Ferrous ion binding to recombinant human H-chain ferritin. An isothermal titration calorimetry study

Iron deposition within the iron storage protein ferritin involves a complex series of events consisting of Fe(2+) binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrous ferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamic properties of Fe(2+) binding to recombinant human H-chain apoferritin (HuHF) by isothermal titration calorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the protein and the principal pathways by which the Fe(2+) travels to the dinuclear ferroxidase center prior to its oxidation to Fe(3+). Calorimetric titrations show that the ferroxidase center is the principal locus for Fe(2+) binding with weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely the His65 containing A-site, preferentially binds Fe(2+). That only one site of the ferroxidase center is occupied by Fe(2+) implies that Fe(2+) oxidation to form diFe(III) species might occur in a stepwise fashion. In dilute anaerobic protein solution (3-5 microM), only 12 Fe(2+)/protein bind at pH 6.51 increasing to 24 Fe(2+)/protein at pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding of Fe(2+) to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe(2+) binding. The total Fe(2+) binding capacity of the protein is reduced in the 3-fold hydrophilic channel variant S14 (D131I+E134F), indicating that the primary avenue by which Fe(2+) gains access to the interior of ferritin is through these eight channels. The binding stoichiometry of the channel variant is one-third that of the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe(2+) binding are similar for the two with an average values (DeltaH degrees = 7.82 kJ/mol, binding constant K = 1.48 x 10(5) M(-)(1) at pH 7.04). Since channel mutations do not completely prevent Fe(2+) binding to the ferroxidase center, iron gains access to the center in approximately one-third of the channel variant molecules by other pathways.     

Metal binding sites of human H‐chain ferritin and iron transport mechanism to the ferroxidase sites: A molecular dynamics simulation study

We study via all atom classical molecular dynamics (MD) simulation the process of uptake of ferrous ions (Fe²⁺) into the human ferritin protein and the catalytic ferroxidase sites via pores (“channels”) in the interior of the protein. We observe that the three‐fold hydrophilic channels serve as the main entrance pathway for the Fe²⁺ ions. The binding sites along the ion pathway are investigated. Two strong binding sites, at the Asp131 and Glu134 residues and two weak binding sites, at the His118 and Cys130 are observed inside the three‐fold channel. We also identify an explicit pathway for an ion exiting the channel into the central core of the protein as it moves to the ferroxidase site. The diffusion of an Fe²⁺ ion from the inner opening of the channel to a ferroxidase site located in the interior region of the protein coat is assisted by Thr135, His136 and Tyr137. The Fe²⁺ ion binds preferentially to site A of the ferroxidase site. © 2013 Wiley Periodicals, Inc.     

Defining metal ion inhibitor interactions with recombinant human H- and L-chain ferritins and site-directed variants: an isothermal titration calorimetry study

Zinc and terbium, inhibitors of iron incorporation in the ferritins, have been used for many years as probes of structure-function relationships in these proteins. Isothermal titration calorimetric and kinetic measurements of Zn(II) and Tb(III) binding and inhibition of Fe(II) oxidation were used to identify and characterize thermodynamically ( n, K, Delta H degrees, Delta S degrees, and Delta G degrees ) the functionally important binding sites for these metal ions in recombinant human H-chain, L-chain, and H-chain site-directed variant ferritins. The data reveal at least two classes of binding sites for both Zn(II) and Tb(III) in human H-chain ferritin: one strong, corresponding to binding of one metal ion in each of the eight three-fold channels, and the other weak, involving binding at the ferroxidase and nucleation sites of the protein as well as at other weak unidentified binding sites. Zn(II) and Tb(III) binding to recombinant L-chain ferritin showed similar stoichiometries for the strong binding sites within the channels, but fewer weaker binding sites when compared to the H-chain protein. The kinetics and binding data indicate that the binding of Zn(II) and Tb(III) in the three-fold channels, which is the main pathway of iron(II) entry in ferritin, blocks the access of most of the iron to the ferroxidase sites on the interior of the protein, accounting for the strong inhibition by these metal ions of the oxidative deposition of iron in ferritin.     

Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala

Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature protein that assembles into functional ferritin shells. The full mature recombinant protein and its S144A mutant were produced to study structural and functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38 Angstrom resolution. The 3D structures of the two proteins resulted very similar to that of human H-ferritin, to which they have high level of sequence identity (approximately 80%). Metal-binding sites were identified in the native crystals and in those soaked in Mn(II) and Zn(II) solutions. The ferroxidase center binds binuclear iron at the sites A and B, and the structures showed that the A site was always fully occupied by Mg(II), Mn(II) or Zn(II), while the occupancy of the B site was variable. In addition, distinct Mg(II) and Zn(II)-binding sites were found in the 3-fold axes to block the hydrophilic channels. Other metal-binding sites, never observed before in H-ferritin, were found on the cavity surface near the ferroxidase center and near the 4-fold axes. Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H-ferritin, except for the difficulty in renaturing to yield ferritin shells and for a reduced ( approximately 41%) rate in ferroxidase activity. This was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H-ferritin. The residue is exposed on a channel that connects the ferroxidase center with the cavity. The finding that the mutation increased both catalytic activity and the occupancy of the B site demonstrated that the channel is functionally important. In conclusion, the present data define the structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin shell.     

Influence of site-directed modifications on the formation of iron cores in ferritin

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.     

Ion accumulation in a protein nanocage: finding noisy temporal sequences using a genetic algorithm

Many pathogenic bacteria are able to survive attack by the host's immune system because of antioxidant systems that mitigate the effects of reactive oxygen species. Dps is a hollow 12-subunit protein nanocage that prevents oxidative damage by oxidizing and sequestering intracellular Fe²⁺; the resulting Fe³⁺ forms an iron oxyhydroxide nanoparticle in the cage interior. Charged sites on the protein nanocage create an electrostatic gradient that guides ions through well-defined pores that connect the cage interior with the surrounding solution and toward nucleation sites on the cage interior. In this study, we use all-atom molecular dynamics to simulate the motion of simple cations into the dodecameric cage formed by the Dps protein from Listeria monocytogenes. Ion trajectories are analyzed by using a novel, to our knowledge, genetic algorithm to determine the temporal sequence of ion-protein interactions. Ions enter Dps through well-defined pores at the ferritinlike C₃ axes, with negatively-charged residues on the outside of the cage forming a fairly well-defined entrance pathway. This method of trajectory analysis may be broadly applicable in situations where the spatial localization of ions or other small molecules is electrostatically driven by a biomolecule.     

The crystal structure of the Dps2 from Deinococcus radiodurans reveals an unusual pore profile with a non-specific metal binding site

The crystal structure of recombinant Dps2 (DRB0092, DNA protecting protein under starved conditions) from the Gram-positive, radiation-resistant bacterium Deinococcus radiodurans has been determined in its apo and iron loaded states. Like other members of the Dps family, the bacterial DrDps2 assembles as a spherical dodecamer with an outer shell diameter of 90 A and an interior diameter of 40 A. A total of five iron sites were located in the iron loaded structure, representing the first stages of iron biomineralisation. Each subunit contains a mononuclear iron ferroxidase centre coordinated by residues highly conserved amongst the Dps family of proteins. In the structures presented, a distinct iron site is observed 6.1 A from the ferroxidase centre with a unique ligand configuration of mono coordination by the protein and no bridging ligand to the ferroxidase centre. A non-specific metallic binding site, suspected to play a regulative role in iron uptake/release from the cage, was found in a pocket located near to the external edge of the C-terminal 3-fold channel.     

Mineralization in Ferritin: An Efficient Means of Iron Storage

Ferritins are a class of iron storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral "ferrihydrite." The eight hydrophilic channels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mammalian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O(2), producing H(2)O(2). The L subunit lacks this site but contains additional glutamate residues on the interior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxidase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a mu-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus minimizing the production of potentially harmful H(2)O(2).     

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.     

Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis

During its metabolism, vanadium is known to become associated with the iron storage protein, ferritin. To elucidate probable vanadium binding sites on the protein, VO2+ binding to mammalian ferritins was studied using site-directed mutagenesis and EPR spectroscopy. VO2+-apoferritin EPR spectra of human H-chain (100% H), L-chain (100% L), horse spleen (84% L, 16% H) and sheep spleen (45% L, 55% H) ferritins revealed the presence of alpha and beta VO2+ species in all the proteins, implying that the ligands for these species are conserved between the H- and L-chains. The alpha species is less stable than the beta species and decreases with increasing pH, demonstrating that the two species are not pH-related, a result contrary to earlier proposals. EPR spectra of site-directed HuHF variants of several residues conserved in H- and L-chain ferritins (Asp-131, Glu-134, His-118 and His-128) suggest that His-118 near the outer opening of the three-fold channel is probably a ligand for VO2+ and is responsible for the beta signals in the EPR spectrum. The data indicate that VO2+ does not bind to the Asp-131 and Glu-134 residues within the three-fold channels nor does it bind at the ferroxidase site residues Glu-62 or His-65 or at the putative nucleation site residues Glu-61,64,67. While the ferroxidase site is not a site for VO2+ binding, mutation of residues Glu-62 and His-65 of this site to Ala affects VO2+ binding at His-118, located some 17 A away. Thus, VO2+ spin probe studies provide a window on structural changes in ferritin not seen in most previous work and indicate that long-range effects caused by point mutations must be carefully considered when drawing conclusions from mutagenesis studies of the protein.     

Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O]_n multimers within the protein cage, en route to the cavity, at sites distributed over ∼50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A_{650 nm}) and on mineral growth (Fe³⁺O-A_{350 nm}), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O]_n transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.     

The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.     

Ferritin M of Cynoglossus semilaevis: an iron-binding protein and a broad-spectrum antimicrobial that depends on the integrity of the ferroxidase center and nucleation center for biological activity

Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, we identified and analyzed the biological activity of a ferritin M subunit (CsFerM) from half-smooth tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of CsFerM is 534?bp and encodes a protein that shares 79.7-86.4% overall sequence identities with the ferritin M subunits of a number of teleosts. In silico analysis identified in CsFerM a eukaryotic ferritin domain with conserved ferroxidase diiron center and ferrihydrite nucleation center. Quantitative real time RT-PCR analysis showed that under normal physiological conditions, expression of CsFerM was highest in liver, moderate in gill, spleen, and muscle, and low in gut, heart, and brain. Following experimental challenge with bacterial pathogens, CsFerM expression was significantly upregulated in kidney, spleen, and liver in time-dependent manners. Biological activity analysis showed that recombinant CsFerM purified from Escherichia coli exhibited apparent iron-binding activity and, when present in the culture medium of six different species of fish bacterial pathogens, completely inhibited bacterial growth. In contrast, a mutant CsFerM that bears alanine substitution at two conserved residues of the ferroxidase diiron center and ferrihydrite nucleation center was abolished in both iron-binding and antimicrobial capacity. These results demonstrate that CsFerM is a biologically active iron chelator with broad-spectrum antibacterial activity, which suggests a role for CsFerM in not only iron storage but also innate immunity. These results also indicate the importance of the conserved iron uptake and mineralization sites to the function of CsFerM.     

Ion binding affinity in the cavity of the KcsA potassium channel

The hydrophobic cell membrane interior presents a large energy barrier for ions to permeate. Potassium channels reduce this barrier by creating a water-filled cavity at the middle of their ion conduction pore to allow ion hydration and by directing the C-terminal "end charge" of four alpha-helices toward the water-filled cavity. Here we have studied the interaction of monovalent cations with the cavity of the KcsA K(+) channel using X-ray crystallography. In these studies, Tl(+) was used as an analogue for K(+) and the total ion-stabilization energy for Tl(+) in the cavity was estimated by measuring its binding affinity. Binding affinity for the Na(+) ion was also measured, revealing a weak selectivity ( approximately 7-fold) favoring Tl(+) over Na(+). The structures of the cavity containing Na(+), K(+), Tl(+), Rb(+), and Cs(+) are compared. These results are consistent with a fairly large (more negative than -100 mV) electrostatic potential inside the cavity, and they also imply the presence of a weak nonelectrostatic component to a cation's interaction with the cavity.     

Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.     

Ferritin structure from Mycobacterium tuberculosis: comparative study with homologues identifies extended C-terminus involved in ferroxidase activity

Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world''s deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments.     

The putative "nucleation site" in human H-chain ferritin is not required for mineralization of the iron core

It is widely believed that the putative nucleation site (Glu61, Glu64, and Glu67) in mammalian H-chain ferritin plays an important role in mineral core formation in this protein. Studies of nucleation site variant A2 (E61A/E64A/E67A) of H-chain ferritin have traditionally shown impaired iron oxidation activity and mineralization. However, recent measurements have suggested that the previously observed impairment may be due to disruption of the ferroxidase site of the protein since Glu61 is a shared ligand of the ferroxidase and nucleation sites of the protein. This study employed a new nucleation site variant A1 (E64A/E67A) which retains the ferroxidase site ligand Glu61. The data (O(2) uptake, iron binding, and conventional and stopped-flow kinetics measurements) show that variant A1 retains a completely functional ferroxidase site and has iron oxidation and mineralization properties similar to those of the wild-type human H-chain protein. Thus, in contrast to previously published literature, this study demonstrates that the putative "nucleation site" does not play an important role in iron uptake or mineralization in H-chain ferritin.     

Structure of the DPS-like protein from Sulfolobus solfataricus reveals a bacterioferritin-like dimetal binding site within a DPS-like dodecameric assembly

The superfamily of ferritin-like proteins has recently expanded to include a phylogenetically distinct class of proteins termed DPS-like (DPSL) proteins. Despite their distinct genetic signatures, members of this subclass share considerable similarity to previously recognized DPS proteins. Like DPS, these proteins are expressed in response to oxidative stress, form dodecameric cage-like particles, preferentially utilize H(2)O(2) in the controlled oxidation of Fe(2+), and possess a short N-terminal extension implicated in stabilizing cellular DNA. Given these extensive similarities, the functional properties responsible for the preservation of the DPSL signature in the genomes of diverse prokaryotes have been unclear. Here, we describe the crystal structure of a DPSL protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Although the overall fold of the polypeptide chain and the oligomeric state of this protein are indistinguishable from those of authentic DPS proteins, several important differences are observed. First, rather than a ferroxidase site at the subunit interface, as is observed in all other DPS proteins, the ferroxidase site in SsDPSL is buried within the four-helix bundle, similar to bacterioferritin. Second, the structure reveals a channel leading from the exterior surface of SsDPSL to the bacterioferritin-like dimetal binding site, possibly allowing divalent cations and/or H(2)O(2) to access the active site. Third, a pair of cysteine residues unique to DPSL proteins is found adjacent to the dimetal binding site juxtaposed between the exterior surface of the protein and the active site channel. The cysteine residues in this thioferritin motif may play a redox active role, possibly serving to recycle iron at the ferroxidase center.     

Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin

Fe(III) storage by ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a di-iron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site was obliterated. However, metal ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions toward the ferroxidase center directly through the protein shell and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted.