From the
Krebs Institute, Department of Molecular
Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United
Kingdom, the ¶Sanger Center, Hinxton Hall,
Cambridge CB10 1SA, United Kingdom, and the
||School of Animal and Microbial Sciences,
University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom
Received for publication, July 22, 2002 , and in revised form, April 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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Most of the known examples of this class of protein have two iron atoms at their dimetal center. Examples include the R2 protein of ribonucleotide reductase (5), the hydroxylase component of the methane monoxygenase (MMO)1 (6), and a homodimeric protein (rubrerythrin) isolated from anaerobic bacteria, which has a di-iron center in its N-terminal domain and an Fe-Cys4 cluster in its C-terminal domain (8). Examples of dimanganese proteins include the dimanganese catalase of some lactobacilli and thermophilic bacteria (3) and a manganese derivative of the R2 protein (9). Binding at the di-iron centers of MMO or R2 leads to oxygen activation and results in either the insertion of an oxygen atom into methane (MMO) (6) or the production of a stable catalytically involved tyrosyl radical (R2) (5). In contrast, hemerythrin binds dioxygen reversibly, whereas the binding of H2O2 to dimanganese catalase results in its catalytic disproportionation to H2O and O2 (3).
The ferritins are a special class of di-iron proteins that have roles both in iron housekeeping and in iron detoxification (1, 10). Iron is an essential element for almost all organisms. Its ability to form complexes such as heme and the iron-sulfur clusters as well as the di-iron enzymes mentioned above endows cells with a variety of functions. On the other hand, free iron is toxic, due, principally, to its part in generating highly reactive oxy-radicals that can damage cell constituents. To provide a "safe haven," ferritins store iron as a relatively inert mineral (ferrihydrite-phosphate or hydrous ferric phosphate) "core," which can be utilized as a source of iron for biosynthesis (1, 10, 11). Ferritins are widespread in nature, being found in eubacteria and archaebacteria as well as in eukaryotes (1). Their molecules are assemblies of 24 structurally equivalent protein chains or subunits related in 432 symmetry and forming a nearly spherical shell (Fig. 1A) with an 80-Å diameter iron storage cavity (7, 1217). The protein subunits are folded as four helix bundles about 27 Å long with a fifth shorter helix lying at about 60° to the bundle (Fig. 1B). The di-iron centers are situated in the middle of the ferritin subunits (Fig. 1B) and are often known as "ferroxidase centers" because of their ability to confer ferroxidase activity (however, some types of ferritin subunit lack the ability to form di-iron centers) (17). Although ferritins characterized by this overall design are found throughout the living kingdom, the sequence identity between members from diverse species may be as low as 12%, and functional variations have arisen from such differences (1, 7).
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It is striking that bacteria possess more than one type of ferritin (1). Many bacteria contain "bacterioferritins," which differ from classical ferritins in that, in addition to the 24 di-iron centers, they also have up to 12 heme groups per molecule (located on the dyad axes between subunit pairs) (18). The function of the heme groups is uncertain, but they may modulate the reductive release of iron from the "core" (19). Escherichia coli has both a bacterioferritin (EcBfr) and a ferritin (EcFtnA). Both are fully functional iron storage proteins with 24 di-iron centers. E. coli also encodes an uncharacterized ferritin-like protein (EcFtnB) with good sequence identity (33%) to EcFtnA (1, 7). However, its dinuclear center ligands are poorly conserved, suggesting that it probably does not function as a normal ferritin.
Several ferritins have been shown to contain more than one type of ferritin chain. In humans, for example, two types of chain, known as H and L, are found to coassemble in proportions that vary with the tissue of origin and body-iron status (10). The H chains contain di-iron centers, but the L chains do not (1012).
The three-dimensional structures of native or recombinant ferritins from human (12, 17), horse (12, 16), rat (17), mouse (20), frog (14, 15), and E. coli (13) have been determined, but most of these are L ferritins, and relatively little high resolution information has been obtained on the structures of the di-iron centers of H-type ferritins. Moreover, the iron centers in the animal H ferritins (e.g. from horse and human) are relatively labile and, upon oxidation, the di-iron tends to move into the iron storage cavity, where it becomes "invisible" to X-rays because the mineral-iron core is complexed in a form that is not specifically orientated to the protein structure (11, 21, 23). In EcFtnA (13), the iron is less labile, allowing the structure of the iron-containing protein to be compared with the metal-free and Zn2+ complex forms (zinc being used as a redox-stable alternative to Fe2+). The corresponding high resolution structures reveal the ligand geometry of the di-iron centers and of a novel, nearby "third" site (Fig. 1C) (13, 23). Structures of the Mn2+ derivatives of EcBfr (18) and the Ca2+ derivative of recombinant human H chain ferritin (HuHF) (17) have been determined at medium and high resolution, respectively. In the present study, the structures of five variant EcFtnA molecules and their Fe(III) and Zn(II) derivatives are analyzed. Each variant bears a single substitution (Ala for Glu) in one of the metal ligands. The results provide new insights into the functional behavior of the metal sites in EcFtnA and their relationships to those in other ferritins. Fig. 2 displays schematic representations of the metal centers of EcFtnA, EcBfr, and HuHF. It can be seen that the metal centers of the two E. coli ferritins differ in several respects. First, the di-iron atoms in EcFtnA are bridged by a single carboxylate (Glu50), whereas EcBfr has two bridging carboxylates (Glu51 and Glu127). Second, Glu130, which is a ligand of FeB in EcFtnA, is replaced in EcBfr by His130. Third and most dramatically, EcFtnA has an additional iron site, FeC, not present in EcBfr. Fig. 2 also shows that the di-iron center in HuHF differs from those in both EcFtnA and EcBfr. Like EcFtnA, it has a single bridging carboxyl (Glu62) with a Gln (Gln141) at a position equivalent to that of Gln127 in EcFtnA and the bridging Glu127 of EcBfr. Another difference is the site B ligand, Glu61, which occupies a sequence position differing from those of the Glu130 ligand of EcFtnA or His130 of EcBfr. Note that Glu61 is equivalent in sequence position to one of the site C ligands in EcFtnA (Glu49), but HuHF has no metal in this position because of changes in the carboxylate ligands (Glu129 and Glu130 to Lys143 and Ala144, respectively).
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Differences in behavior of these three types of ferritin iron center have been described and must reflect the observed variation in structure. The most studied of the mammalian ferritins are horse spleen ferritin, which is a heteropolymer co-assembled from both H and L chains in various proportions, and the single recombinant H or L homopolymers from human, horse, or mouse which have been expressed and assembled separately. When a pair of Fe2+ atoms at the dinuclear centers of HuHF or horse spleen ferritin binds a dioxygen molecule, the two Fe2+ atoms are oxidized to two Fe3+ with the production of H2O2 and the formation of µ-oxo-bridged diferric centers (22, 2426).
Surprisingly, the diferric centers are quite labile. The ferric atoms can migrate into the storage cavity of the same or even another molecule, where they form ferrihydrite by hydrolytic polymerization (11, 22, 25, 27). When this happens, two processes are possible; the original dinuclear centers can be reutilized, or iron can be oxidized directly on the growing core surface by a 4Fe2+/O2 reaction (11, 22, 25). The function of the L subunit seems to be to provide a site that favors the four-electron reduction of dioxygen. This site consists of clusters of Glu residues located on the L-subunit inner surface and acts as a nucleation center for the formation of mineral, thus stimulating its deposition within the inner cavity (7, 16). EcBfr and EcFtnA each consist of just one type of subunit with features more reminiscent of H subunits than L subunits (1, 13, 17, 18). Nevertheless, measurements of Fe(II) oxidation stoichiometry give values of 3.54.0 Fe(II)/O2 for both proteins (2830). In the case of EcBfr, this stoichiometry seems to result from a combination of two processes: the reduction of O2 to H2O2 in an initial di-Fe(II) oxidation, followed by the rapid consumption of the H2O2 thus produced in another di-Fe(II) oxidation, with both reactions giving Fe(III)-O-Fe(III) dimers as their products (29). Interestingly, the ferric dimers produced by EcBfr are much less mobile (more stable) than those formed in HuHF.
The behavior of EcFtnA differs from that of EcBfr or HuHF. Both kinetic and Mössbauer spectroscopic measurements indicate that Fe(II) atoms at the unique C site are oxidized along with those at the A and B sites, which, as in both EcBfr and HuHF, are linked by µ-oxo-bridges (3033). New evidence is presented here for additional metal (Zn2+) sites, which might enable the concerted oxidation of four Fe(II) by one O2 molecule. The structural data also indicate that the presence of Fe3+ at site C stabilizes Fe3+ at the A and B sites such that the tendency of Fe3+ at these sites to migrate into the cavity is decreased. Thus, inactivation of site C converts EcFtnA into a more HuHF-like ferritin.
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EXPERIMENTAL PROCEDURES |
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Crystallization and Data CollectionCrystals of all five EcFtnA variants were grown at 20 °C by microdialysis against 20 mM piperazine-HCl buffer (pH 5.2) containing 1 mM EDTA and 0.10.6 M NaCl.
Crystals grew after 1 week, and x-ray analysis showed that they all belonged to the space group I4 with cell parameters a = b = 130.0 ± 0.7 Å, c = 173.0 ± 0.7 Å (for individual cell dimensions, see Tables I and II) and with six subunits in the asymmetric unit. For the metal soaking experiments, crystals were transferred into identical solutions to those in which they had grown except that EDTA was omitted and either ammonium ferrous sulfate or zinc chloride was added to a concentration of 10 mM. Metal soaks were carried out for 3 h before crystal mounting and data collection.
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X-ray data were typically collected to a resolution of 2.2 Å from a
single crystal at room temperature. All data sets were obtained using a
rotating anode with a copper target and recorded on a MAR image plate
detector. They were processed using DENZO
(35) and merged and scaled
using ROTAVATA and AGROVATA
(36). Most data sets were more
than 95% complete, with a multiplicity of at least 2.0 and with more than 70%
of the reflection intensities greater than 3 . However, for some of the
metal-soaked crystals, the diffraction limit was slightly less, as indicated
in Tables I and
II along with individual data
processing statistics.
Structure Solution and RefinementThe coordinates of the
wild-type EcFtnA soaked in iron
(13) were used as the starting
model for solving the structures of each variant. In each case, the model was
edited to replace the relevant glutamate residue by alanine and all iron atoms
and water molecules were removed prior to refinement. Each subsequent model
was then refined with the program TNT
(37) with cycles of rigid
body, xyz, and then xyz with B factor refinement. After convergence, (3
Fo - 2
Fc)exp(ic)
electron density maps were calculated, and to these the individual models were
rebuilt using the program FRODO
(38), and the known metal
binding sites (13) were
inspected for the presence of metal. After further refinement, additional
solvent molecules were added using the program PEAKMAX
(36) until all high electron
density features had been explained. In some cases, "solvent
molecules" with unusually low B factors and shorter than expected ligand
distances were reinterpreted as low occupancy metal sites. Iterative cycles of
model building and refinement gave final models containing
200 solvent
molecules and overall reliability indices (R) of <20%. The
geometry of each final model was checked using PROCHECK
(39), and the individual
refinement statistics are given in Tables
I and
II.
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RESULTS |
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A notable feature of the wild-type protein shown in Fig. 3A is the hydrogen bond network linking glutamates 17, 50, and 94, Tyr24, His53, Gln127, and two water molecules W1 and W2, with hydrogen bond distances lying between 2.48 and 3.07 Å. Inevitably, this network is disrupted by the glutamate substitutions, which also lead to loss of one or both of the water molecules, although the Tyr24OHGlu94OE1 and Gln127NH2Glu94OE2 bonds are retained in all except E94A and E17A (where Glu94 has moved). Tyr24, which lies in a hydrophobic pocket, remains in a similar conformation throughout. Some of the glutamate side chains, in contrast, exhibit a considerable degree of flexibility, especially Glu130 (the MB-MC bridging ligand) and the three site MC ligands, Glu49, Glu126, and Glu129, which lie on the inner surface of the protein shell. Movement of Glu50 in E17A leads to the loss of W1, whereas the usual Glu50 side chain position is taken by a water molecule in E50A.
In principle, Gln127 could be acting as either donor or acceptor when hydrogen bonded to Glu94. The latter situation would be possible only if Glu94 is protonated. However, Glu94 would be expected to be negatively charged in the metal derivatives of Figs. 4, AD, and 6, A and C, yet all of these show hydrogen bonding between Gln127 and Glu94. Hence, in these structures, Gln127 must have its amino group directed toward OE1 of Glu94. If Gln127 is orientated in the same way in the wild type metal-free structure of Fig. 3A, then it follows that Glu94 is deprotonated here too. To account for the remainder of the hydrogen-bonded network observed in Fig. 3A it further follows that both Glu17 and Glu50 are protonated, leaving a single negative charge on the carboxylate cluster (the side chain of Glu130 being turned away). His53ND1 must also be protonated, and if it is also positively charged, then the negative charge on Glu94 would be balanced. In the alternative arrangement of Gln127 with Glu94 protonated, then either Glu17 or Glu50 must be protonated, and the other must be negatively charged. His53ND1 would receive a proton from W2, and the cluster would have a net charge of 1-. The first suggested arrangement is probably to be preferred.
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Loss of a site MA or MB ligand in all of the variants except E49A would interfere with this charge balance as well as lowering the affinity of one or both of the metal sites. In EcFtnA, W2 is not fully present, and this water is absent from E49A so that the network shown in Fig. 3A is incomplete in these proteins. Not shown in this figure for clarity is residue His46, which can occupy alternative positions in which it is hydrogen-bonded either to Glu94 or to Glu130 (13).
Zn2+ and Fe3+ Derivatives of EcFtnA and Its VariantsTo obtain metal derivatives, the crystals were soaked in either zinc chloride or ferrous ammonium sulfate. Zn2+, being a known inhibitor of Fe2+ binding and oxidation (40), was used as a probe for Fe2+ because of the ease of Fe2+ oxidation. For the same reason, the iron was assumed to be Fe3+ after the 3-h aerobic soak in the ferrous salt solution. Indeed, iron oxidation was confirmed in some variants by the development of a brown color due to iron core ferrihydrite formation.
Zn2+ Binding SitesAs described previously (13), the Zn2+ derivative of wild-type EcFtnA contained only two bound Zn2+ atoms, situated 3.43 Å apart at sites MA and MB of the dinuclear center. There was no Zn2+ at site MC (Fig. 4A). Three of the variants (E17A, E49A, and E130A) showed Zn2+ with high occupancy at sites MA and MB, and two variants (E17A and E130A) had significant density near the site MC identified in the wild-type Fe3+ derivative (13) (Fig. 4, BF). On refinement, the occupancy of site MC in both variants was only 0.4 relative to that of either MA or MB. Unlike wild-type EcFtnA, three additional low occupancy Zn2+ sites MD, ME, and MF were observed in variants E17A, E49A, and E130A and these are displayed in Fig. 5. Zn2+ ligand distances are listed in Table III, together with ZnA2+ZnB2+ distances and Zn2+ occupancies.
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The stereochemistries of ZnA2+ and
ZnB2+ in variants E17A, E49A, and E130A are very similar
to those of wild-type (Fig. 4B, C,
F, and A, respectively, and
Table III) with a notable
swinging inwards of Glu130 to ligate Zn2+ in both the
E49A and wild-type structures and with Glu50 forming a
syn-syn bridge in all four structures. His53 is a
ZnA2+ ligand in the four proteins. In the position
occupied by Glu17 in the other proteins, E17A has an electron
density peak at a distance of 2.17 Å from
ZnA2+, which was modeled as a chloride ion, and in E130A
the missing site MB ligand, Glu130, is replaced by a
water molecule. Common to the Zn2+ derivatives of all three
variants as well as of wild type is the presence of electron density between
ZnA2+ and ZnB2+, which is
interpreted as a water molecule, W1, that acts as a second bridging ligand.
Thus, the ligand geometries in the four di-Zn2+ structures are
roughly tetrahedral for both Zn2+ atoms, although wild-type
ZnA2+ has an additional weak ligand, water W2, at an
average distance of 2.67 Å, whereas the second glutamate oxygen,
Glu94OE2, is 2.70 Å from ZnB2+. The
ZnA2+ZnB2+ separations are
3.40 Å in variant E49A and 3.43 Å in E130A, with the distance in
E17A being reduced to 3.32 Å (Table
III). Another feature of the four structures is the maintenance of
Tyr24OHGlu94OE1 and
Gln127NH2Glu94OE2 hydrogen bonds.
Unlike the four di-Zn2+ structures, variant E50A shows no bound Zn2+, presumably because the absence of the ZnA2+ZnB2+ bridging ligand destabilizes both dinuclear sites for divalent metal binding (Fig. 4D). There was no density either for the water replacing Glu50 or for two water molecules seen in metal-free wild type (Fig. 3A), and the hydrogen bond network was further disrupted by side chain movement. In contrast to E50A, site MA is well occupied in E94A, although the occupancy of site MB is low. The refined occupancies for sites MA and MB are 0.88 and 0.49, respectively. The coordination around ZnA2+ is identical to that in wild type, but in the absence of Glu94 a new electron density peak was seen. This was refined as a water molecule.
Three new minor Zn2+ positions not seen in wild-type EcFtnA were found in E17A, E49A, and E130A although not in the other two variants. These sites are shown for E17A in Fig. 5. The first, site MD (occupancy 0.63), lies toward the inside of the protein shell on the interface between subunits related by a 3-fold axis. The ligation of ZnD2+ by Asp63 of one subunit and by His128 of its symmetry-related neighbor replaces an ion-pair interaction between the same two amino acid side chains, and two other electron density peaks are assigned to Cl- ions (Fig. 5A). This Zn2+ lies roughly equidistant from the dinuclear center of the two subunits (direct distances of 15.1 and 17.8 Å). The second Zn2+ (site ME, occupancy 0.48) is attached to His93 toward the outside of the shell with two Cl- ions nearby (Fig. 5B) and lies at about 11 Å from the dinuclear center. The third Zn2+ (site MF, occupancy 0.56), which is present on only 8 of the 24 subunits, is located at intermolecular crystal contacts and involves Glu74 of one molecule and His106 of another as ligands, with Cl- as a third ligand (Fig. 5C).
Fe3+ Binding SitesThe three Fe3+ sites located in wild-type EcFtnA are displayed in Fig. 6A, and the equivalent regions of the ferrous ammonium sulfate-soaked variants can be seen in Fig. 6, BF. As discussed previously (13), iron is bound at both sites MA and MB in the wild-type structure, but with the MB site occupancy only 0.68-fold that of MA. Another notable observation is the strong occupation (0.91) of site MC by Fe3+. The FeC3+ is linked to FeB3+ by a long (5.79-Å) Glu130 bridge in an anti-anti conformation (Fig. 6A).
Variant E17A showed low occupancy (0.42) for site MC (Fig. 6B), with no metal present in either MA or MB. Thus, the affinity of site MB for Fe3+ is evidently low in the absence of any Fe3+ in site MA. No iron core electron density was seen, but iron core formation was evident from the brown color of the crystals, which showed that iron oxidation and hydrolysis had taken place. To examine the possibility that iron could have been present in sites MA and MB at earlier times, shorter crystal soaks of 1 and 10 min were investigated. However, no iron was seen in the dinuclear sites at either time, and the occupancy of site MC was even lower than after 3 h. Only one of the variants (E49A) that exhibited dinuclear Zn2+ binding showed Fe3+ occupation of the MA and MB sites, and the occupancy of MB by Fe3+ was greater (0.95) than in wild type. In E49A, only a site MC ligand had been removed, with the consequence that there was no iron binding at MC and that the MB-MC bridging ligand Glu130 had changed its orientation to ligate only FeB3+ (Fig. 6C).
In the Fe3+ derivative of E50A (Fig. 6D), which showed no Zn2+ binding, a strong peak of electron density was found at the MA site, giving an occupancy of 0.79 on refinement. Very weak density was also found at MC (0.32). Another difference between Zn2+ and Fe3+ binding was observed with variant E94A. Here Zn2+ was bound strongly at site MA (0.88), less strongly at MB (0.49), and not at MC (Fig. 4E), whereas Fe3+ was found at MA and MC in E94A with low occupancies (0.39 and 0.55, respectively) but not at MB (Fig. 6E). No bound Fe3+ was observed at any of the specific protein sites in variant E130A, although the presence of Fe3+ in the iron core was again evidenced by the brown color of the crystals. None of the six Fe3+ derivatives examined showed metal density in the minor Zn2+ binding sites MD, ME, or MF or at any other non-specific site.
In all three sites (MA, MB, and MC) of wild-type EcFtnA, the Fe3+ has approximately octahedral coordination except that water W3 (not seen in the Zn2+ derivative) was rather far from the iron atoms (3.05 and 2.87 Å from FeA3+ and FeB3+, respectively, indicating very weak coordination). Fe3+ is ligated by Glu17, His53, and another water (W2), which, like W2 of the metal-free proteins, is hydrogen-bonded to Gln127OE1. Glu50 bridges FeA3+ and FeB3+ in a syn-syn conformation as in the di-Zn2+ complex. A second electron density bridge, which in the latter was assigned as a water molecule, in the di-Fe3+ protein is likely to be O2- or OH- (32, 33). An O2- bridge along with the four negatively charged carboxylate ligands would compensate for the six positive charges on the iron atoms, and the observed Fe3+W1 bond distances of 1.98 Å (Table IV) are consistent with such a bridge, unless W3 had also lost a proton. However, the Glu127OE1-W1 distance of 2.9 Å is indicative of hydrogen bonding through an OH- proton. This would give the cluster a net charge of 1-. Although the hydrogen bonds linking Tyr24, Glu94, and Gln127 remain intact, the hydrogen atoms in the remainder of the network in Fig. 3A are lost from Glu17, His53, and Glu50 as a consequence of metal binding.
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There are some notable differences between the coordination of FeA3+ and FeB3+ in variant E49A compared with wild type (Fig. 6, C and A, respectively). The two iron atoms are further apart (3.43 Å instead of 3.24 Å as in wild-type) (Table IV), and W3 is missing. In the absence of FeC3+, Glu130 moves closer to FeB3+ such that the average bond length of Glu130OE1-FeB3+ (2.03 Å) is much shorter than in wild-type (2.38 Å), and this, combined with the shortening of Glu94OE2-FeB3+ (1.79 instead of 2.15 Å) may explain the increased occupancy of site MB (Table IV). The binding of FeB3+ by Glu130 involves considerable side chain movement, and the co-ordination of FeC3+ in wild type (and in E94A) also necessitates realignment of the carboxyl ligands Glu126, Glu129, and Glu49 to a lesser extent. In E94A and in wild type, FeC3+ has similar geometry with two water ligands (W4 and W5) in addition to monodentate ligation by the four glutamates. The coordination of FeA3+ in E50A (Fig. 6D) is unusual compared with that in wild type or E49A. In addition to the usual protein ligands, Glu17 and His53, there are two well ordered water molecules, W1 (at 2.02 Å) and W2 (at 2.08 Å), and these are hydrogen-bonded to both OE1 and OE2 of E94 in a syn-syn conformation and also to S31 and N141, respectively (not shown). A small movement of Glu94 is required to give the arrangement described, and this is accompanied by a small movement of Tyr24 to maintain its hydrogen binding. In E50A, as in E94A in which there is no FeB3+, Glu130 binds only FeC3+ along with its two other carboxyl ligands Glu49 and Glu126. Glu129 is not a ligand in E50A, presumably because only three glutamate ligands are required to neutralize the Fe3+ charge, and its side chain points away from the metal site. In E94A, the side chain of Glu129 has alternative positions, although in one position it would seem partially to ligate FeC3+.
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DISCUSSION |
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Importance of the Dinuclear CenterNot only do the x-ray data presented here confirm the localization of ferroxidase activity to the dinuclear sites, MA and MB, but the observed functional effects of the individual substitutions are explicable by the changes in metal binding. Thus, the most drastic alterations in the binding of both Zn2+ and Fe3+ produced by the substitutions E50A and E94A also give the largest reduction in the rates of Fe2+ oxidation (30, 34). Hence the x-ray analysis supports the conclusion that fast oxidation depends on the presence of a pair of Fe2+ atoms enabling the transfer of two electrons to dioxygen. The observed oxidation rate with E49A is only slightly less than that with wild-type (30), and the binding of both divalent and trivalent metals is similar in the two proteins. Thus, substitution of the site MC ligand, Glu49, leads to a loss of metal from this site (Figs. 4C and 6C) without greatly diminishing the rate of oxidation (30). Hence, the presence of iron in site MC is not essential for fast oxidation, and in this regard it may be noted that human H chain ferritin, which has no site MC, oxidizes iron as efficiently as EcFtnA (30). Although no Fe3+ was observed in sites MA or MB of variants E17A or E130A (Fig. 6), both sites were fully occupied by Zn2+ (Fig. 4). This, together with the known ability of Zn2+ to displace both Fe2+ and Fe3+ from these sites, supports the conclusion that Fe2+ oxidation takes place pairwise in these variants. In the iron-containing derivatives, it is inferred that oxidation must have occurred before x-ray data were collected but that the loss of MA or MB site ligands, respectively, from these variants had greatly reduced their affinity for Fe3+ and enabled Fe3+ to leave these sites for core formation in the protein shell. Kinetic studies indicated that the Fe2+ oxidation rate was diminished with E17A and (to a small extent) with E130A compared with wild-type, but such losses in oxidative efficiency were not comparable with those found for variants E50A or E94A.
Coupled Dimetal PairsA feature common to wild type and variants in which both the MA and MB sites are occupied by Zn2+ or Fe3+ is the presence of bridging electron density coupling the metal ions. This is likely to be a water molecule in the charge-neutral complex formed by two Zn2+ and four carboxylate ligands. However, Mössbauer spectroscopic data indicate that a µ-oxo-bridged Fe3+ dimer is formed immediately after Fe2+ oxidation and that, with time, this is transformed into a species having different parameters, which may be a µ-hydroxo-coupled Fe3+ pair (32, 33). The x-ray data indicate the presence of such a bridge in both E49A and wild type without clearly distinguishing the type of coupling between the Fe3+ atoms. Indeed, a mixture of O2- and OH- bridges is a possibility.
The Role of Iron in Site CAlthough it has been inferred that Fe2+ must bind at site MC in wild-type prior to its oxidation along with Fe2+ at MA and MB (32, 33), the first direct evidence for the binding of a divalent metal at this position is given in Fig. 4, B and F. With ligation to the protein only by Glu49 and Glu126, the binding is weak in the variants E17A and E130A, and no ZnC2+ was observed in wild-type or any of the other variants. It has been noted previously (13) that a strong electron density peak appears at site MC in the wild-type Fe3+ derivative (Fig. 6A). Weaker binding is now also observed in E94A, E50A, and E17A (Fig. 6). The absence of FeC3+ from E49A and E130A emphasizes the importance of the ligands that have been changed. The electron density for Glu126 is always well ordered and correctly orientated for metal binding, indicating that it is also an essential ligand, whereas Glu129, which is often disordered, is only brought into play when required to form a stable, charge-neutral complex as in wild type, where Glu130 bridges both FeC3+ and FeB3+.
The Zn2+ and Fe3+ binding data displayed in Figs.
4,5,6
provide structural underpinning for several other observations. Mössbauer
spectroscopic data indicate that Zn2+, which binds only weakly, if
at all, at site C, is unable to displace monomeric (site MC)
Fe3+, although it is able to compete with both Fe2+ and
Fe3+ for at least one of the dinuclear sites
(33). Of greater functional
relevance is the finding that the blue diferric peroxo intermediate, seen in
wild type when oxygen binds to a di-Fe2+ pair at the dinuclear
center, is increased in intensity in variants E49A, E126A, and E130A
(31). This suggests that site
MC competes with sites MA and MB for
Fe2+ binding and hence that there are molecules in which all three
sites are occupied by Fe2+. Although no such measurements were made
for E49A, it has been observed that both wild-type and variant E17A gave an
oxidation stoichiometry of 3.5 Fe2+/O2, whereas
E130A and E126A gave a ratio of only 1.8 Fe2+/O2
(30). Thus, the available data
suggest that when there is significant binding of Fe2+ at site
MC and when this iron can be coupled through Glu130 to
FeB3+ and then through Glu50 to
FeA2+, at least three electrons can be transferred to a
dioxygen molecule bound at the dinuclear center.
The current study suggests a further possibility that binding of Fe2+ at one of the minor sites, MD or ME (Fig. 5), would enable dioxygen to acquire a fourth electron, leading to its complete reduction to water. These sites lie at "through-bond" distances of about 24 and 18 Å, respectively, from dinuclear site MB or at "through-space" distances of 15 and 11 Å. Although only Zn2+ has been seen at MD or ME (and only in some variants), it is conceivable that Fe2+ could bind transiently to one of these sites in wild type but that Fe3+ would not remain bound. In this connection, it may be significant that His93, a site ME ligand, is conserved and lies next in sequence to the FeB3+ ligand Glu94 and that a site MD ligand (His128) is also conserved in microbial nonheme ferritins and lies next in sequence to the Gln127 of the dinuclear center.
Formation of the Iron CoreThe occupancy of FeB3+ is noticeably greater in E49A, in which no Fe3+ is observed at site MC, than in wild-type EcFtnA, and the Glu130OE1FeB3+ bond is shortened (Table III). These observations suggest that the long anti-anti MB-MC bridge through E130 not only provides electronic connection between sites MC and MB in wild type but also loosens the binding of Fe3+ at MB. The looser binding could enable the movement of Fe3+ out of its oxidation site into the iron storage cavity for ferrihydrite nucleation. However, this suggestion needs further investigation, since kinetic measurements (performed in solution) have indicated that the dinuclear site of E49A is more readily vacated than that of the wild-type protein (30). On the other hand, the crystallographic data for EcFtnA indicate that, when Glu130 is absent, the dinuclear center no longer provides stable binding for Fe3+, and the iron now forms hydrolytic polymers that turn the crystals brown. The ease of core formation shown here for E130A and in solution for other site MC variants (30), imply that site MC is not an iron core nucleation center. It is not clear whether any other residues promote ferrihydrite nucleation in EcFtnA, but electron micrographs and the iron staining of electropherograms show that the mineral forms within the protein shell (1). The fact that no electron density attributable to mineral was detected does not mean that the cavity was empty but must mean that the microcrystalline structure of the mineral of EcFtnA, as in other ferritins (21), is not specifically orientated with respect to that of the protein.
In conclusion, genetically engineered substitutions of dinuclear center ligands show drastic effects on iron or zinc binding and negligible effects on the structure as a whole. The structural analysis, which is the first such study for any ferritin, now provides a sound structural basis for the conclusion, based previously on solution studies, that binding of Fe2+ pairs at these centers is responsible for their rapid oxidation. The binding of iron at a third site (MC) near the di-iron pair (MA and MB), which is observed only in bacterial ferritin, could allow the coupled oxidation of a third Fe2+. Moreover, iron binding at one of three even more remote sites (MDMF), observed here for the first time, may enable the oxidation of four Fe2+ by one dioxygen molecule with the production of water.
Relationship with Other Ferritins and MetalloproteinsThe
results given here provide an interesting comparison with the other ferritins
of Fig. 2. Although comparable
structural data are not available for HuHF or EcBfr variants, Glu
substitutions equivalent to E17A and E94A have been made in these proteins
(E27A and E107A, respectively, in HuHF and E18A and E94A in EcBfr). In all
three ferritins, the site B ligand substitution enormously increases the
t of oxidation as does the site A ligand change in
HuHF (24,
28) and EcBfr
(28,
42). Although considerable,
the effect of the E17A substitution in EcFtnA is less marked than the site A
change in the other two ferritins. Interestingly, the equivalent substitutions
Q127E in EcFtnA and Q141E in HuHF had little observed effect on the
t
of oxidation, whereas the converse substitution
in EcBfr, E127Q, drastically reduced the oxidation rate
(28). It seems that both
carboxyl bridges are essential for the stability of the center in this
ferritin. Of further interest is the comparison of the effects of the E130A
and E49A substitutions in EcFtnA with HuHF (which has Ala144 at the
Glu130 position) and with the HuHF-E61A variant (equivalent to E49A
in EcFtnA). The first results to note are that neither E49A in EcFtnA nor E61A
in HuHF, site C and site B ligands, respectively, gave a large diminution in
the Fe(II) oxidation rates, and the same is true of the site B ligand change
E130A in EcFtnA (28,
30). Of further interest is
the finding that in the Zn2+ derivative of the EcFtnA variant
E130A, the site C ligand, Glu49, does not swing in to replace
Glu130 as a site B ligand although no metal is bound at site C
(Fig. 4F), whereas in
the Fe3+ derivative of E49A, in which Fe3+ is lost from
site C, Glu130 moves closer to Fe3+ at site B
(Fig. 6F and
Table IV). In HuHF,
Glu61 was observed in two orientations in crystals grown from
solutions containing CaCl2, one directed toward site B and a second
toward the inner surface of the protein, where it ligated a Ca2+
(17) together with residue
Glu64 (at a weak "C site" different from site C in
EcFtnA). If this Fe(II) is bound at site C, then it is not oxidized along with
the Fe(II) at sites A and B, but it was suggested that movement of
Glu61 might shepherd Fe3+ from site B to site C in the
cavity for ferrihydrite nucleation
(17). This evidently does not
happen in EcFtnA, since Glu49 is not a site B ligand
(Fig. 1C), and
although Glu49 is a site C ligand, site C is not part of a
ferrihydrite nucleation site in this ferritin
(30). Although divalent
Zn2+ ions are found at sites A and B in the EcFtnA variant bearing
the substitution E130A (Fig.
4F), this change leads to the loss of Fe3+
from all three sites (Fig.
6F) and hence to increased ferrihydrite core deposition.
In this respect, the EcFtnA variant more closely resembles HuHF than does wild
type EcFtnA, and again, the observed change in oxidation stoichiometry from
3.54.0 to 1.8 Fe(II)/O2 given by the E130A substitution
shows another HuHF-like property
(30). Thus, although when
Fe3+ is present at site C the lengthening of the
Glu130FeB3+ bond may lead to the loss
of some Fe3+ from site B, the use of Glu130 instead of
Glu61 at site B seems to stabilize iron at the di-iron site. The
sequence differences between HuHF and EcFtnA may result from a greater and
more immediate demand for iron storage in the mammalian ferritin, although
initially this occurs at the expense of the production of hydrogen peroxide
when its center is used for Fe(II) oxidation.
A further common feature of some of the dinuclear centers is the presence
of an outer sphere tyrosine residue (Figs.
2,
3,
4,
5,
6). A tyrosine similar in
position to those depicted in Fig.
2 is found in both rubrerythrin
(8) and dimanganese catalase
(3), where it is a conserved
feature, and in both proteins the tyrosine is hydrogen-bonded to a site B
carboxyl ligand as in the ferritins (e.g.
Tyr24Glu94 in EcFtnA)
(Fig. 2). In dimanganese
catalase, it is suggested that this tyrosine may function as a "safety
valve," allowing the enzyme to form a relatively reversible free radical
following hyperoxidation of the cluster, thus protecting the active site and
preventing permanent oxidative damage to the protein. In the R2 protein, a
conserved tyrosine, which is hydrogen-bonded to a site A ligand (usually Asp)
forms a phenoxyl free radical when the reduced center is oxidized by dioxygen
and participates in an electron transfer chain leading to the reduction of
ribonucleotide (4,
5). In both EcFtnA and HuHF,
the Y24F/Y34F change leads to a 4.5-fold increase in the
t of Fe(II) oxidation
(28), but the conservation of
this residue in the FtnB of E. coli
(1) and in mammalian L chains
(10) could mean that its role
is largely structural. However, tyrosyl radical production occurs in HuHF but
not in recombinant horse L ferritin, and it is further associated with
ferroxidase activity through the finding that a variant (E62K and H65G)
lacking ferroxidase activity did not give the EPR radical signal
(43). The tyrosyl radical
signal was also absent from the HuHF-Y34F variant but not from those bearing
change Y137F, Y29F, or Y32F, clearly implicating Tyr34 as its
source (43). However, the low
level of tyrosyl radical production (about one per 300 Fe2+
oxidized) implies that it is not of critical importance to the
protein-catalyzed mechanism of iron oxidation in this ferritin.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 44-114-222-2738; Fax:
44-114-272-8697; E-mail:
t.stillman{at}sheffield.ac.uk.
1 The abbreviations used are: MMO, methane monoxygenase; EcFtnA, non-heme
ferritin of E. coli; EcBfr, E. coli bacterioferritin; HuHF,
human H chain ferritin.
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REFERENCES |
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