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Structural and Mechanistic Studies of a Stabilized Subunit Dimer Variant of Escherichia coli Bacterioferritin Identify Residues Required for Core Formation*

  • Steve G. Wong
    Footnotes
    Affiliations
    Departments of Biochemistry and Molecular Biology

    Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Stacey A.L. Tom-Yew
    Footnotes
    Affiliations
    Microbiology and Immunology
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  • Allison Lewin
    Affiliations
    School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Nick E. Le Brun
    Affiliations
    School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Geoffrey R. Moore
    Affiliations
    School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Michael E.P. Murphy
    Affiliations
    Microbiology and Immunology
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  • A. Grant Mauk
    Correspondence
    Recipient of a Canada Research Chair. To whom correspondence should be addressed. Tel.: 604-822-3719; Fax: 604-822-6860;
    Affiliations
    Departments of Biochemistry and Molecular Biology

    Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Author Footnotes
    * This work was supported by a Canadian Institutes of Health Research/Canadian Blood Services partnership grant (to A. G. M.), Canadian Institutes of Health Research Grant MOP-49597 (to M. E. P. M.), and Grants 83/B14704 and BB/D001943/1 from the Biotechnology and Biological Sciences Research Council (to G. R. M. and N. E. L.). This work was also supported in part by a grant from the Canadian Foundation for Innovation to the University of British Columbia Laboratory of Molecular Biophysics, an infrastructure support grant from the Michael Smith Foundation for Health Sciences to the University of British Columbia Centre for Blood Research and the University of British Columbia Laboratory of Molecular Biophysics, and funds from the United States Department of Energy Office of Basic Energy Sciences and Office of Biological and Environmental Research, the National Institutes of Health, the National Center for Research Resources, the Biomedical Technology Program, and the National Institute of General Medical Sciences.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.
    1 Recipient of a Graduate Studentship from the Canadian Institutes of Health Research Strategic Training Program in Transfusion Science.
    2 Recipient of an National Science and Engineering Research Council Postgraduate Studentship.
Open AccessPublished:May 13, 2009DOI:https://doi.org/10.1074/jbc.M901747200
      Bacterioferritin (BFR) is a bacterial member of the ferritin family that functions in iron metabolism and protects against oxidative stress. BFR differs from the mammalian protein in that it is comprised of 24 identical subunits and is able to bind 12 equivalents of heme at sites located between adjacent pairs of subunits. The mechanism by which iron enters the protein to form the dinuclear (ferroxidase) catalytic site present in every subunit and the mineralized iron core housed within the 24-mer is not well understood. To address this issue, the properties of a catalytically functional assembly variant (E128R/E135R) of Escherichia coli BFR are characterized by a combination of crystallography, site-directed mutagenesis, and kinetics. The three-dimensional structure of the protein (1.8 Å resolution) includes two ethylene glycol molecules located on either side of the dinuclear iron site. One of these ethylene glycol molecules is integrated into the surface of the protein that would normally be exposed to solvent, and the other is integrated into the surface of the protein that would normally face the iron core where it is surrounded by the anionic residues Glu47, Asp50, and Asp126. We propose that the sites occupied by these ethylene glycol molecules define regions where iron interacts with the protein, and, in keeping with this proposal, ferroxidase activity decreases significantly when they are replaced with the corresponding amides.
      Bacterioferritin (BFR)
      The abbreviations used are: BFR
      bacterioferritin
      MES
      4-morpholineethanesulfonic acid
      TEV
      tobacco etch virus.
      4The abbreviations used are: BFR
      bacterioferritin
      MES
      4-morpholineethanesulfonic acid
      TEV
      tobacco etch virus.
      is a prokaryotic form of ferritin that has been identified in a number of bacteria (
      • Stiefel E.I.
      • Watt G.D.
      ,
      • Stiefel E.I.
      • Grossman M.J.
      • Hinton S.M.
      • Minak-Bernero V.
      • George G.N.
      • Prince R.C.
      • Bare R.E.
      • Watt G.D.
      ,
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      ). Despite low sequence similarity with eukaryotic ferritins, the three-dimensional structures and functional properties of BFRs from Escherichia coli, Rhodobacter capsulatus, Desulfovibrio desulfuricans, and Azotobacter vinelandii (
      • Cobessi D.
      • Huang L.S.
      • Ban M.
      • Pon N.G.
      • Daldal F.
      • Berry E.A.
      ,
      • Dautant A.
      • Meyer J.B.
      • Yariv J.
      • Précigoux G.
      • Sweet R.M.
      • Kalb A.J.
      • Frolow F.
      ,
      • Frolow F.
      • Kalb A.J.
      • Yariv J.
      ,
      • Liu H.L.
      • Zhou H.N.
      • Xing W.M.
      • Zhao J.F.
      • Li S.X.
      • Huang J.F.
      • Bi R.C.
      ,
      • Macedo S.
      • Romão C.V.
      • Mitchell E.
      • Matias P.M.
      • Liu M.Y.
      • Xavier A.V.
      • LeGall J.
      • Teixeira M.
      • Lindley P.
      • Carrondo M.A.
      ,
      • Swartz L.
      • Kuchinskas M.
      • Li H.
      • Poulos T.L.
      • Lanzilotta W.N.
      ,
      • van Eerde A.
      • Wolterink-van Loo S.
      • van der Oost J.
      • Dijkstra B.W.
      ) are remarkably reminiscent of those reported for mammalian ferritins. For example, BFRs are oligomeric proteins comprised of 24 subunits (∼18 kDa each) that catalyze oxidation of Fe2+ by dioxygen (ferroxidase activity) to promote formation of a mineralized iron core that can contain as many as 2700 iron atoms/ 24-meric molecule (
      • Baaghil S.
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      ). On the other hand, those BFRs that have been characterized differ from the mammalian proteins in that the 24 subunits are identical, and each possesses a catalytic dinuclear iron center that is referred to as the ferroxidase site (in mammalian ferritins, only the H-chains possess such catalytic sites). The pairwise arrangement of BFR monomers within the 24-mer creates 12 binding sites for heme, commonly protoheme IX but iron-coproporphyrin III in D. desulfuricans BFR, in which a methionyl residue on the surface of adjacent BFR monomers provides an axial ligand to create a b-type heme-binding site with bismethionine axial coordination (
      • Andrews S.C.
      • Le Brun N.E.
      • Barynin V.
      • Thomson A.J.
      • Moore G.R.
      • Guest J.R.
      • Harrison P.M.
      ,
      • Cheesman M.R.
      • Thomson A.J.
      • Greenwood C.
      • Moore G.R.
      • Kadir F.
      ). Although a functional role for the heme of BFR has not been identified, the functional role of BFR is believed to be in iron storage and detoxification (
      • Bou-Abdallah F.
      • Lewin A.C.
      • Le Brun N.E.
      • Moore G.R.
      • Chasteen N.D.
      ), thereby protecting against oxidative stress (
      • Vasil M.L.
      • Ochsner U.A.
      ).
      The subunits of BFR are arranged to form eight 3-fold channels and six 4-fold channels. These channels have been proposed as possible entry and exit routes for iron incorporation into or release from the central iron core. For human ferritin, the 3-fold channel plays a significant role in the transport of iron into the iron core (
      • Levi S.
      • Santambrogio P.
      • Corsi B.
      • Cozzi A.
      • Arosio P.
      ), but a similar role for this channel in BFR has not been demonstrated.
      The dinuclear ferroxidase site located within each subunit binds two iron atoms. Coordination of these iron atoms involves Glu51 and Glu127 as bridging ligands for both irons, Glu18 and His54 as ligands for FE1, and Glu94 and His130 as ligands for FE2. Previous studies of E. coli BFR have demonstrated that the ferroxidase center is essential for core formation and that core formation involves at least three kinetically distinguishable phases (
      • Baaghil S.
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      ,
      • Le Brun N.E.
      • Wilson M.T.
      • Andrews S.C.
      • Guest J.R.
      • Harrison P.M.
      • Thomson A.J.
      • Moore G.R.
      ). Phase 1 involves the very rapid reversible binding of two Fe2+ ions to each of the 24 dinuclear ferroxidase centers and can be studied by monitoring small changes in the spectrum of the bound heme. Phase 2 occurs in the presence of dioxygen (or an alternative oxidant such as hydrogen peroxide) and involves the rapid oxidation of each di-Fe2+ center to form an intermediate that is probably an oxo- or hydroxo-bridged di-Fe3+ center. In the presence of Fe2+ exceeding the amount required to saturate the ferroxidase centers, a slower reaction, Phase 3, is observed in which a large ferric oxyhydroxo mineral is synthesized within the protein cavity. The change in absorbance at 340 nm that is observed during aerobic addition of Fe2+ to apo-BFR results from Phases 2 and 3 but is influenced by the kinetics of Phase 1. Although Phases 1 and 2 are well characterized, less is known about Phase 3. This phase probably involves the interaction of Fe2+ (or Fe3+) with amino acid residues on the inner surface of the ferritin oligomer, as part of a complex and poorly defined process known as nucleation. Further information on this phase of core formation is now required.
      An assembly variant of E. coli BFR (E128R/E135R) has been shown previously to form stable subunit dimers that bind one equivalent of protoheme IX and not to form higher order oligomers (
      • Malone S.A.
      • Lewin A.
      • Kilic M.A.
      • Svistunenko D.A.
      • Cooper C.E.
      • Wilson M.T.
      • Le Brun N.E.
      • Spiro S.
      • Moore G.R.
      ). Each monomer in this minimal functional unit can form a dinuclear iron center that catalyzes the formation of a minimal iron core comprised of four to six iron atoms before precipitating (
      • Malone S.A.
      • Lewin A.
      • Kilic M.A.
      • Svistunenko D.A.
      • Cooper C.E.
      • Wilson M.T.
      • Le Brun N.E.
      • Spiro S.
      • Moore G.R.
      ,
      • Kilic M.A.
      • Spiro S.
      • Moore G.R.
      ). The overall kinetics of Fe2+ oxidation observed on addition of Fe2+ to this variant are similar to those observed for wild-type BFR but have not been reported in detail. Nevertheless, the properties of this variant are of interest because the minimal structural unit that it forms constitutes a potentially important experimental model for evaluating detailed mechanistic features of BFR function. Simplification of the oligomeric structure of the protein as represented by this variant form of BFR makes the inner surface of the protein as accessible to bulk solvent as the outer surface, thereby removing any kinetic influences of the channels present in the 24-mer protein.
      The present paper reports detailed kinetic and structural studies that validate this dimeric variant of BFR as a model of the minimal functional unit of wild-type BFR. In addition, the crystallographic structural data suggest a likely functional role of acidic inner surface residues in iron core formation that led to construction of a family of variants of the stable subunit dimer involving replacement of Glu47, Asp50, and Asp126 by site-directed mutagenesis. Kinetic studies of these additional variants confirm a functional role for these residues and lead to the proposal of a model of BFR action.

      DISCUSSION

      The three-dimensional structure of the E128R/E135R variant unambiguously confirms the dimeric structure of the BFR subunit dimer and establishes that the three-dimensional structures of the individual monomers are nearly identical to those of monomers in wild-type BFR (
      • van Eerde A.
      • Wolterink-van Loo S.
      • van der Oost J.
      • Dijkstra B.W.
      ) (root mean square deviation = 1.43 Å over all 158 Cα atoms). One notable difference, however, can be observed in the position of the small C-terminal α-helix, which forms the intersubunit 4-fold channels in the wild-type protein; removal of the intersubunit contact leads to a small repositioning of this helix relative to the four-helix bundle. Omitting 15 residues from the C-terminal α-helix and using only residues 1–143 resulted in a root mean square deviation of 0.48 Å. The remarkable structural similarity between the stabilized subunit dimer and a subunit dimer from the wild-type 24-mer protein indicates that the main features of the structure of the subunit dimer do not require the higher order assembly of the wild-type protein.
      Cryoprotection of crystals with ethylene glycol serendipitously resulted in integration of one ethylene glycol molecule into a pocket that opens on the outer surface of the monomer and provides access to one-half of the dinuclear center. A second ethylene glycol molecule is integrated into a corresponding pocket that is accessed from the inner surface of the monomer and provides access to the other half. The ethylene glycol molecule integrated into the outer surface of the protein is located in a position that corresponds to that of a so-called ferroxidase pore that has been observed in the structure of D. desulfuricans (
      • Macedo S.
      • Romão C.V.
      • Mitchell E.
      • Matias P.M.
      • Liu M.Y.
      • Xavier A.V.
      • LeGall J.
      • Teixeira M.
      • Lindley P.
      • Carrondo M.A.
      ) and that was suggested to be the route by which iron enters the ferroxidase site and iron core of that protein. Thus, ethylene glycol appears to be a suitable probe for hydrated Fe2+ despite their differing shape and size. In the current structure, residue Asn17 is located close to the ethylene glycol on the inner surface, so the two positions described above for this residue probably result from the presence of the ethylene glycol molecule.
      Iron has been proposed previously to be translocated from the dinuclear iron site to the iron core by movements of residues His59 and Glu131 in D. desulfuricans BFR and residues Glu47 and His130 in A. vinelandii BFR (
      • Liu H.L.
      • Zhou H.N.
      • Xing W.M.
      • Zhao J.F.
      • Li S.X.
      • Huang J.F.
      • Bi R.C.
      ,
      • Swartz L.
      • Kuchinskas M.
      • Li H.
      • Poulos T.L.
      • Lanzilotta W.N.
      ). The observation of a similar conformational change by residues Glu47 and His130 in the current structure suggests that a structural change of this type upon metal ion release from, or partial occupancy of, the ferroxidase center is characteristic of this family of proteins. Frolow and Kalb (
      • Frolow F.
      • Kalb A.J.
      ) have reported that the uranyl derivative of E. coli BFR exhibits a similar movement of Glu47 and His130 for the binding of uranyl in the dinuclear iron site. A significant body of mechanistic data on the E. coli BFR protein indicates that following the Phase 2 oxidation of the di-Fe2+ ferroxidase center, a stable bridged di-Fe3+ center is generated (
      • Le Brun N.E.
      • Wilson M.T.
      • Andrews S.C.
      • Guest J.R.
      • Harrison P.M.
      • Thomson A.J.
      • Moore G.R.
      ,
      • Kilic M.A.
      • Spiro S.
      • Moore G.R.
      ). Further high resolution structural data on iron-bound forms of the ferroxidase center are now required to further address the fate of Fe3+ following oxidation at the ferroxidase center.
      The ethylene glycol on the inner surface of the E128R/E135R variant is close to the negatively charged residues Glu47, Asp50, and Asp126 that have been speculated previously as participating either in guiding of Fe2+ to the unoccupied dinuclear iron center or nucleation of the iron core (
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      ,
      • Baaghil S.
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      ,
      • Malone S.A.
      • Lewin A.
      • Kilic M.A.
      • Svistunenko D.A.
      • Cooper C.E.
      • Wilson M.T.
      • Le Brun N.E.
      • Spiro S.
      • Moore G.R.
      ), although clear evidence that these residues are involved in such processes has not been reported until now. To evaluate the potential functional role of these residues in the E128R/E135R variant, the ferroxidase kinetics of variants in which residues Glu47, Asp50, and Asp126 were replaced individually and in combination by the corresponding amides were investigated. Stopped flow measurements established that the E47Q and E47Q/D50N/D126N variants possess markedly decreased Phase 2 iron oxidation activity relative to the wild-type protein and the D50N and D126N variants. These results clearly support a significant role for Glu47 in the ferroxidase activity of BFR. Because Glu47 is not a ferroxidase center ligand, the mechanistic origin of this effect is not clear. For example, Glu47 could play a role in guiding iron to the ferroxidase center, which may result simply from the closer proximity of this residue to FE2 than is the case for Asp50 or Asp126 as noted above, although the difference in orientation of this residue in the current structure and in protein with both irons present (
      • van Eerde A.
      • Wolterink-van Loo S.
      • van der Oost J.
      • Dijkstra B.W.
      ) may also be a factor. Both D50N and D126N exhibit diminished reactivity during Phase 3 of Fe2+ oxidation. This result supports a role for Asp50 and Asp126 in the nucleation step of BFR iron core formation. Notably, these modifications of the electrostatic properties of the inner surface of the dimeric variant are sufficient to impede oxidation of Fe2+ in the absence of changes to the corresponding outer surface of the protein adjacent to the outer ferroxidase pore.
      To evaluate the possibility that the acidic residues studied in the current work might participate similarly in the ferroxidase activity of other bacterioferritins, the sequences of several members of this protein family are aligned (
      • Larkin M.A.
      • Blackshields G.
      • Brown N.P.
      • Chenna R.
      • McGettigan P.A.
      • McWilliam H.
      • Valentin F.
      • Wallace I.M.
      • Wilm A.
      • Lopez R.
      • Thompson J.D.
      • Gibson T.J.
      • Higgins D.G.
      ) in Fig. 8. From this analysis, it is clear that each of these residues is highly but not absolutely conserved, so it seems highly likely that the functional contributions of these residues observed in the present study would also be observed for these other species of the protein. In those unusual cases where one of these acidic residues is not conserved, an adjacent acidic residue may occupy the same stereochemical position, but confirmation of this possibility must await detailed characterization of these other proteins.
      Figure thumbnail gr8
      FIGURE 8Alignment of amino acid sequences of several bacterioferritins. The acidic residues replaced in the current work are indicated by arrows. The intensity of the background shading is proportional to the extent of conservation.

      Acknowledgments

      We thank Dr. David S. Waugh (National Cancer Institute-Frederic Cancer Research and Development Center) for the plasmid for expression of the TEV protease and Dr. Susanne Ludwiczek for samples of the purified TEV protease.

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