Crystal Structure of Plant Ferritin Reveals a Novel Metal Binding Site That Functions as a Transit Site for Metal Transfer in Ferritin*

Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-Å resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu173 and carbonyl oxygen of Thr168, which correspond, respectively, to Glu140 and Thr135 of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

Ferritin is a class of iron storage proteins distributed ubiquitously in plants, mammals, and bacteria. This protein class plays an important role in storage and detoxification of excess iron in a living cell. Although the primary sequences of ferritins vary, their three-dimensional structures of 24 structurally equivalent subunits assembled into a cage-like oligomer and related by 4-, 3-, and 2-fold symmetry axes (1-3) are highly conserved. Each subunit has four helices (A-D), each of which is composed of ϳ30 amino acid residues, along with a fifth short helix (E) having 10 residues or more, resulting in the formation of a "four-helix bundle." In general, the outer shell diameters of the spherical oligomeric ferritin composed of 24 subunits are ϳ120 Å, whereas the inner cavity diameters are ϳ80 Å. Up to 4500 iron atoms are deposited in the inner cavity of ferritin (1)(2)(3). To date, three major metal binding sites have been identified in ferritin. These exhibit some common aspects related to the iron storage mechanism: (i) they exist around the 3-fold symmetry channel, (ii) constitute a ferroxidase center in the four-helix bundle, and (iii) constitute a nucleation site facing the inner cavity of ferritin (4 -7). It has been suggested that iron atoms pass through the hydrophilic channels that traverse the ferritin shell around the 3-fold symmetry axes of oligomeric ferritin (8 -11). This funnel-like channel is surrounded by hydrophilic residues, such as aspartic acid and glutamic acid, which have been demonstrated to serve as ligands for metal ions, such as ferrous, calcium, or zinc ions (4,5). With respect to the ferroxidase center and nucleation center, two distinct subunits, heavy (H) 2 and light (L) chains, are seen in mammalian ferritin (12)(13)(14). The H chain possesses the ferroxidase site (15,16), which is positioned inside the 4-helix bundle of each subunit and is responsible for oxidation of ferrous iron atoms to produce the -oxo-bridged Fe(III) species. The ferroxidase site is composed of six residues: Glu 27 , Tyr 34 , Glu 62 , His 65 , Glu 107 , and Gln 141 , which coordinate with metal ions directly or indirectly. Crystallographic analyses of ferritin in vertebrates (4,5,17), insects (18), and bacteria (7,19) revealed an extremely high degree of similarity among the structures of these ferroxidase centers. On the other hand, the L-type ferritin lacks the ferroxidase center, but possesses the nucleation site. The site is composed of three or four glutamate residues facing the inner cavity of the ferritin shell, and facilitates ferrihydrite nucleation (20,21). In mammals, the H and L chains form a heteropolymer and cooperatively share the role of iron storage in vivo (14).
Recently, multiple copies of the ferritin gene were identified from various plant species (22)(23)(24)(25)(26)(27)(28)(29)(30). It has been suggested that their expression was differentially regulated via various environmental stresses or metal overload (25,(31)(32)(33)(34)(35)(36), and are sometimes subjected to different types of processing (26,30). In contrast to the case of the H and L chains of mammalian ferritin, all of the identified plant ferritin subunits have similar primary sequences, especially in their deduced ferroxidase center and nucleation site. Although the primary structures of plant ferritins are basically well conserved with those of mammalian ferritins ( Fig. 1), plant ferritins show two further differences: first, plant ferritin possesses the N-terminal extension regions, which can be divided into two parts based on their functions (37). The upstream one is called a "transit peptide" responsible for transportation of the polypeptide chain to plastid, whereas the downstream one is called an "extension peptide" (EP) with an unknown function (38). Several reports have suggested that EP helps to stabilize the oligomeric protein shell (39 -41). Second, plant ferritins can incorporate and store iron without the differentiation of subunits. As mentioned above, all plant ferritins possess both a ferroxidase center and a nucleation site. These observations led us to hypothesize that a crystallographic analysis of plant ferritin would offer new insight into the pathway of metal ion transfer from the iron entry channel via the ferroxidase center to the nucleation site and inner cavity of the ferritin shell. With respect to the three-dimensional structures of plant ferritin, Lobréaux et al. (42) presented the deduced structure of ferritin from pea (Pisum sativum) using a computer simulation. They predicted that the main chain structure of plant ferritin can be superimposed onto human H chain ferritin up to 1 Å of the root mean square distance (42). Thus, it can be considered that its overall structure, including structure of the 3-fold symmetry channel, ferroxidase center, and nucleation site, is highly conserved. However, the actual metal binding sites and precise orientation of side chains of metal ligands have remained unclear. On the other hand, we have attempted to crystallize plant ferritin from soybean (SFER1) to obtain the real structure of these active sites; however, the diffraction data from the crystal were not sufficient to provide high resolution structural analysis (43).
In this study, to address the question of the metal ion pathway in the ferritin oligomer, we have crystallized a recombinant plant ferritin subunit from soybean (SFER4). The high resolution x-ray crystallographic analysis of this soybean ferritin subunit has revealed a novel metal binding site between the 3-fold symmetry channel and ferroxidase center. The deletion of this site resulted in a delay of the iron oxidation/nucleation process, suggesting that this site functions as a transit site for metal translocation in ferritin. Based on these results, coupled with the extremely high conservation of this site, we propose that this site is part of the metal ion pathway widely distributed in ferritin from various species.

EXPERIMENTAL PROCEDURES
cDNA Cloning and Protein Preparation-Cloning of the soybean ferritin SFER4 cDNA (GenBank TM number AB062756) was performed as described previously (44). The SFER4 cDNA fragment containing NcoI and BamHI restriction sites was cloned into pET21d (Novagen, San Diego, CA) through restriction sites NcoI and BamHI using a PCRbased method. The E173A SFER4 variant was obtained by oligonucleotide site-directed mutagenesis using pET_SFER4 as a template. The DNA sequence of the resulting plasmid pET_ SFER4 and pET_SFER4-E173A was verified by automated DNA sequencing (ABI PRISM 3100, Applied Biosystems, Foster City, CA). The constructs were then introduced into Escherichia coli strain BL21(DE3). The positive transformants of each construct were grown at 37°C on LB medium supplemented with 50 mg/liter of carbenicillin, and protein expression was induced with 100 M isopropyl ␤-D-1-thiogalactopyranoside when the cell density reached an A 600 of 0.6. Eventually, the cells were harvested by centrifugation after 3 h of induction and resuspended in Buffer A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.15 M NaCl, 0.1 mM p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 0.2 M pepstatin, 0.5 g/liter leupeptin) to a concentration of 40 g (fresh weight bacteria)/liter, followed by disruption by sonication. The supernatant of the resulting crude extract was collected by centrifugation and fractionated by 50 -60% saturation of ammonium sulfate. The pellet was resuspended in Buffer B (20 mM Tris-HCl, pH 7.8, 1 mM EDTA) and dialyzed against the same buffer. The protein solution was applied to an ion-exchange column (Q-Sepharose 26/10, GE Healthcare, Piscataway, NJ), followed by elution with 0 -0.5 M NaCl gradient. Finally, the protein solution was concentrated and purified on a gel filtration column (Superdex 200pg 16/60, GE Healthcare), equilibrated with Buffer C (10 mM Tris-HCl, 0.15 M NaCl). The resulting SFER4 and SFER4-E173A were electrophoretically pure (data not shown). The protein concentrations were estimated by A 280 , using molar absorptivity (⑀ M ) of 17,420 cm Ϫ1 M Ϫ1 , calculated from amino acid composition (45).
Crystallization, Data Collection, and Processing-Initial crystals of native SFER4 were obtained after about 2 weeks (Wizard I; B-Bridge, Mountain View, CA) using the hanging drop vapor diffusion method. The optimized crystallization drops were prepared by mixing equal volumes of mother liquid composed of 15% PEG 1000, 0.2 M calcium acetate, 0.1 M imidazole, pH 7.8, and protein solution containing 10 mg/ml of recombinant SFER4. Orthorhombic crystals of SFER4 (space group P2 1 2 1 2; a ϭ 222.610, b ϭ 220.886, c ϭ 122.452 Å; V M ϭ 2.61 Å 3 /Da for 24 monomers per asymmetric unit) of ϳ0.5 ϫ 0.2 ϫ 0.2 mm appeared within 3 weeks at 20°C. The crystallization condition of the E173A variant was the same as that of the native crystal. Diffraction data of the native and E173A crystals were collected to 1.8-and 1.9-Å resolution at 77 K at the SPring-8 beamline 44XU after flash cooling with 30% of 2-methyl-2,4-pentanediol as a cryoprotectant. Data were processed, merged, and scaled with HKL-2000 (HKL Research, Charlottesville, VA) (46). Data processing statistics are shown in Table 1.
Structure Determination and Refinement-The structure of native SFER4 was determined by molecular replacement using the Molrep program (47) in CCP4i suit 1.4.4 (48) using the structure of bullfrog M ferritin (PDB code 1MFR) (17) as a search model. All water molecules and calcium atoms were removed from the search model prior to molecular replacement. Refinement was performed with 24-fold non-crystallographic symmetry using the REFMAC5 program (49) in CCP4i suit 1.4.4 (48). The structure was visualized and rebuilt using COOT 0.5.2 (50) and further modified on -weighted (2͉F o ͉Ϫ͉F c ͉) and (͉F o ͉ Ϫ ͉F c ͉) electron density maps. Water molecules were added using the COOT autofind function with a lower cutoff of 3 in the ͉F o ͉ Ϫ ͉F c ͉ map. Metal ions were positioned into higher (͉F o ͉ Ϫ ͉F c ͉) residual densities and according to shorter bond distances with neighboring water molecules or other protein ligands. A Bijvoet difference Fourier map calculated from the final protein model was used to identify the metal binding sites. The final refinement was carried out using PHENIX software (51). The structure of the E173A variant was determined using the final model of the native SFER4, and the refinement strategy was the same as that for the native. Figs. 2-6 and 8 were produced by PyMOL (DeLano Scientific, San Carlos, CA).
Iron Uptake-Apoferritins of native SFER4 and the E173A variant were obtained using the methods described by Chasteen and Theil (52) with some modifications. Purified ferritins were dialyzed against 50 mM HEPES-Na buffer, pH 7.0, containing 1% thioglycolic acid, followed by successive changes of HEPES-Na buffer with (0.1%) or without thioglycolic acid. The protein was then dialyzed against Tris-HCl buffer containing 13 g/liter of Chelex 100 (Bio-Rad Laboratories) and 0.15 M NaCl, and finally dialyzed against 10 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. The concentration of purified proteins was determined using the absorbance at 280 nm.
Reactions examining iron uptake by native and the E173A variant were performed in 10 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl with a Fe 2ϩ /ferritin molar ratio of 100:1, 40:1, and 20:1 (100, 40, and 20 M ferrous ammonium sulfate and 1 M ferritin) at 25°C. Iron oxidation/nucleation by each type of ferritin was monitored by measuring the absorbance at 310 nm, the generation of -oxo-bridged Fe(III) species (16, 54) using a UV spectrophotometer (UV160, Shimadzu, Kyoto, Japan), and the resulting absorbance every 10 s for kinetic analysis. After each Fe(II) addition, several spectra were taken until the absorbance increased no further. The molar absorptivity of 4570 (cm Ϫ1 M Ϫ1 ), which was calculated for -oxo bridged Fe(III) in a iron excess condition on horse spleen ferritin by Yang et al. (21), was used to calculate the kinetic parameters. The kinetics data were analyzed with Excel 2007 software (Microsoft, Redmond, WA). The initial rates of iron oxidation were obtained from the linear A 1 term of a third-order polynomial fitted to the experimental data (21,55). The kinetic parameters were calculated by Lineweaver-Burk plots.
Data Deposition-Coordinates of plant ferritin SFER4 and its variant E173A have been deposited at the RCSB Protein Data Bank with accession codes 3A68 and 3A9Q, respectively.

Model Quality and Overall Structure of Plant Ferritin
(SFER4) Subunit-The overall coordinate error of the final model, estimated from Luzzati plots, was 0.18 Å. Root mean square deviations of bond lengths and angles from ideality as calculated by the PHENIX program are listed in Table 1. These values are well within accepted limits, indicating that all of the structures have tight stereochemical constraints. A Ramachandran plot of the final structure showed that 98.43 and 1.57% of all residues were in the most favored and allowed regions, respectively. Refinement statistics are shown in Table 1.
The final model contained the whole oligomeric protein shell composed of 24 subunits with 4642 residues, 4570 waters, 136 Ca 2ϩ ions, and 18 acetate ions. Like the structures of ferritins from vertebrates and bacteria, the structure of plant ferritin, SFER4, has a cage-like hollow shell composed of 24 subunits, which are related by 4-, 3-, and 2-fold symmetry (Fig. 2). There are four 3-fold axes and three 4-fold axes traversing the shell of the assembled 24-mer, resulting in eight 3-fold channels and six 4-fold channels. In the present model for SFER4, each subunit excludes the N-terminal 11 to 13 residues and the C-terminal 5 residues, because they have no electron density. Exceptionally, chain E excludes the first 17 residues. The N-terminal EP (from the N terminus to Thr 30 in this model) forms a loop and a short helix that lies on the C-helix in a neighboring 3-fold symmetry mate (counterclockwise) (Fig. 2a), although the side chains of amino acids in the region are relatively disordered and have high temperature factors (Fig. 2). The interactions between the EP and the conserved helical region of an adjacent chain were formed mainly by apolar contacts, except for an ion pair formed between Glu 20 and Lys 146 . Table 2 shows an example of side chains and residues related to the interactions between the EP of chain G and the helical region of chain O.  Fig. 3a.
Calcium ions on the outer surface of the ferritin shell had seven coordinated waters, two of which were hydrogen bonded to the carboxylate oxygen atoms of Asp 43 and Glu 46 , and one of which was hydrogen bonded to the acetate anion located between the side chains of Arg 38 and Lys 108 . The acetate anions were also usually hydrogen bonded to the main chain nitrogen of Ser 35 . These outer calcium ions were not related to the intermolecular contact, in contrast to vertebrate ferritin (4 -7). Intermolecular contacts were formed mainly by hydrogen bonds or salt bridges between the main chains or side chains of residues positioned around the end of the C-helix (Ser 152 , Thr 155 , Lys 156 , and Asn 157 ) and the side chains of Lys 40 , Asp 43 , Glu 44 , and Ser 47 positioned around the start of the A-helix.
3-Fold Symmetry Axis-The hydrophilic channel penetrating along the 3-fold symmetry axis is considered the iron entry channel to the inner cavity of the ferritin shell (8 -11). The channel of soybean ferritin, SFER4, had similar coordination with mammalian ferritins. As for the 3-fold channel of SFER4, the channel is lined with Asp 164 , Glu 167 , and Thr 168 of three symmetrically related subunits (Fig. 3a, residues shown in yellow), corresponding to Asp 131 , Glu 134 , and Thr 135 of the human H chain. In the crystal structure of SFER4, two electronic densities of calcium ions are found around the 3-fold symmetry axis in the bottleneck of the funnel-like channel leading to the cavity. One calcium ion positioned deeper inside the 3-fold symmetry channel is coordinated by OEs in the side chains of two symmetrically related Glu 167 , one OD of Asp 164 and three waters (Fig. 3b). On the other hand, the other calcium ion, positioned outside the channel, is coordinated by an OE of Glu 167 and three to four waters. The structure around the 3-fold channel composed of chain L, M, and T was unambiguously determined, whereas the outer calcium ions and its ligands in other channels were sometimes disordered. In the channel composed of chains L, M, and T, the inner calcium ion was coordinated by two ODs of Asp 164 in chains L and T, two OEs of Glu 167 in chains L and M, and three waters. On the other hand, the outer calcium is coordinated by two OEs of Glu 167 of chains L and M plus four coordinated waters (Fig. 3b).
4-Fold Symmetry Axis-The E-helices of vertebrate ferritins lie roughly parallel with the 4-fold axis, making a hydrophobic channel traverse the shell. In the homo 24-mer of human H and L chains, this channel is formed by three layers. In the case of the H chain, the inner chain is lined with four histidines, whereas the middle and outer chains are each lined with four leucines. On the other hand, the channel in the human L chain is lined with 12 leucines, making it highly hydrophobic. In contrast, this channel in plant ferritin SFER4 is formed by histidine side chains (Fig. 4a) composed of two layers (Fig. 4b), because the side chain of Met 203 , which corresponds to the His 173 or Leu 169 positions in the inner layer of the human H chain or L chain, does not form the inner layer of the 4-fold channel. The two layers of SFER4 are composed of two histidines (His 195 and His 199 ) and their 4-fold symmetry related mates (Fig. 4b). Thus, the channel is lined by eight histidines. The NDs in the side chains of outer histidines (His 195 ) are directed to the outer surface of the 4-fold channel, whereas the NEs of His 195 electrostatically interact with the NDs of the inner histidine (His 199 ) in a symmetrically related subunit (Fig. 4b). Consequently, the NEs of inner histidines are directed to the inner cavity of the protein shell. The distance between the two nitrogen atoms in histidine side chains is ϳ2.9 Å. No electronic peaks of metal ion are seen around the channel, in contrast to other kinds of ferritins (17,56).
Deduced Ferroxidase Center and Nucleation Center-In general, the ferroxidase center, located inside the four-helix bundle of each subunit, contains two metal binding sites, named A and B. It was suggested that the occupancy of site A is higher than that of site B (4). Concerning the SFER4, the ferroxidase center is composed of Glu 56 , Tyr 63 , Glu 91 , His 94 , Glu 140 , and Gln 174 , corresponding to Glu 27 , Tyr 34 , Glu 62 , His 65 , Glu 107 , and Gln 141 , respectively, of the human H chain ferritin (Fig. 1). In the native crystal structure of SFER4, metal binding was seen only in site A, although all amino acid residues of the ferroxidase center are conserved and the overall structure of this site is nearly identical. The metal binding site A is coordinated by Glu 56 , Glu 91 , His 94 , and three waters (Fig. 5a), showing that the coordination of calcium ion in this site is nearly identical with metal binding site A of the human H chain.
The putative nucleation center of SFER4 is composed of glutamates facing the inner cavity, Glu 86 , Glu 89 , Glu 90 , and Glu 93 ,  corresponding to Glu 53 , Glu 56 , Glu 57 , and Glu 60 of the human L chain (Fig. 1). In the present model Glu 90 has an alternative conformation, and these four residues tend to be disordered, as seen in crystallographic analysis of other L chains. The electron densities with high intensity (8 to 10 ) are seen in the vicinity of the side chains of Glu 93 and Glu 96 . Some of the peaks exhibited weak anomalous signals; therefore, they were considered calcium ions. In addition to these residues, Asp 71 , Glu 86 , Glu 89 , Glu 90 , Glu 100 , Glu 169 , Glu 173 , Glu 176 , and Glu 183 are also facing the inner cavity, rendering it acidic. Calcium ions are also seen in the vicinity of Glu 183 , Asp 71 , and Lys 179 , all of which are facing the inner cavity.
Novel Metal Binding Site-A novel metal binding site is seen intermediate between the channel and ferroxidase center binding sites 3-fold. The site, which is positioned at the bottom of the 3-fold symmetry channel and facing the inner cavity of the ferritin shell, is coordinated by the OE of Glu 173 , carbonyl oxygen of Thr 168 , and four waters (Fig. 6). The distances from two OEs of Glu 173 and the carboxyl oxygen of Thr 168 are 2.48, 2.53, and 2.32 Å. This calcium ion binding site is seen clearly in all chains of the SFER4 crystal structure. The distance of this calcium ion from the inner calcium ion in the 3-fold symmetry axis is 9.7 Å, whereas that from the ferroxidase site A is 12.4 Å.
Biochemical Property of the E173A Mutant-To assess the function of the Glu 173 residue of SFER4, the E173A mutant, in which Glu 173 was replaced with alanine, was prepared by the E. coli expression system, and ferroxidase/nucleation activity was compared with the native SFER4. The proper oligomeric formation of the E173A mutant was verified by gel filtration (data not shown). UV spectral absorption around 305-330 nm has been traditionally used to monitor the ferroxidase activity of ferritins. When adding ferrous ions to ferritin possessing a ferroxidase center such as the human H chain, the absorbance around 310 nm, which is characteristic of -oxo-bridged Fe(III) dimers (21,57), increases as a result of the Fe(II) oxidation. Fig. 7 shows the time-dependent change in absorbance at 310 nm, when ferrous ions were added to SFER4 and the E173A mutant at molar ratios of 1:20 (20 M ferrous ions was added to 1 M of the SFER4 and E173A) (Fig. 7a) and 1:100 (Fig. 7b). The results show delays in the formation of the -oxo-bridged Fe(III) species in the E173A mutant compared with SFER4 in all conditions tested here at ferritin/iron ratios of 1:20, 1:40, and 1:100, whereas the final absorbencies were nearly identical (Fig. 7). The kinetic parameters were calculated as follows: K m (SFER4) ϭ 31.

DISCUSSION
Since 1991, when the three-dimensional structure of human H-type ferritin was solved (4), many structures of ferritin from various species have been investigated and iron storage mechanisms have been proposed. In the present study, we reported the three-dimensional structural model and novel metal binding site of plant ferritin from soybean. The results showed that the overall structure of plant ferritin, SFER4, was highly conserved among known structures of ferritins. It was proved that part of the plant-specific EP formed a loop and a short helix, although this region was highly disordered. This observation supports the prediction by Lobréaux et al. (42) that this region of pea ferritin would form a short helix named the P-helix, and it would lie on the outer surface of the assembled ferritin shell. They also predicted that the P-helix was folded back to the surface of the original subunit. However, the present model shows that this N-terminal helix is interacting with a neighboring subunit on the shell surface (see Fig. 2 and Table 2). It has been suggested that the N-terminal EP stabilizes the whole oligomeric conformation of plant ferritin (39 -41). The interactions between the EP and the shell surface were formed mainly via apolar contacts. The model demonstrates that seven residues contribute to the interactions, in which four residues (Leu 135 , Leu 139 , Leu 142 , and Lys 146 ) are members of the C-helix (Table 2). Thus, it can be considered that the EP stabilizes the protein shell by maintaining the conformation of the C-helix. It has been suggested that interaction between the conserved Leu 110 in the C-helix and Leu 134 in the D-helix of the bullfrog H chain, which correspond to Leu 147 and Leu 171 of SFER4 (Fig. 1), plays a crucial role in stabilization of the 3-fold channel and that the mutation of these residues enhances the iron release from the ferritin mineral core (58 -60). The EP may contribute to tight packing of the 3-fold pore conformation by stabilizing the C-helix. This hypothesis is based on the faster rate of iron incorporation of plant ferritin with EP deletion than that of the native protein (40). It is possible that the removal of EP accelerates not only the rate of iron incorporation but also that of iron release from the ferritin shell. Concerning the function of the EP, Li et al. (61) recently suggested that the EP has iron binding capacity and facilitates iron incorporation into the ferritin shell. However, there is no metal binding site in the EP of the present   Another unique feature of plant ferritin is its 4-fold symmetry channel. The present model of SFER4 indicates that this narrow channel is lined with eight tightly packed histidine side chains, resulting in formation of a hydrogen bond network from inside to outside the ferritin shell (Fig. 4). In contrast, in mammalian ferritins the channel is lined with 12 leucines or 8 leucines plus 4 histidines (4,5). Takahashi and Kuyucak (62) suggested that the 4-fold channel functions as a proton channel that facilitates hydrogen ion transfer in and out of the human H ferritin shell. As a result of the ferroxidase reaction, the proton concentration in the inner cavity increases, which leads to protonation of NEs of the His 199 side chain facing the inner cavity. The proton can be transferred by sequential protonation/deprotonation of His 199 and His 195 side chains to be exported from the shell. Thus, from a structural point of view, it can be considered that the 4-fold symmetry axis of plant ferritin is more feasible than that of the mammalian ferritin if this channel functions as a pathway for proton transfer. The histidine residues (His 195 and His 199 ) lining the channel are highly conserved among plant ferritins, suggesting that the histidine cluster forming the 4-fold channel is a common feature of plant ferritin species.
The structures of the key sites for the iron storage process of ferritin, the 3-fold iron entry channel, the ferroxidase center, and the nucleation center were proven to be highly conserved among the SFER4 and vertebrate ferritins. In the present model of SFER4, metal binding in the ferroxidase site was seen only in site A (see Fig. 5a). In general, the ferroxidase center is composed of two metal binding sites, A and B (4,5,63). However, x-ray crystallographic analysis of non-heavy atom derivatives indicated that site B was sometimes vacant and that an alkalineearth metal such as calcium or magnesium bound only to site A (56,57). Because many studies concerning ferroxidase activity of plant ferritin have suggested that plant ferritin species have ferroxidase activity comparable with that of mammalian or bacterial ferritin (30,40,64), the ferroxidase site of SFER4 can be considered functional.
Recently, the roles of the carboxyl side chains facing the inner cavity of ferritins have been discussed. It was suggested that the acidic side chains play a critical role in Fe(III) nucleation on the cavity surface (14,65,66). In contrast, Bou-Abdallah et al. (67) demonstrated that the core formation rate was not affected by deletion of the inner glutamate residues (Glu 64 and Glu 67 ), which were considered the nucleation site of human H ferritin. In the present model of SFER4, in addition to the traditional nucleation site Glu 93 /Glu 96 , a calcium ion was seen in the vicinity of Asp 71 and Glu 183 facing the inner cavity. Asp 71 and Glu 183 of SFER4 correspond to Asp 42 and Asp 150 of the human H chain (Fig. 1). It is possible that this site functions as the second nucleation site for Fe(III) core formation and that the traditionally  identified nucleation site is not the sole nucleation site. In fact, these residues, Asp 42 and Asp 150 , were maintained in the experiments performed by Bou-Abdallah (67). Furthermore, Levi et al. (14) suggested that disruption of Asp 42 was required to abolish the iron incorporation activity of human H chain ferritin. These observations also support the hypothesis that the putative nucleation site is not the sole nucleation site. The contribution of each acidic side chain on the cavity surface should be determined.
The pathway from the iron entry channel via the ferroxidase center to the nucleation site has been investigated by x-ray crystallographic analysis and site-directed mutagenesis (4,7,56,68,69). In the present SFER4 model, two calcium ions were seen around the iron entry channel (Fig. 3b), showing that amino acid residues related to the coordination with calcium ions were Asp 164 and Glu 167 , which correspond to Asp 131 and Glu 134 of human H chain, and to Asp 127 and Glu 130 of human L chain, respectively (Fig. 1). Grainer et al. (68) suggested that the alternative conformation of Asp 127 and Glu 130 in the human L chain were important in assisting the metal movement from outside to inside through the channel. Similarly, Toussaint et al. (56) observed metal binding to Cys 130 positioned at the entrance to the 3-fold channel and suggested that the alternative configuration of this residue facilitated metal entry into the channel. As for the movement from the ferroxidase site to the nucleation site, x-ray crystallographic analysis of human H chain has shown that Glu 61 had an alternative conformation and served to transport metal ions from the ferroxidase site to the nucleation site (4,5). Furthermore, Stillman et al. (7) indicated that the alternative conformation of His 46 of ferritin from E. coli (EcFTN), which is a highly conserved amino acid residue among prokaryotic ferritin, enabled the metal to move from the ferroxidase center. In the present model of plant ferritin SFER4, a novel metal binding site, Glu 173 , was discovered between the two well defined metal binding sites: the iron entry channel and the ferroxidase site (Fig. 3a). To date, few studies have described the pathway from the metal binding site in the 3-fold channel to the ferroxidase center. We postulate that this residue, Glu 173 of the SFER4, functions as a transit site from the metal entrance to the ferroxidase site. This assumption was supported by the difference in ferroxidase activity of native SFER4 and the E173A mutant, whose ferroxidase site was not disrupted by the mutation (Fig. 5b). The K m value for iron oxidation of the mutant was increased to 2.2-fold compared with that of native SFER4. The delay in iron oxidation in the E173A mutant was more prominent at low iron concentrations (ferritin/iron ϭ 1:20) (Fig. 7a), whereas the difference in activity diminished as the iron concentration increased (ferritin/iron ϭ 1:100) (Fig. 7b). These results, together with the fact that Glu 173 is not involved in iron oxidation in the ferroxidase site (Fig. 5a), indicate that the side chain of this residue is required for mechanical transfer of the metal ions from the entry channel to the ferroxidase site, especially under ordinary physiological conditions in vivo. For instance, iron atoms enter from the 3-fold channels and interact with Glu 167 and/or Asp 164 and are then translocated from Asp 164 or Thr 168 to Glu 173 . In the crystal structure of native SFER4, calcium ions are seen coordinated by the side chain of Glu 173 and the main chain oxygen of Thr 168 (Fig. 6). In another orientation, the side chain of Glu 173 can interact with the side chains of Gln 174 and His 94 , which are part of the ferroxidase site of SFER4. On the other hand, in the crystal structure of the E173A variant, no metal ion was seen adjacent to Ala 173 . But, a hydrophilic route emerged between Thr 168 (deep in the entry channel) and the ferroxidase site. This hydrophilic route crossing the D-helix is positioned on the main chain oxygen of Thr 168 and Glu 169 , which do not hydrogen bond with the main chain nitrogen of Gly 172 and Ala 173 because of the kink in the D-helix (Tyr 170 ) (Fig. 8). This hydrophilic route of the mutant may enable the diffusion of metal ions from the entry channel to the ferroxidase site, especially in the presence of a high concentration of metal ions (Fig. 7b).
The Glu 173 of SFER4 is conserved not only among all known members of plant ferritins such as soybean, pea, maize, tobacco, and Arabidopsis (22,25,28,30,44), but also among ferritins from eukaryotes, except the mouse L chain ferritin (Fig. 1). In the well characterized structure of human H ferritin, conformation around Glu 140 (Glu 173 of the SFER4) is nearly identical to that of SFER4. Therefore, Glu 140 of human H ferritin can also contribute to the mechanical transfer of metal ions. However, further investigation into the function of the glutamate residue using vertebrate ferritins is required. In prokaryotic ferritin, this glutamate residue is also conserved in non-heme binding bacterial ferritin, such as EcFTN (from E. coli) (Fig. 1). In contrast, the corresponding glutamate is substituted with a lysine (Lys 140 ) in the mouse L chain. Santanbrogio et al. (53) suggested that this substitution causes the low iron incorporation activity of the mouse L chain compared with the human L chain, which has a Glu 140 , corresponding to the Glu 173 of SFER4. This observation also supports the significance of the Glu 173 side chain in iron traffic in ferritin.
Thus, it is possible that the function of this residue, Glu 173 of SFER4, as a transit site from the iron entry channel to the ferroxidase site, is related to a universal mechanism in iron translocation in ferritin. This finding provides evidence that a common pathway does exist from the iron entry channel to the ferroxidase center in ferritin molecules in various living kingdoms.