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Structural Basis for Iron Mineralization by Bacterioferritin Allister Crow,† Tamara L

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Structural Basis for Iron Mineralization by Bacterioferritin Allister Crow,† Tamara L Published on Web 04/24/2009 Structural Basis for Iron Mineralization by Bacterioferritin Allister Crow,† Tamara L. Lawson,‡ Allison Lewin, Geoffrey R. Moore, and Nick E. Le Brun* Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, UniVersity of East Anglia, Norwich NR4 7TJ, U.K. Received November 30, 2008; E-mail: [email protected] Abstract: Ferritin proteins function to detoxify, solubilize and store cellular iron by directing the synthesis of a ferric oxyhydroxide mineral solubilized within the protein’s central cavity. Here, through the application of X-ray crystallographic and kinetic methods, we report significant new insight into the mechanism of mineralization in a bacterioferritin (BFR). The structures of nonheme iron-free and di-Fe2+ forms of BFR showed that the intrasubunit catalytic center, known as the ferroxidase center, is preformed, ready to accept Fe2+ ions with little or no reorganization. Oxidation of the di-Fe2+ center resulted in a di-Fe3+ center, with bridging electron density consistent with a µ-oxo or hydro bridged species. The µ-oxo bridged di-Fe3+ center appears to be stable, and there is no evidence that Fe3+species are transferred into the core from the ferroxidase center. Most significantly, the data also revealed a novel Fe2+ binding site on the inner surface of the protein, lying ∼10 Å directly below the ferroxidase center, coordinated by only two residues, His46 and Asp50. Kinetic studies of variants containing substitutions of these residues showed that the site is functionally important. In combination, the data support a model in which the ferroxidase center functions as a true catalytic cofactor, rather than as a pore for the transfer of iron into the central cavity, as found for eukaryotic ferritins. The inner surface iron site appears to be important for the transfer of electrons, derived from Fe2+ oxidation in the cavity, to the ferroxidase center. Bacterioferritin may represent an evolutionary link between ferritins and class II di-iron proteins not involved in iron metabolism. Introduction shown to function as a gated site for the transfer of Fe3+ into the cavity following oxidation5,6 and is required for core The ability to store iron in a bioavailable and safe form is a nucleation.6 central feature of the strategy Nature has evolved to overcome Bacterioferritins (BFRs), which are unique among ferritins the dual problem of poor bioavailability of iron and the potential in that they contain up to 12 b-type heme groups,7 are composed of the free metal to catalyze the formation of extremely reactive of 24 H-chain-like subunits. However, the BFR ferroxidase 1 radical species. The function of iron storage is fulfilled by the center is distinct from those of other H-chain subunits, bearing Publication Date (Web): April 24, 2009 | doi: 10.1021/ja8093444 ferritin family of proteins, in which subunits are arranged in a a much closer relationship to the centers of class II di-iron highly symmetric fashion to form an approximately spherical proteins such as ribonucleotide reductase and soluble methane protein coat surrounding a hollow center. Large amounts of iron monooxygenase.7,8 These structural differences result in distinct can be stored within the central cavity, in the form of a ferric- mechanistic and functional properties. For example, spectro- Downloaded by CARNEGIE MELLON UNIV on September 22, 2009 | http://pubs.acs.org oxy-hydroxide mineral core.2,3 scopic and kinetic studies showed that the oxidized form of the Mineralization occurs when iron, as Fe2+, is taken up by the ferroxidase center of Escherichia coli BFR is stable and is 9-11 protein, oxidized to Fe3+, and hydrated. In 24-meric ferritins, a required throughout core formation. dinuclear metal ion binding site, known as the ferroxidase center, To gain further insight into the mechanism of mineralization which is found within H-chain-type subunits (but not in in BFR, we have applied a combination of X-ray crystallography L-chains, the other major subunit type), is key to this activity.4 and kinetic methods. We report the high resolution structural In eukaryotic H-chain ferritins the ferroxidase center has been (5) Bou-Abdallah, F.; Zhao, G. H.; Mayne, H. R.; Arosio, P.; Chasteen, N. D. J. Am. Chem. Soc. 2005, 127, 3885–3893. † Current address: Department of Biological Chemistry, John Innes (6) Zhao, G. H.; Bou-Abdallah, F.; Arosio, P.; Levi, S.; Janus-Chandler, Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. C.; Chasteen, N. D. Biochemistry 2003, 42, 3142–3150. ‡ Current address: Department of Biochemistry and Molecular Biology, (7) Frolow, F.; Kalb, A. J.; Yariv, J. Nat. Struct. Biol. 1994, 1, 453–460. University of British Columbia, Life Sciences Centre, 2350 Health Sciences (8) Le Brun, N. E.; Andrews, S. C.; Guest, J. R.; Harrison, P. M.; Moore, Mall, Vancouver, BC V6T 1Z3 Canada. G. R.; Thomson, A. J. Biochem. J. 1995, 312, 385–392. (1) Lewin, A.; Moore, G. R.; Le Brun, N. E. Dalton Trans. 