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.     

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.     

Iron Translocation into and out of Listeria innocua Dps and Size Distribution of the Protein-enclosed Nanomineral Are Modulated by the Electrostatic Gradient at the 3-fold ��Ferritin-like�� Pores

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called “ferritin-like” because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.The widely distributed bacterial Dps proteins (1, 2) belong to the ferritin superfamily and are characterized by strong similarities (3) but also distinctive differences with respect to “canonical” ferritins, the ubiquitous iron storage, and detoxification proteins found in biological systems. The structural resemblance is apparent in the overall molecular assemblage because both Dps proteins and ferritins are shell-like oligomers constructed from four-helix bundle monomers. However, Dps proteins are 12-mers of identical subunits that assemble with 23 symmetry, whereas ferritins are built by 24 highly similar or identical subunits related by 432 symmetry. The functional similarities consist in the common capacity to remove Fe(II) from cytoplasm, catalyze its oxidation, and store Fe(III) thus produced in the protein cavity, wherefrom the metal can be mobilized when required by the organism. However, ferritins use molecular oxygen as iron oxidant with the production of hydrogen peroxide, whereas Dps proteins prefer hydrogen peroxide, which is typically about 100-fold more efficient than molecular oxygen (1). This difference is of major importance because it renders Dps proteins capable of removing concomitantly Fe(II) and H₂O₂ whose combination leads to the production of reactive oxygen species via Fenton chemistry (4). This capacity confers H₂O₂ resistance and hence may be a virulence factor in certain pathogens (e.g. Campylobacter jejuni, Streptococcus mutans, and Porphyromonas gingivalis) because the H₂O₂ burst represents one of the first defense lines of the host during infection (5–7).Key to a full understanding of the iron uptake and release processes at a molecular level is the route by which iron enters and exits the protein shell. In both 24-subunit ferritins and 12-subunit Dps proteins, the subunit assemblage creates pores across the protein shell that put the internal cavity in communication with the external medium. In ferritins there are two types of pores: largely hydrophobic ones along the axes with 4-fold symmetry and hydrophilic ones along the axes with 3-fold symmetry. The latter channels are funnel-shaped, with the smaller opening toward the protein cavity, and are lined with conserved glutamic and aspartic residues located in the narrow region of the funnel (8). These 3-fold pores were recognized to provide the route for iron entry into the protein cavity soon after resolution of the horse ferritin x-ray crystal structure (9). Later site-directed mutagenesis studies defined the role of specific residues (Asp¹³¹ and Glu¹³⁴) that line the pore (10, 11), whereas electrostatic calculations related the passage of iron to the existence of a gradient that drives metal ions toward the protein interior cavity (12, 13). More recently, the 3-fold symmetry pores were shown to be involved also in the exit process of iron from the protein cavity. Thus, in H-frog ferritin used as model system, iron exit is affected by local protein unfolding promoted by site-specific mutagenesis of individual amino acid residues (14, 15), by the use of chaotropes (16), and by means of selected peptides designed to bind at these channels (17).In Dps proteins, the protein shell is breached by two types of pores along the 3-fold axes, one type is formed by the N-terminal portion of the monomers and bears a strong similarity to the typical 3-fold channels of 24-subunit ferritins in that it is funnel-shaped, hydrophilic, and lined by conserved, negatively charged residues. It was therefore named “ferritin-like” and assumed to be involved in iron entry into the protein cavity upon resolution of the Listeria innocua x-ray crystal structure (18). The other type of pore is formed by the C-terminal ends of the monomers and was called “Dps type” because it is created at a subunit interface that is unique to Dps proteins. Although somewhat variable in length and in the size of the openings, the Dps type pore is mainly hydrophobic in nature (19).The present paper investigates the role of the ferritin-like pores in the iron uptake and release processes in Dps proteins using the well characterized L. innocua Dps (LiDps) as a model system (18, 20–22). In LiDps, the ferritin-like pores contain a set of three aspartate residues, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ that would be encountered in succession by a metal ion that is attracted by the electrostatic gradient they create and moves down the funnel-shaped pore toward the protein cavity (Fig. 1). Asp¹³⁰, which is located in the narrowest part of the funnel, is conserved significantly among Dps proteins (∼ 80%), whereas Asp¹²¹ and Asp¹²⁶ are less conserved (Fig. 1). Such considerations were used in the design of site-specific variants D121N, D126N, D130N, and D121N/D126N/D130N to elucidate the function of the ferritin-like pores in Dps proteins.Open in a separate windowFIGURE 1.Dps proteins sequences and conservation of the aspartate residues that line the 3-fold ferritin-like pores in L. innocua Dps. A, alignment of multiple Dps sequences from different bacteria: LiDps, non-heme iron-binding ferritin (L. innocua Clip11262]; EcDps, DNA-binding protein Dps (E. coli); HpDps, neutrophil-activating protein (Helicobacter pylori); YpDps, ferritin family protein (Yersinia pestis Angola); GtDps, DNA-protecting protein (Geobacillus thermodenitrificans NG80–2); RmDps, ferritin, and Dps (Ralstonia metallidurans CH34); AtDps, DNA protection during starvation conditions (Agrobacterium tumefaciens str. C58); TeDps Dps family DNA-binding stress response protein (Thermosynechococcus elongatus BP-1); PaDps, putative DNA-binding protein, Dps (Psychrobacter arcticus 273-4); TfDps, hypothetical protein Tfu_0799 (Thermobifida fusca YX); PhDps, DNA-binding DPS protein (Pseudoalteromonas haloplanktis TAC125); SoDps, Dps family protein (Shewanella oneidensis MR-1); BaDps1, general stress protein 20U (Bacillus anthracis str. Ames); BaDps2 general stress protein (B. anthracis str. Ames); LlDps, non-heme iron-binding ferritin (Lactococcus lactis subsp. lactis Il1403); VcDps, DPS family protein (Vibrio cholerae MZO-3); and StDps, DNA-binding ferritin-like protein (oxidative damage protectant) (Streptococcus thermophilus LMD-9). Residues forming the Dps catalytic center are highlighted in pale blue (His³¹, His⁴³, Asp⁵⁸, and Glu⁶² in LiDps); 3-fold pores aspartate residues are highlighted in yellow and marked in bold type. Alignment has been created with ClustalW2 (34). B, view of the junction of three monomers forming the 3-fold ferritin-like pore. C, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ aspartate residues comprised the pore area. D, three-dimensional view of the pore colored by charge. Red, negatively charged residues; blue, positively charged residues; white, uncharged residues. E, schematic representation of the vertical section of the pore. The images were created with PyMol (35).The results demonstrate that iron uses the LiDps ferritin-like pores to enter and leave the protein shell and hence that these pores have the same role as the structurally similar 3-fold channels in 24-subunit ferritins. LiDps residue Asp¹³⁰ is the most important determinant of the negative electrostatic gradient because of its location in the narrow part of the pores. Importantly, the data show for the first time that the electrostatic gradient at the pores modulates cooperativity in the iron uptake process and influences the size distribution of the iron core (23). The effect of the electrostatic gradient can be explained in terms of the electrostatic interaction effects between the fixed negative charges of the aspartate residues at the pores and the mobile positive charges of iron ions.     

Assessment of iron toxicity using a luminescent bacterial test with an Escherichia coli recombinant strain

The toxic effect of the Fe²⁺ and Fe³⁺ ions on the luminescent recombinant Escherichia coli strain with the luxCDABE operon was studied in short- and long-term experiments. At 30-min exposure of bacteria to the iron ions, the effective concentrations of Fe²⁺ and Fe³⁺ resulting in acute toxicity (EC₅₀) were 8.5 and 1.3 mg/L, respectively. In the long-term (24 h) experiment, during active bacterial growth, the toxicity index for Fe²⁺ and Fe³⁺ was 65.5 and 62.8, respectively. Addition of the iron ions into the medium did not suppress growth, although it inhibited luminescence. Comparative analysis of the short- and long-term experiments made it possible to assess iron toxicity at the concentrations from 0.5 to 20 mg/L (as calculated for the Fe²⁺ and Fe³⁺ ions). Iron ions were found to affect only the reactions that were not vitally important for the cell. At the same time, they had no negative effect on the genetic mechanisms and protein synthesis, thus indicating non-specific toxicity of Fe²⁺ and Fe³⁺.     

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C 4 cage axes

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe₂O₃·H₂O minerals from Fe²⁺ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe²⁺ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe²⁺/O₂ catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe²⁺ + O₂ protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe₂O₃·H₂O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe²⁺ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.     

Linoleic acid peroxidation initiated by Fe-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work evaluates linoleic acid peroxidation reactions initiated by Fe³⁺-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe³⁺ ions from freshly prepared solutions. The compounds responsible for the Fe³⁺-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe³⁺ ions and the Fe³⁺-reducing compounds showed that the rate of O₂ consumption during peroxidation was proportional to the Fe³⁺-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe³⁺-reducing compounds formed during wood biodegradation by C. subvermispora can mediate lignin degradation through linoleic acid peroxidation.     

Protein- and Metal-dependent Interactions of a Prominent Protein in Mussel Adhesive Plaques

The adhesive plaques of Mytilus byssus are investigated increasingly to determine the molecular requirements for wet adhesion. Mfp-2 is the most abundant protein in the plaques, but little is known about its function. Analysis of Mfp-2 films using the surface forces apparatus detected no interaction between films or between a film and bare mica; however, addition of Ca²⁺ and Fe³⁺ induced significant reversible bridging (work of adhesion W_ad ≈ 0.3 mJ/m² to 2.2 mJ/m²) between two films at 0.35 m salinity. The strongest observed Fe³⁺-mediated bridging approaches the adhesion of oriented avidin-biotin complexes. Raman microscopy of plaque sections supports the co-localization of Mfp-2 and iron, which interact by forming bis- or tris-DOPA-iron complexes. Mfp-2 adhered strongly to Mfp-5, a DOPA-rich interfacial adhesive protein, but not to another interfacial protein, Mfp-3, which may in fact displace Mfp-2 from mica. In the presence of metal ions or Mfp-5, Mfp-2 adhesion was fully reversible. These results suggest that plaque cohesiveness depends on Mfp-2 complexation of metal ions, particularly Fe³⁺ and also by Mfp-2 interaction with Mfp-5 at the plaque-substratum interface.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

Gated pores in the ferritin protein nanocage

Properties of ferritin gated pores control rates of FMNH₂ reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, and are discussed in terms of: (1) the conserved pore gate residues (ion pairs: arginine 72, aspartate 122, and a hydrophobic pair, leucine 110-leucine 134), (2) pore sensitivity to heat at temperatures 30 °C below that of the nanocage itself, and (3) pore sensitivity to physiological changes in urea (1-10 mM). Conditions which alter ferritin pore structure/function in solution, coupled with the high evolutionary conservation of the pore gates, suggest the presence of molecular regulators in vivo that recognize the pore gates and hold them either closed or open, depending on biological iron need. The apparent homology between ferrous ion transport through gated pores in the ferritin nanocage and ion transport through gated pores in ion channel proteins embedded in cell membranes, make studies of water soluble ferritin and the pore gating folding/unfolding a useful model for other gated pores.     

Correlation of proton release and ultraviolet difference spectra associated with metal binding by transferrin

A variety of metal ions can bind to the iron-transport protein, transferrin, at two specific sites. For each metal ion, a carboxylate anion is concomitantly bound. Six metal ions which were examined fall into two classes based on proton release and ultraviolet spectral changes which accompany binding to the protein. Class II ions, which include Cu²⁺ and Zn²⁺, release approximately 2 H⁺/metal bond. Class III ions, which include Fe³⁺, Ga³⁺, Al³⁺, and VO²⁺, release approximately 3 H⁺/metal bound. The increase in absorbance near 242 nm, characteristic of tyrosine ionization, has the ratio 0.55–0.75 for class II:class III ions. Both Fe³⁺ and Cu²⁺ form metal-transferrin-oxalate complexes in the presence of excess C₂O₄^2?. Fe³⁺ releases close to 3 H⁺/metal whether forming oxalate or bicarbonate complexes with transferrin. Binding of Cu²⁺ to transferrin releases 2 H⁺/metal in the presence of C₂O^2?₄ or HCO₃^?. Since equal numbers of H⁺/metal are released for both anions, it is likely that the bicarbonate ion does not lose its proton, and remains as HCO₃^? in transferrin. These results are interpreted in terms of possible combinations of ligands at the metal binding sites.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.Abbreviations MAPK mitogen-activated protein kinase - MCO metal ion-catalyzed oxidation - kinase F_A the activating factor of ATP.Mg-dependent protein phosphatase - I₂ inhibitor-2 - EDTA ethylenediaminetetraacetic acid - MBP myelin basic protein     

The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K_d of 3.7 × 10⁻⁸ m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H₂O₂-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.     

Unraveling Geometrical Site Confinement in Highly Efficient Iron‐Doped Electrocatalysts toward Oxygen Evolution Reaction

Introduction of iron in various catalytic systems has served a crucial function to significantly enhance the catalytic activity toward oxygen evolution reaction (OER), but the relationship between material properties and catalysis is still elusive. In this study, by regulating the distinctive geometric sites in spinel, Fe occupies the octahedral sites (Fe³⁺_(Oh)) and confines Co to the tetrahedral site (Co²⁺_(Td)), resulting in a strikingly high activity (η_{j = 10 mA cm}^?2 = 229 mV and η_{j = 100 mA cm}^?2 = 281 mV). Further enrichment of Fe ions would occupy the tetrahedral sites to decline the amount of Co²⁺_(Td) and deteriorate the OER activity. It is also found that similar tafel slope and peak frequency in Bode plot of electrochemical impedance spectroscopy indicate that Co²⁺_(Td) ions are primarily in charge of water oxidation catalytic center. By means of electrochemical techniques and in situ X‐ray absorption spectroscopy, it is proposed that Fe³⁺_(Oh) ions mainly confine cobalt ions to the tetrahedral site to restrain the multipath transfer of cobalt ions during the dynamic structural transformation between spinel and oxyhydroxide, continuously activating the catalytic behavior of Co²⁺_(Td) ions. This material‐related insight provides an indication for the design of highly efficient OER electrocatalysts.     

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.     

Protein Scission by Metal Ion–Ascorbate System

About 14 proteins were tested for specific oxidative scission catalyzed by metal ions in the presence of ascorbate and oxidizing agents (O₂ or hydrogen peroxide). Only four of them were degraded by Fe³⁺/Fe²⁺- ascorbate, twelve – by Cu²⁺/Cu⁺-ascorbate and two proteins (α- and β-caseins) were degraded by Pd²⁺ ions. The rate and the intensity of degradation are very different for various proteins. For the most of tested proteins only a small fraction of molecules was degraded. None of them was degraded completely. Two possible reasons of protein stability against oxidative degradation may be proposed as follows: either there is no metal binding site in a protein molecule, or metal binding ligands of protein undergo a rapid oxidative modification and the metal ion is released from the binding site. Human growth hormone was cut specifically at two sites by Cu²⁺/Cu⁺-ascorbate system. At least one of amino acid residues of this protein was modified by formation of reactive carbonyl.     

Non-reductive iron release from horse spleen ferritin using desferoxamine chelation

The rate of Fe³⁺ release from horse spleen ferritin (HoSF) was measured using the Fe³⁺-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction. The overall two-phase reaction was resolved into three kinetic events: 1) a rapid first-order reaction in HoSF (k₁); 2) a second slower first-order reaction in HoSF (k₂); and 3) a zero-order slow reaction in HoSF (k₃). The zero-order reaction was independent of DES concentration. The two first-order reactions had a near zero-order dependence on DES concentration and were independent of pH from 6.8 to 8.2. The two first-order reactions accounted for 6-9 rapidly reacting Fe³⁺ ions. Activation energies of 10.5 ± 0.8, 13.5 ± 2.0 and 62.4 ± 2.1 kJ/mol were calculated for the kinetic events associated with k₁, k₂, and k₃, respectively. Iron release occurs by: 1) a slow zero-order rate-limiting reaction governed by k₃ and corresponding to the dissociation of Fe³⁺ ions from the FeOOH core that bind to an Fe³⁺ binding site designated as site 1 (proposed to be within the 3-fold channel); 2) transfer of Fe³⁺ from site 1 to site 2 (a second binding site in the 3-fold channel) (k₂); and 3) rapid iron loss from site 2 to DES (k₁).     

Cation Penetration through Isolated Leaf Cuticles

The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS⁺ ≥ Rb⁺ > K⁺ > Na⁺ > Li⁺. The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K⁺ and H₂O at pH above 9 were of similar magnitude. At pH 5.5 H₂O penetration was not affected but that of K⁺ was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites.