). Such results emphasize contributions of Asp¹²⁷ carboxylates around the Fe²⁺ channel exits (Fig. 3), to directing Fe²⁺ substrate to each of the three catalytic sites in the middle of the helix bundles that form the ion channels. With the addition of six or more Fe²⁺/active site, the inhibitory effect of D127A is partly reversed; DFP formation is restored and by 10 s after adding Fe²⁺, long after DFP decay, the mineral growth (A_{350 nm}) reaches WT values (Fig. 2B). Such results suggest that nonspecific Fe²⁺ binding sites, compete with the active sites for binding in the absence of Asp¹²⁷, but are saturated when enough Fe²⁺ is added. Apparently, Asp¹²⁷ is too far away to alter reactions after oxidation and during mineral growth.

FIGURE 3.

Distribution of metal ion in ferritin protein cages to three active sites depends on the structure of the ion channels at the 3-fold cage axes. Three Mg²⁺ ions (hexahydrated) are observed bound to the Asp¹²⁷ carboxylate and Glu¹³⁰ carbonyl from each of the three frog-M ferritin near the ion channel exits into the ferritin cavity; the view is from inside the cavity into the ion channel toward the external pore opening in the exterior cage surface. In addition, in the center of the ion channel are three of the four hexahydrated Mg²⁺ in the line that stretches from the outside of the cage to the cavity entrance (10). The figure was made from Protein Data Bank ID code 3KA3; Mg, green spheres; coordinated water, red spheres; fragment of backbone of helix 3 of subunit forming the iron channel subunit 1, green; subunit 2, magenta; subunit 3, azure.

To show that the effects of altering pore residues Glu¹³⁰ and Asp¹²⁷ were selective for Fe²⁺ catalysis, we studied rates of mineral dissolution (NADH/FMN reduction + bipyridyl chelation of Fe²⁺ ions after dissolution and exit from the cage), because other conserved Fe²⁺ ion channel residues, e.g. Arg⁷², Leu¹¹⁰, Asp¹²², and Leu¹³⁴, near the outside end of the channel, altered mineral reduction (20, 21). Once the hydrated ferric oxide mineral was formed in WT, D127A and E130A ferritins, the caged mineral was dissolved, and Fe²⁺ released at the same rates (supplemental Fig. S2) among all of the proteins. Such results emphasize the selectivity of Glu¹³⁰ and Asp¹²⁷ contributions to Fe²⁺ active site access and the reaction with dioxygen.

Conserved Residues in the Fe³⁺ Nucleation Channel of Ferritin, Identified by NMR Spectroscopy, Affect Ferritin Function

Residues in the postoxidation channels, identified by NMR in the presence and absence of Fe³⁺ (11), influence Fe²⁺/O₂ reaction rates at the active sites (Fig. 4, A and B). 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 Fe³⁺ paramagnetic effects indicated that the residues were ∼5 Å away from Fe²⁺ oxidized in situ (11). Note that the apparent absence of carboxylate residues in the Fe³⁺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 ¹³C chemical shifts for Glu and Asp, the narrow dispersion between Asn/Asp or Gln/Glu, and the effects of α-helix bundle structure (low chemical shift dispersion). Thus, carboxylate ligands in the channels may simply remain among those residues for which assignments remain to be made. Alternatively, when Fe³⁺O moves through the channels, charge balance could be maintained, e.g. by proton loss from water coordinated to Fe³⁺ in the Fe³⁺O species.

FIGURE 4.

Ala²⁶ near the active site destabilizes the DFP catalytic intermediates in ferritin. Single turnovers (48 Fe²⁺/nanocage; 2 Fe²⁺/oxidoreducatse site) by wild-type frog-M ferritin (WT) and site-directed amino acid substitutions variants were monitored, ΔA_{650 nm} (DFP) and ΔA_{350 nm} (Fe³⁺O, which includes DFP, diferric oxo, Fe³⁺O tetramers, and mineral), after rapidly (<5 ms) mixing equal volumes ferritin cage protein with ferrous sulfate solutions, described in Fig. 2 and under “Experimental Procedures.” The results are averages from two to three preparations of protein analyzed four to six times each, and the error is the S.D. Significantly different from WT, *, p < 0.0001 or **, p < 0.006 (t test). A, initial rates of DFP formation (ΔA_{650 nm}/s). B, initial rates of DFP decay. C, frog-M ferritin nanocage structure viewed from the outside, with the 4-fold axis near the center (Protein Data Bank ID code 1MFR). The residues near the 4-fold pores are shown in red or blue spheres. D, part of a single subunit of the frog-M ferritin showing the ferroxidase center and the position of residue Ala²⁶ nearby.

Residue Ala²⁶ was identified as a part of the iron nucleation channel in the ferritin solution NMR study with and without Fe³⁺O species (11). In addition, we used covariation analysis, with the methods in Ref. 39 and analyzing ∼350 sequences of ferritins that were either catalytically active (His) or inactive (Leu). Residue 26 is part of a covariation network of 15 residues distributed among helices 1, 2, 3, and 4, coincident with catalytic activity. Ser²⁶ occurs in wild-type, catalytically inactive, frog L ferritin, emphasizing the functional importance of Ala²⁶ in ferroxidation and iron nucleation. For this reason and because of increased hydrophilicity and similar size of serine to alanine, the A26S substitution was studied in the well characterized frog-M, an H-type ferritin.

The position and orientation of Ala²⁶ in the 4-helix bundle close to the active site at the entry of the nucleation channel suggest a role in catalysis and product release (Fig. 4D). Moreover, the A26S substitution had no effect on Fe²⁺ oxidation (DFP formation, A_{650 nm}) in the first catalytic cycle. However, when more Fe²⁺ was added, inhibition of oxidation was significant (p < 0.0001) (Fig. 4A). In contrast to oxidation, DFP decay was sensitive to inhibition (p < 0.0001) even in the first catalytic turnover (Fig. 4B). Such observations show that Ala²⁶ participates in product (Fe³⁺-O-Fe³⁺) release, which explains the stabilization of the DFP intermediate in A26S, even during the first catalytic cycle (Fig. 4B), and indicate slow movement of the Fe³⁺O product after the first turnover. The impact of abnormal product decay in A26S is further emphasized by examining the less specific A_{350 nm} (Fig. 5). Fe³⁺O formation was inhibited 25% during the first catalytic turnover (supplemental Table S1), contrasting with DFP formation, which was only affected after the second addition of substrate (96 Fe²⁺/cage). The A_{350 nm} was always lower in A26S than WT (Fig. 5) and even after 1 h had not reached the levels in WT protein, suggesting different properties of the Fe³⁺O nuclei. Thus, in addition to a role for Ala²⁶ in the rates of product release from the Fe²⁺/O₂ oxidoreductase center, the change in the entry to the nucleation channel changes the spectral properties of the growing mineral nuclei (11), possibly by altering hydration of the hydroxo bridges.

FIGURE 5.

Ala²⁶, a residue near the entry of the Fe³⁺ nucleation channel, influences properties of growing ferritin mineral nuclei. Fe²⁺ was added in aliquot of 48 Fe²⁺/nanocage, under the conditions described in Fig. 2 and under “Experimental Procedures.” The absorbance between 300 and 450 nm (Fig. 2D), which increases during and after DFP decay (>0.1 s), is a combination of DFP, diferric oxo, and other ferric oxo multimers. Many studies analyze ferritin activity/mineralization at 305 or 320 nm (19, 20, 27, 28); we use A_{350 nm} to avoid contributions from protein side chains. When DFP dominates the Fe³⁺O spectrum, Ala²⁶ and WT are the same; only after DFP decays, when diferric oxo/hydroxo and larger multinuclear species begin to form, is the A_{350 nm} absorbance difference detectable. The results are averages from two to three preparations of protein analyzed four to six times each, and the error is the S.D. Significantly different from WT, *, p < 0.0001 (Student's t test).

Thr¹⁴⁹ in helix 4, and nearby Val⁴² in the loop connecting helices 1 and 2 of a neighboring subunit, are 15–20 Å from the active site residues Asp¹⁴⁰ or Gln¹³⁷ and near the Fe³⁺O channel exits into the cavity, around the 4-fold axes (Figs. 1B and 4C). Note that in the NMR experiment (11), Thr¹⁴⁹ and Val⁴² were not affected by Fe³⁺ oxidized in situ until 144 Fe²⁺ were added (6 Fe²⁺/active site) with additional broadening of NMR signal when 192 Fe²⁺ were added (8 Fe²⁺/active site). Cysteine was chosen as the substitution for threonine because a Fe³⁺-O interaction, if it existed, would be changed by the substitution of sulfur for oxygen. Glycine was chosen as the substitution for valine to change the hydrophobicity in the loop around Val⁴². Compared with WT ferritin, rates of Fe²⁺ oxidation, monitored as ΔA_{650 nm} or ΔA_{350 nm} decreased significantly (p < 0.001) in V42G and T149C ferritins at all of the catalytic cycles analyzed (cycles 1–4), (Fig. 4A and supplemental Table S2); DFP stability also decreased compared with WT (p < 0.0001 for V42G and p < 0.006 for T149C) (Fig. 4B). In addition, in V42G the development of the broad absorbance band for Fe³⁺O (ΔA_{350 nm}/s) lagged behind WT after mixing (supplemental Table S2). The threonine/cysteine substitution and valine/glycine substitution likely cause sufficient conformational changes throughout the channel from the active site to the channel exits to alter Fe³⁺O passage through the channel.

None of the amino acid substitutions studied, A26S, T149C, and V42G, like D127A and E130A, had any effect on mineral dissolution after adding reductant (FMN/NADH) with bipyridyl as an Fe²⁺ reporter. Such results indicate the separation of residue-specific protein-Fe interactions during iron entry and exit.

DISCUSSION

Ferritin ferric oxo minerals are built up from the diferric oxo products of catalysis in a multistep process that depends in part on the protein cage itself (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 (∼20 Å) Fe³⁺O nucleation channels (Fig. 1, A–C). However, negatively charged residues in the ion entry channels form an electrostatic environment favorable to moving Fe²⁺ ions to active sites (2–4). The carboxylate residues appear to have additional functions. For example, the three Asp¹²⁷ residues from each subunit appear to also distribute Fe²⁺ substrate to each of the three active sites in the subunits that form the entry channels around the 3-fold axes (Fig. 3). Moreover, the three Glu¹³⁰ residues, which form a constriction in the iron entry channel, may control not only metal ion access but also metal ion selectivity, blocking entry of large, divalent cations. Together, the structural results identified new potential functions or new amino acid-iron interactions with potential contributions to ferritin protein function (10, 11), that we studied here by making amino acid substitutions at conserved positions (Ala²⁶, Val⁴², Asp¹²⁷, Glu¹³⁰, and Thr¹⁴⁹) and by covariation between catalytically active (H) and inactive (L) ferritins (39).

Substitutions of Ala²⁶, Val⁴², Asp¹²⁷, Glu¹³⁰, and Thr¹⁴⁹ all changed ferritin function during the first four catalytic cycles when mineral nuclei are still in the protein cage and before mineral growth; in the case of Glu¹³⁰ and Asp¹²⁷, the effects were different (Fig. 2) as predicted using the new structural data (10). The functional effects fell into four categories: (i) Glu¹³⁰, where all three carboxylates are close together and the channel is constricted, controls Fe²⁺ access to the active sites (DFP formation); (ii) three proximal carboxylate residues, Asp¹²⁷, one from each subunit, regulate Fe²⁺ distribution to three active sites; (iii) Ala²⁶ near the active site at the entry to the Fe³⁺ nucleation channel, regulates release of product, diferric oxo, and, thus, formation of the Fe³⁺O tetramer between two Fe³⁺O dimers; (iv) hydrophilic and hydrophobic residues in and around the Fe³⁺ nucleation channel influence Fe²⁺ oxidation and turnover. The specificity of the residues for mineral formation was shown by the absence of any effects on mineral dissolution in the ferritins with amino acid substitutions at Ala²⁶, Val⁴², Asp¹²⁷, Glu¹³⁰, or Thr¹⁴⁹.

Three Glu¹³⁰ carboxylates form a constriction in the middle of the eight Fe²⁺ entry channels, one from each subunit that creates each channel and controls Fe²⁺ access to the active sites for DFP formation. Substitution of the negatively charged glutamate carboxylates with neutral alanine created a protein with no significant DFP formation (Fig. 2, A and C), as if the catalytic sites were inactive. E130A behaved like L ferritin, which has no catalytic sites and where mineral formation is attributed to chelating effects of conserved, clusters of carboxylate ligands on the inner surface of the protein (27, 28, 40). The diameter of the Fe²⁺ channels at the Glu¹³⁰ constriction is ∼5.4 Å. Fe²⁺ hexahydrate has a diameter of ∼4.5 Å. Multiple small ions such as Mg²⁺ line up in the channels at and below the Glu¹³⁰ constriction whereas a single, larger Co²⁺ ion is “stuck” at Glu¹³⁰ in protein cocrystals (10). In addition to attracting the ions into the channel by the high concentration of negative charge, the close fit of Fe²⁺ at the Glu¹³⁰ channel constriction suggests that Glu¹³⁰ also exerts some metal ion selectivity.

The Asp¹²⁷ residues in the Fe²⁺ entry channels, one from each ferritin subunit that form the channels, surround the channel exits into the mineral cavity (Figs. 1B and 3). Asp¹²⁷ influences Fe²⁺ access to the three active sites because substitution with alanine prevented formation of the DFP intermediate, when only small numbers of Fe²⁺ were added. The amino acid substitutions D127A and E130A had no detectable effect on Fe release (supplemental Fig. S2).

However, when more Fe²⁺ was added (6 Fe²⁺/active site) DFP was detected (Fig. 2, A and C). The rescue of catalytic activity in D127A, by adding more Fe²⁺ substrate, contrasts with E130A and has several possible explanations, although in each case the electrostatic potential in the channels decreases with the substitution of alanine for each of the carboxylates. First, it may take at least 6 Fe²⁺ to eliminate competition from the carboxylate chelating residues on the inner surface (40). Second, with such a large number of Fe²⁺ ions, the dependence on subunit-subunit cooperativity around the channel may be overcome (Fe²⁺ binding at the active site, monitored by MCD/CD had a Hill coefficient of 3 (19) that could only be explained by subunit-subunit interactions). Third, in a recent structure of Mg²⁺-ferritin cocrystal, metal ions were bound to the three Asp¹²⁷ carboxylates around each channel exit on the inner surface of the cage (Fig. 3), pointing toward the three active sites nearest each channel (10). A reasonable model that fits the available data is that Fe²⁺ after passing the Glu¹³⁰ “filter” is directed by Asp¹²⁷ to each of the three active sites in the subunits that make up each pore. Binding of Fe²⁺ at the active sites, thus, depends on: (i) the orientation of Fe²⁺ by Asp¹²⁷ toward each of the three active sites in the subunits that form each pore, indicating the functional significance of cage assembly geometry (Fig. 3); (ii) conformational changes that enhance the coordinated filling of three active sites around each entry pore; and (iii) tight binding of Fe²⁺ at the active sites that outcompetes binding to clusters of carboxylate residues on the inner surface of the cavity (40).

Ala²⁶, close to the active site at the beginning of the Fe³⁺ nucleation channel (Fig. 4D), enhances reaction/product release because rates of oxidation and decay were decreased in A26S ferritin cages, except for the first catalytic cycle when DFP formation was normal but decay was still slower. The importance of conserved Ala²⁶ was indicated by proximity to the diferric oxo dimer (<5 Å) in the NMR experiment when 48 Fe²⁺/cage were added (2 Fe²⁺/active site) (11), as well as covariation analysis (39) of catalytically active (H-type) and inactive (L-type) ferritin subunits. In addition, when the A_{350 nm} is measured after the DFP intermediate has been converted to the diferric oxo precursor (<1 s), contributions of A26S to slower reactions such as mineral growth itself are observed (Fig. 5). Ala²⁶ may also influence Fe³⁺O tetramer formation in the channels, observed by magnetic susceptibility (11), because the full effect of A26S is not observed until 96 Fe²⁺ ions (4 Fe³⁺O/channel) are present (data not shown). Possibly the steric resistance or potential hydrogen bond interaction of the serine, which replaces alanine in the A26S ferritin, not only inhibits diferric oxo product release from the catalytic sites but also inhibits the reaction of two Fe³⁺O dimers to form Fe³⁺oxo tetramer in the nucleation channel, possibly by interfering with the correct orientation of Fe³⁺O dimers, after they leave the active sites.

Val⁴² and Thr¹⁴⁹, situated near each other and near the exits of the Fe³⁺ nucleation channels, at the cavity entrance, are ∼25 Å from Fe²⁺ entry channel exits into the cavity. They most likely influence Fe²⁺/O₂ oxidoreductase catalysis and Fe³⁺O mineral nucleation through effects on subunit conformations along the long axes of each subunit bundle and at subunit-subunit interfaces, because the Val⁴² close to [Fe³⁺O]_n is in another subunit adjacent to the one with Thr¹⁴⁹ (Fig. 1B). A role for uncharged residues in moving ions through channel proteins has recently been demonstrated in a Kv 2.1 channel (41). Because the Val⁴² and Thr¹⁴⁹ are in different subunits (11), interactions between pairs of subunits may be key in moving Fe³⁺O through ferritin nucleation channels. Such an idea is supported by the inhibitory effects of subunit cross-links between pairs of ferritin subunits on Fe³⁺O/mineral formation (42).

An active role of H ferritin subunits in ferritin mineral nucleation and ordered iron mineral growth (11) provides an explanation for the physiologically significant and different combinations of catalytically active (H) and inactive (L) ferritin subunits in animal ferritins and the coincident variations in iron mineral order (43, 44). Ordered/more crystalline ferritin iron minerals will dissolve more slowly (release iron more slowly) than disordered ferritin minerals, which explains why increased L ferritin subunits increased cell proliferation: iron was released faster for critical iron proteins such as ribonucleotide reductase, which is rate-limiting for DNA synthesis. Decreased L subunits deceased cell proliferation (43). Moreover, heart ferritin, which functions in highly oxygenated tissue, has more H subunits, more antioxidant activity, and more crystalline (slow iron release) mineral (44). However, liver ferritin, which must release iron for other tissues, has a large number of L subunits and less ordered mineral. The disordered growth of ferritin iron minerals with large numbers of L subunits such as that from liver, is attributable to protein-independent, inorganic reactions (45).

Conserved amino acids, both hydrophilic and hydrophobic, along the entire length of each ferritin four α-helix subunit bundle contribute to function in ferritin protein nanocages, based on the new data in this report, and contrasting with earlier studies that have focused on the functions of localized clusters of conserved amino acids related, e.g. to catalysis or mineralization (5, 38). Previously, conserved amino acids in ferritin that were not in localized clusters such as the oxidoreductase sites were assigned roles in folding and stabilizing the unique ferritin cage structure, with the exception of those around the 3-fold channels shown to regulate Fe²⁺ release from the mineral and the cage (5) or those theoretically shown to create local electrostatic gradients for Fe²⁺ entry through the ion channels (32). The amino acids studied here, which all have functional effects on iron oxidation and mineralization, are distributed over a distance of ∼50 Å. From the Fe²⁺ entry channels, formed by three subunits at the 3-fold cage axes, to the nucleation channel exits, at the other end of the subunit 4-helix bundles around the 4-fold cage axes, where mineral nuclei emerge, conserved, ferritin subunit amino acid residues influence the process of iron biomineralization. How these studies relate to the ferritins with different active sites and/or cage locations as in bacteria and archaea is not known (46, 47). However, the results here suggest that the unusually high cage symmetry in ferritins, especially in the more highly evolved eukaryotic ferritins, contributes to function emphasizing the role of cage geometry itself in function.