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Mechanism of Ferrous Iron Binding and Oxidation by Ferritin from a Pennate Diatom*

  • Stephanie Pfaffen
    Affiliations
    From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and
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  • Raz Abdulqadir
    Affiliations
    the Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom
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  • Nick E. Le Brun
    Affiliations
    the Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom
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  • Michael E.P. Murphy
    Correspondence
    To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
    Affiliations
    From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and
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  • Author Footnotes
    * This work was supported by a Natural Sciences and Engineering Research Council of Canada discovery grant and the Canadian Foundation for Innovation (to M. E. P. M.) and Biotechnology and Biological Sciences Research Council Grant BB/I021884/1 (to N. E. L. B.).
Open AccessPublished:April 02, 2013DOI:https://doi.org/10.1074/jbc.M113.454496
      A novel ferritin was recently found in Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65–2.2-Å resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.
      Background: Ferritin stores iron by ferroxidation to form a mineral core, enabling diatom blooms upon iron input.
      Results: Ferrous iron binds solely to ferroxidase site A anaerobically. Ferroxidation kinetics has two observed phases.
      Conclusion: Ferrous iron and dioxygen binding to the di-iron ferroxidase site is stepwise.
      Significance: Iron storage by ferritins requires a coordinated binding of iron and dioxygen.

      Introduction

      Ferritin is a ubiquitous, iron storage, and detoxifying protein found in mammals, plants, and many microorganisms. Ferritin is a 24-mer that forms a hollow sphere, takes up soluble ferrous iron, oxidizes it at di-iron ferroxidase centers, and stores the iron oxide mineral in its central cavity. Iron is subsequently released upon demand of the organism's metabolism (
      • Carrondo M.A.
      Ferritins, iron uptake and storage from the bacterioferritin viewpoint.
      ,
      • Liu X.
      • Theil E.C.
      Ferritins: dynamic management of biological iron and oxygen chemistry.
      ). Mammalian ferritins are heteropolymers of two homologous monomers that can store thousands of iron atoms in the central cavity. The heavy (H)
      The abbreviations used are: H, heavy; PmFTN, P. multiseries ferritin; EcBFR, E. coli bacterioferritin; EcFtnA, E. coli ferritin; TCEP, tris(2-carboxyethyl)phosphine; Fe (5), Fe (45), Fe (75), Fe (2 h), Fe (4 h), and Fe (o.n.) refer to the structures of PmFTN from crystals soaked in solutions of ferrous iron for 5 min, 45 min, 75 min, 2 h, 4 h, and overnight, respectively; Zn (1 h), refers to the structure of PmFTN from crystals soaked in Zn(II) for 1 h.
      chain contains the ferroxidase site, whereas the light chain promotes iron core formation (
      • Hempstead P.D.
      • Yewdall S.J.
      • Fernie A.R.
      • Lawson D.M.
      • Artymiuk P.J.
      • Rice D.W.
      • Ford G.C.
      • Harrison P.M.
      Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution.
      ). In contrast, bacterial ferritins are homopolymers in which the monomer contains both the ferroxidase site and the mineral nucleation sites. The homologous bacterioferritins additionally contain a heme group between two monomers that functions principally in iron release (
      • Yasmin S.
      • Andrews S.C.
      • Moore G.R.
      • Le Brun N.E.
      A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral.
      ). Storage of iron is the main function of ferritins, and detoxification of iron and reactive oxygen species is a secondary protective function utilized under extreme oxidative stress (
      • Theil E.C.
      ). However, whether these roles constitute the primary function of bacterioferritins remains to be proven (
      • Carrondo M.A.
      Ferritins, iron uptake and storage from the bacterioferritin viewpoint.
      ,
      • Liu X.
      • Theil E.C.
      Ferritins: dynamic management of biological iron and oxygen chemistry.
      ).
      Recently, ferritin homologs were identified in five species of pennate diatoms but not in other stramenopiles using a PCR approach (
      • Marchetti A.
      • Parker M.S.
      • Moccia L.P.
      • Lin E.O.
      • Arrieta A.L.
      • Ribalet F.
      • Murphy M.E.
      • Maldonado M.T.
      • Armbrust E.V.
      Ferritin used for iron storage in bloom-forming marine pennate diatoms.
      ). A phylogenetic analysis of diatom ferritin sequences with those from both prokaryotes and eukaryotes showed that diatom ferritins are clearly distinct from other eukaryotic ferritins but may be weakly associated with prokaryotic ferritins (
      • Marchetti A.
      • Parker M.S.
      • Moccia L.P.
      • Lin E.O.
      • Arrieta A.L.
      • Ribalet F.
      • Murphy M.E.
      • Maldonado M.T.
      • Armbrust E.V.
      Ferritin used for iron storage in bloom-forming marine pennate diatoms.
      ). Diatoms are unicellular photosynthetic organisms that play a major role in global primary production and carbon sequestration into the deep ocean (
      • Falkowski P.G.
      • Barber R.T.
      • Smetacek V.V.
      Biogeochemical controls and feedbacks on ocean primary production.
      ). In many offshore areas of the open ocean, primary productivity and therefore CO2 uptake from the atmosphere is limited due to iron availability. These regions are sporadically pulsed with new iron inputs from dust or upwelling deep waters. Pennate diatoms readily bloom upon such iron additions and continue to grow and divide after iron levels return to a low and ambient level (
      • Martin J.H.
      • Fitzwater S.E.
      Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic.
      ). The expression of ferritin is thought to facilitate the blooming of pennate diatoms after iron fertilization in the open ocean.
      A crystal structure of recombinant, iron-soaked ferritin derived from the pennate diatom Pseudo-nitzschia multiseries (PmFTN) was resolved at 1.95-Å resolution (
      • Marchetti A.
      • Parker M.S.
      • Moccia L.P.
      • Lin E.O.
      • Arrieta A.L.
      • Ribalet F.
      • Murphy M.E.
      • Maldonado M.T.
      • Armbrust E.V.
      Ferritin used for iron storage in bloom-forming marine pennate diatoms.
      ). The structure confirmed the characteristic ferritin ferroxidase center, monomeric fold, and spherical assembly. Nevertheless, the ferroxidase center found in PmFTN shows key differences from those of other ferritins of known structure. Typical eukaryotic H chain ferritins have a di-iron ferroxidase center (
      • Bertini I.
      • Lalli D.
      • Mangani S.
      • Pozzi C.
      • Rosa C.
      • Theil E.C.
      • Turano P.
      Structural insights into the ferroxidase site of ferritins from higher eukaryotes.
      ); however, three iron atoms are observed in and around the PmFTN ferroxidase center: one is found in ferroxidase site B, and the other two are positioned toward the core. An unexpected finding was that the ferroxidase site A is occupied by a water molecule. The iron atom found at site B is coordinated by three glutamate residues (Glu-48, Glu-94, and Glu-130) conserved in all ferritins. A unique site C is found in PmFTN at which iron is coordinated by only one glutamate residue (Glu-44). A glutamate is found at position 44 only in diatoms and cyanobacteria, and moreover, no third iron site is found in human H chain ferritin or other eukaryotic ferritins.
      To get a better understanding of the ferroxidase reaction and iron binding in PmFTN, we have determined the x-ray structures of several PmFTN crystals soaked for various durations in ferrous iron and zinc sulfate under aerobic and anaerobic conditions. Furthermore, stopped-flow kinetic analysis was applied to determine reaction phases of the ferroxidase reaction and to understand the iron oxidation mechanism in PmFTN.

      DISCUSSION

      Iron storage ferritins are the archetype of the ferritin-like superfamily of proteins, which includes the enzymes ribonucleotide reductase and methane monooxygenase. A common feature is a catalytic di-iron site where dioxygen is reduced. In ferritin, Fe(II) is the source of electrons, and the resulting oxidized Fe(III) is deposited in the mineral core. A key question in ferritin function is the mechanism of iron mobilization in and out of the ferroxidase site and the core. Iron was visualized in the ferroxidase site (sites A and B) and a third site C of PmFTN. The residues interacting with the iron in site A (Glu-15, Glu-48, and His-51) are conserved with those of all characterized eukaryotic and prokaryotic ferritins. In addition to Glu-48, Glu-94 coordinates iron in site B and is also conserved.
      A third iron site is generally not associated with ferritin ferroxidase sites from vertebrates; however, a site C has been observed in some non-heme ferritins from bacteria and archaea, for example in E. coli (EcFtnA) (
      • Treffry A.
      • Zhao Z.
      • Quail M.A.
      • Guest J.R.
      • Harrison P.M.
      How the presence of three iron binding sites affects the iron storage function of the ferritin (EcFtnA) of Escherichia coli.
      ,
      • Stillman T.J.
      • Hempstead P.D.
      • Artymiuk P.J.
      • Andrews S.C.
      • Hudson A.J.
      • Treffry A.
      • Guest J.R.
      • Harrison P.M.
      The high-resolution x-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe3+ and Zn2+ derivatives.
      ), Pyrococcus furiosus (
      • Tatur J.
      • Hagen W.R.
      • Matias P.M.
      Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus.
      ), and Archaeoglobus fulgidus (
      • Johnson E.
      • Cascio D.
      • Sawaya M.R.
      • Gingery M.
      • Schröder I.
      Crystal structures of a tetrahedral open pore ferritin from the hyperthermophilic archaeon Archaeoglobus fulgidus.
      ). Nevertheless, these sites differ from that of PmFTN in terms of the number and origin of glutamate residues. Of these residues, Glu-130 is in common, is observed to bridge the iron atom of sites B and C (Fig. 3), and is a conserved residue in prokaryotic ferritins. In the PmFTN structure soaked aerobically for more than 4 h, Glu-130 is a ligand to iron atoms in sites B and C. In contrast, in the earlier PmFTN structure soaked in iron for 10 min by Marchetti et al. (
      • Marchetti A.
      • Parker M.S.
      • Moccia L.P.
      • Lin E.O.
      • Arrieta A.L.
      • Ribalet F.
      • Murphy M.E.
      • Maldonado M.T.
      • Armbrust E.V.
      Ferritin used for iron storage in bloom-forming marine pennate diatoms.
      ), the iron in site C is coordinated by only Glu-44. In all known prokaryotic ferritins, Glu-44 is substituted by histidine. The equivalent His-46 in EcFtnA is proposed to orient Glu-130 so it can bind iron ions in sites B and C as well as gating the passage of the metal through these sites (
      • Stillman T.J.
      • Hempstead P.D.
      • Artymiuk P.J.
      • Andrews S.C.
      • Hudson A.J.
      • Treffry A.
      • Guest J.R.
      • Harrison P.M.
      The high-resolution x-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe3+ and Zn2+ derivatives.
      ). Glu-44 and Glu-130 in PmFTN may have a similar function of gating the passage of iron from site B to site C.
      As the aerobic iron soaking time increases from minutes to hours, iron is observed first in site B followed by site A and eventually occupies all three sites (Table 2). In contrast, only site A is occupied by Fe(II) in crystals of PmFTN under anaerobic conditions even though the crystals were exposed to 2 mm Fe(II) for over an hour (Fig. 2A). This observation is in contrast to short (∼1-min) aerobic Fe(II) soaking experiments with frog ferritin in which both sites A and B are occupied (
      • Bertini I.
      • Lalli D.
      • Mangani S.
      • Pozzi C.
      • Rosa C.
      • Theil E.C.
      • Turano P.
      Structural insights into the ferroxidase site of ferritins from higher eukaryotes.
      ). Interestingly, the interiron distance in some subunits of frog ferritin is comparable with that observed with Cu(II) as a proxy for Fe(II) (∼4.3 Å). Longer exposure to ferrous iron results in a shortening of the di-iron interatomic iron distance to ∼3.1 Å. In PmFTN, iron occupancy at both sites is only observed after prolonged iron exposure, and the site A and B intermetal distance of less than 3.8 Å decreases only slightly with time to 3.6 Å, suggesting that in the structures where iron is bound to both sites it is in the Fe(III) state. Note that with the data presented here we are not able to directly determine the oxidation state of iron bound to the crystals under aerobic conditions, including that in site B in Fe (5). A single high affinity and two low affinity Fe(II) binding sites were identified in P. furiosus ferritin by calorimetry in the absence of dioxygen (
      • Honarmand Ebrahimi K.
      • Bill E.
      • Hagedoorn P.L.
      • Hagen W.R.
      The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.
      ). Site-directed mutagenesis was used to propose assignment of the high affinity site to site A, consistent with our anaerobic crystallographic observations in PmFTN.
      Kinetic measurements of PmFTN iron oxidation revealed an extremely rapid initial oxidation phase involving the binding and oxidation of two ferrous iron. The first order dependence of the rate of ferroxidase center oxidation on the concentration of Fe(II) demonstrates a close link between binding and oxidation events such that they cannot be distinguished. Thus, oxidation occurs immediately upon Fe(II) binding to PmFTN, and the binding event can be viewed as the slow step of the reaction. This is in contrast to previous reports of ferritins in which binding and oxidation are considered to be kinetically distinct events. Measurement of Fe(II) binding kinetics is not generally straightforward, although it was possible for EcBFR because Fe(II) binding caused a perturbation of absorbance due to the heme groups. In that case, Fe(II) binding occurred on a much shorter time scale than the subsequent Fe(II) oxidation. Interestingly, Fe(II) binding to EcBFR occurred with a second order rate constant of 2.5 × 105 m−1 s−1 (at 30 °C) (
      • Treffry A.
      • Zhao Z.
      • Quail M.A.
      • Guest J.R.
      • Harrison P.M.
      Dinuclear center of ferritin: studies of iron binding and oxidation show differences in the two iron sites.
      ), a value similar to that measured here (at 25 °C) for PmFTN-catalyzed Fe(II) oxidation.
      An oximetric assay previously showed that the ferroxidase reaction of PmFTN is associated with consumption of dioxygen in a ratio of 1.9 ± 0.2 Fe(II):O2 (
      • Marchetti A.
      • Parker M.S.
      • Moccia L.P.
      • Lin E.O.
      • Arrieta A.L.
      • Ribalet F.
      • Murphy M.E.
      • Maldonado M.T.
      • Armbrust E.V.
      Ferritin used for iron storage in bloom-forming marine pennate diatoms.
      ). Furthermore, addition of catalase to the assay solution resulted in the stoichiometric regeneration of O2, indicating production of H2O2 by the ferroxidase reaction as seen with ferritins (
      • Yang X.
      • Chen-Barrett Y.
      • Arosio P.
      • Chasteen N.D.
      Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins.
      ) but not bacterioferritins (
      • Yang X.
      • Le Brun N.E.
      • Thomson A.J.
      • Moore G.R.
      • Chasteen N.D.
      The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin.
      ) or Dps ferritins (
      • Yang X.
      • Chiancone E.
      • Stefanini S.
      • Ilari A.
      • Chasteen N.D.
      Iron oxidation and hydrolysis reactions of a novel ferritin from Listeria innocua.
      ). In contrast, the oxidation stoichiometry of EcFtnA is 3–4 Fe(II) ions per O2, which is suggested to be a consequence of the binding of three iron atoms (one at site C) and a possible fourth iron at an unknown metal site, leading to reduction of O2 to water rather than hydrogen peroxide (
      • Treffry A.
      • Zhao Z.
      • Quail M.A.
      • Guest J.R.
      • Harrison P.M.
      How the presence of three iron binding sites affects the iron storage function of the ferritin (EcFtnA) of Escherichia coli.
      ). The ferroxidase reaction in EcFtnA is similar to that observed in human H chain ferritin, although it is more complex due to the third iron in site C (
      • Stillman T.J.
      • Hempstead P.D.
      • Artymiuk P.J.
      • Andrews S.C.
      • Hudson A.J.
      • Treffry A.
      • Guest J.R.
      • Harrison P.M.
      The high-resolution x-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe3+ and Zn2+ derivatives.
      ). Site C, however, is not essential for ferroxidase activity in EcFtnA as site C variants showed only a slight decrease in the overall oxidation rate but the expected stoichiometry of two ferrous iron per dioxygen (
      • Treffry A.
      • Zhao Z.
      • Quail M.A.
      • Guest J.R.
      • Harrison P.M.
      How the presence of three iron binding sites affects the iron storage function of the ferritin (EcFtnA) of Escherichia coli.
      ). In contrast, although a third iron site is present in PmFTN, a 2:1 Fe:O2 stoichiometry is retained. The kinetic data reported here support the conclusion that only two Fe(II) ions are initially oxidized per subunit. A key structural difference is that site C in PmFTN is only 3.5–3.7 Å from site B, whereas site C in EcFtnA is 7–8 Å from the A/B pair. Third iron binding sites were observed in EcBFR as well as human mitochondrial ferritin (
      • Crow A.
      • Lawson T.L.
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      Structural basis for iron mineralization by bacterioferritin.
      ,
      • Langlois d'Estaintot B.
      • Santambrogio P.
      • Granier T.
      • Gallois B.
      • Chevalier J.M.
      • Précigoux G.
      • Levi S.
      • Arosio P.
      Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala.
      ). However, these iron sites were observed to be at the core surface and are more likely involved in the nucleation/mineralization process rather than in ferroxidase center-catalyzed iron oxidation (
      • Wong S.G.
      • Tom-Yew S.A.
      • Lewin A.
      • Le Brun N.E.
      • Moore G.R.
      • Murphy M.E.
      • Mauk A.G.
      Structural and mechanistic studies of a stabilized subunit dimer variant of Escherichia coli bacterioferritin identify residues required for core formation.
      ).
      Only ferroxidase site A is occupied by ferrous iron in the anaerobic crystal; however, stopped-flow data show saturation of the rapid phase 2 after binding of 2 ferrous iron eq per monomer of PmFTN. Together these results point to stepwise binding of the ferrous iron and dioxygen to the ferroxidase site. A model can be proposed in which one ferrous iron binds to site A followed by the binding of the oxidant. Only when the latter is bound can the second ferrous iron bind to site B. Thus, at the moderate iron concentrations (2 mm) used for soaking experiments, a second Fe(II) ion is not observed at the center in the absence of the oxidant (dioxygen). We note that a similar model was proposed for the two Dps proteins from Bacillus anthracis (
      • Schwartz J.K.
      • Liu X.S.
      • Tosha T.
      • Diebold A.
      • Theil E.C.
      • Solomon E.I.
      CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers.
      ). These are 12-mer (mini)ferritins that contain intersubunit dinuclear ferroxidase centers that are distinct from those of the 24-mer (maxi)ferritins but nevertheless share some common features. For PmFTN, such a model accounts for the observed rate dependence on Fe(II) because once the second Fe(II) binds oxidation can proceed immediately. Thus, ferrous iron binding to site B of the ferroxidase site is proposed to be the rate-determining step.
      Evolution of a rapid dioxygen-driven reaction is of value to an organism living in an iron -limited environment. Under low oxygen conditions, a PmFTN 24-mer would sequester at most 24 ferrous ions, making more iron available for metabolic processes. In the presence of dioxygen and an environmental iron pulse, pre-existing PmFTN would rapidly store newly accumulated iron, preventing the generation of reactive oxygen species and providing a buffer for induction of PmFTN expression.
      Zn(II) is an inhibitor of EcFtnA and is proposed to compete with ferrous iron for the dinuclear center and consequently inhibit oxidation at these sites (
      • Treffry A.
      • Zhao Z.
      • Quail M.A.
      • Guest J.R.
      • Harrison P.M.
      The use of zinc(II) to probe iron binding and oxidation by the ferritin (EcFtnA) of Escherichia coli.
      ). In the crystal structure, Zn(II) does bind to sites A and B but is not observed in site C (
      • Stillman T.J.
      • Hempstead P.D.
      • Artymiuk P.J.
      • Andrews S.C.
      • Hudson A.J.
      • Treffry A.
      • Guest J.R.
      • Harrison P.M.
      The high-resolution x-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe3+ and Zn2+ derivatives.
      ). Nonetheless, from the Fe:O2 stoichiometry of the EcFtnA reaction, all three sites were proposed to bind Fe(II) during catalysis. Crystal structures of Zn(II) complexes of EcBFR and human mitochondrial ferritin have Zn(II) bound at sites A and B of the ferroxidase center, consistent with a proposed model of Fe(II) binding (
      • Langlois d'Estaintot B.
      • Santambrogio P.
      • Granier T.
      • Gallois B.
      • Chevalier J.M.
      • Précigoux G.
      • Levi S.
      • Arosio P.
      Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala.
      ,
      • Willies S.C.
      • Isupov M.N.
      • Garman E.F.
      • Littlechild J.A.
      The binding of haem and zinc in the 1.9 Å x-ray structure of Escherichia coli bacterioferritin.
      ). However, anaerobic Fe(II) complexes for these systems are not available in the literature. We have directly compared Zn(II) and Fe(II) binding in PmFTN (Fig. 2). Zn(II) bound to sites A, B, and C in contrast to the two sites observed in EcFtnA. Furthermore, overall Zn(II) occupancy of the three metal sites resembles iron bound in the presence of dioxygen in PmFTN rather than mimicking Fe(II) binding. Thus, the use of Zn(II) and likely other metal ions as analogs of Fe(II)/Fe(III) may not identify the correct binding sites in other ferritins.
      Two models have been proposed for the mechanism of ferroxidation by ferritins and bacterioferritins. In one model, the ferroxidase site functions as a substrate site as seen in human H ferritin (
      • Treffry A.
      • Hirzmann J.
      • Yewdall S.J.
      • Harrison P.M.
      Mechanism of catalysis of Fe(II) oxidation by ferritin H chains.
      ), EcFtnA (
      • Bauminger E.R.
      • Harrison P.M.
      • Hechel D.
      • Nowik I.
      • Treffry A.
      Mossbauer spectroscopic investigation of structure-function relations in ferritins.
      ), and frog M ferritin (
      • Hwang J.
      • Krebs C.
      • Huynh B.H.
      • Edmondson D.E.
      • Theil E.C.
      • Penner-Hahn J.E.
      A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay?.
      ). Ferrous iron binds to the ferroxidase site, and after oxidation, ferric iron rapidly migrates to the mineral core. In a second model, first described for EcBFR (
      • Crow A.
      • Lawson T.L.
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      Structural basis for iron mineralization by bacterioferritin.
      ) and P. furiosus ferritin (
      • Tatur J.
      • Hagen W.R.
      The dinuclear iron-oxo ferroxidase center of Pyrococcus furiosus ferritin is a stable prosthetic group with unexpectedly high reduction potentials.
      ), the ferroxidase site is a stable di-iron site that functions as a cofactor after the binding of 2 eq of ferrous iron per subunit. Additional ferrous ions are then added directly to the mineral core, and the ferroxidase site functions solely in oxygen or peroxide reduction. Recently, Honarmand et al. (
      • Honarmand Ebrahimi K.
      • Bill E.
      • Hagedoorn P.L.
      • Hagen W.R.
      The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.
      ) proposed a unifying mechanism in which the Fe(III) product at the ferroxidase site remains bound to the ferroxidase site but is rapidly displaced by incoming Fe(II). A prediction of this revised model is the observation of a fully Fe(III)-loaded ferroxidase site in crystals after prolonged soaking in Fe(II). The ferroxidase site of PmFTN was not fully occupied after soaking aerobic crystals in ferrous iron for 45 min, suggesting that iron movement occurred at the ferroxidase site during iron loading with 2 mm ferrous iron over an extended time period. Small absorbance changes immediately following oxidation of Fe(II) at the ferroxidase center are consistent with this conclusion. Nevertheless, for PmFTN, the rapid oxidation of ferrous iron was not regenerated upon up to 20 h incubation, indicating that iron remains present at least in part at the ferroxidase site. Thus, if Fe(III) is displaced, the subsequent iron oxidation is much slower than the initial oxidation. Thus, the mechanism of mineralization in PmFTN appears to be more complex with partial iron migration to the core.
      Our data indicate that site A of the ferroxidase center has a higher affinity than site B for Fe(II) under anaerobic conditions. The two distinct kinetic phases observed after the second addition of 48 Fe(II) ions may also be related to slow iron migration to the core likely involving the third iron binding site (site C) and perhaps other sites along a path to the cavity. In frog ferritin, the transit of Fe(III) from the ferroxidase center to the cavity has been shown to occur via a pathway through the subunit toward a 4-fold channel (
      • Turano P.
      • Lalli D.
      • Felli I.C.
      • Theil E.C.
      • Bertini I.
      NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin.
      ). Site C may function to direct Fe(III) along a different path to the mineral core. In EcBFR, the two ferroxidase center sites were fully occupied after 2.5 min of soaking, and occupancy was not affected following oxidation, suggesting that a distinct mechanism is in operation (
      • Crow A.
      • Lawson T.L.
      • Lewin A.
      • Moore G.R.
      • Le Brun N.E.
      Structural basis for iron mineralization by bacterioferritin.
      ).

      Acknowledgments

      Portions of this work were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the United States Department of Energy Office of Science by Stanford University. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the United States Department of Energy Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393), and the National Center for Research Resources (Grant P41RR001209).

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