2005, 3597– (9) Le Brun, N. E.; Wilson, M. T.; Andrews, S. C.; Guest, J. R.; Harrison, 3610. P. M.; Thomson, A. J.; Moore, G. R. FEBS Lett. 1993, 333, 197– (2) Andrews, S. C. Iron storage in bacteria; Academic Press: 1998; Vol. 202. 40, pp 281-351. (10) Yang, X.; Le Brun, N. E.; Thomson, A. J.; Moore, C. R.; Chasteen, (3) Theil, E. C. AdV. Enzymol. Relat. Areas Mol. Biol. 1990, 63, 421– N. D. Biochemistry 2000, 39, 4915–4923. 449. (11) Baaghil, S.; Lewin, A.; Moore, G. R.; Le Brun, N. E. Biochemistry (4) Chasteen, N. D. Metal Ions Biol. Sys. Vol. 35, 1998, 35, 479-514. 2003, 42, 14047–14056. 6808 9 J. AM. CHEM. SOC. 2009, 131, 6808–6813 10.1021/ja8093444 CCC: $40.75 2009 American Chemical Society Fe Mineralization by Bacterioferritin ARTICLES characterization of nonheme iron free, di-Fe2+, and µ-oxo were applied throughout refinement and the same set of reflections 3+ bridged di-Fe forms of the catalytic ferroxidase center and a (5%) were used in calculating Rfree in all structures. For structures novel iron site located on the inner surface of the protein, of crystals soaked in metal solutions, bound metals were identified relatively close to the ferroxidase center, in which the iron is by a combination of map-based techniques including the following: ligated by only two residues, His46 and Asp50. Site-directed analysis of electron density maps in the refined structures; anomalous-difference Fourier analysis (using X-ray data collec- variants of BFR lacking one or both of these residues are ted at the relevant absorption edge); isomorphous-difference Fourier significantly disrupted in their capacity to form an iron core analysis; and, where metal-binding was detected, omit map analysis but are essentially unaffected in terms of their ferroxidase center showing “unbiased” electron density for the bound ligands. activities. A model is discussed in which both the inner surface Anomalous difference, isomorphous difference, and omit maps were iron site and the ferroxidase center are essential for mineralization. calculated with FFT,20 and 12-fold NCS-averaged versions of these maps were created using COOT. Metal-soak data sets were scaled Experimental Section to the apo data set using SCALEIT and isomorphous difference maps calculated using the difference in observed amplitudes Strains, Growth Media, and Site-Directed Mutagenesis. between metal-soaked and apo data sets with phases derived from - 11 Escherichia coli strains JM109 and AL1 (BL21(DE3) bfr ) were the apo-BFR structure; 2|Fo| - |Fc| omit maps used phases and |Fc| used for site-directed mutagenesis and expression, respectively, and values derived from models in which the bound metal ions were grown at 37 °C in LB (Luria-Bertani) broth, on LA plates omitted. Structural figures were produced using PYMOL21 and consisting of LB broth with 1.25% (w/v) agar. Ampicillin, where annotated with GIMP. Atomic coordinates have been deposited in appropriate, was used at a concentration of 100 mg/L. Site directed the Protein Data Bank (PDB ID codes: 3E1J (apo), 3E1L mutants encoding H46A and D50A BFR were generated using a (Phosphate-soak), 3E1O (Zn2+-soak), 3E1P (Zn2+/Fe2+-soak), 3E1 whole-plasmid method, employing pALN1 (a pET21a derivative M (Fe2+-short soak), 3E1N (Fe2+-long soak)). containing wild-type bfr, generated by ligating the NdeI and EcoRI Kinetic Methods. BFR catalyzed Fe2+ oxidation was measured fragment from pGS75810 into pET21a (Novagen) cut with the same at 340 nm using either a conventional UV-visible spectrophotom- enzymes) as a template. Resulting plasmids encoding H46A BFR eter (Perkin-Elmer λ35 or Hitachi U2900), for which Fe2+ additions (pTLN8) and D50A BFR (pALN39) were confirmed by sequencing were made using a microsyringe (Hamilton), or by using a stopped- (MWG Biotech). A pET21a-derived construct (pTLN10) encoding flow apparatus (Applied Photophysics DX17MV). For multiple Fe2+ the double variant H46A/D50A was obtained directly from Gen- additions, oxidation was allowed to go to completion (constant Script (New Jersey, USA). A340 nm value) before centrifuging samples (10 000 rpm, 5 min) to Protein Purification and the Removal of Nonheme Iron. Wild- remove any Fe3+ not associated with the protein prior to subsequent type, H46A, D50A, and H46A/D50A BFRs were prepared as additions. Rates of phase 3 Fe2+ oxidation were calculated from 11 11 previously described. Nonheme iron present on purification was initial, linear increases in A340 nm per unit time. removed by treatment with sodium dithionite and bipyridyl.12 Heme contents of nonheme iron-free proteins, determined through the Results and Discussion -1 heme Soret absorbance intensity, using ε418 nm ) 107 000 M - The Metal-Free Ferroxidase Center is Preformed. cm 1,9 were found to be <1 for wild-type BFR, ∼1.5 for H46A BFR was and D50A BFRs, and ∼1.7 for H46A/D50A BFR.
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