Simonetta Stefanini

The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site.

Ferritin is characterized by a highly conserved architecture that comprises 24 subunits assembled into a spherical cage with 432 symmetry. The only known exception is the dodecameric ferritin from Listeria innocua. The structure of Listeria ferritin has been determined to a resolution of 2.35 Å by molecular replacement, using as a search model the structure of Dps from Escherichia coli. The Listeria 12-mer is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely the negatively charged channels along the three-fold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition. The electron density map shows 12 iron ions on the inner surface of the hollow core, at the interface between monomers related by two-fold axes. Analysis of the nature and stereochemistry of the iron-binding ligands reveals strong similarities with known ferroxidase sites. The L. innocua ferritin site, however, is the first described so far that has ligands belonging to two different subunits and is not contained within a four-helix bundle.

A Novel Non-heme Iron-binding Ferritin Related to the DNA- binding Proteins of the Dps Family in Listeria innocua*

A multimeric protein that behaves functionally as an authentic ferritin has been isolated from the Gram-positive bacterium Listeria innocua The purified protein has a molecular mass of about 240,000 Da and is composed of a single type of subunit (18,000 Da). L. innocua ferritin is able to oxidize and sequester about 500 iron atoms inside the protein cage. The primary structure reveals a high similarity to the DNA-binding proteins designated Dps. Among the proven ferritins, the most similar sequences are those of mammalian L chains that appear to share with L. innocua ferritin the negatively charged amino acids corresponding to the iron nucleation site. In L. innocua ferritin, an additional aspartyl residue may provide a strong complexing capacity that renders the iron oxidation and incorporation processes extremely efficient. This study provides the first experimental evidence for the existence of a non-heme bacterial ferritin that is related to Dps proteins, a finding that lends support to the recent suggestion of a common evolutionary origin of these two protein families.

Iron and proteins for iron storage and detoxification

Iron is required by most organisms, but is potentially toxic due to the low solubility of the stable oxidation state, Fe(III), and to the tendency to potentiate the production of reactive oxygen species, ROS. The reactivity of iron is counteracted by bacteria with the same strategies employed by the host, namely by sequestering the metal into ferritin, the ubiquitous iron storage protein. Ferritins are highly conserved, hollow spheres constructed from 24 subunits that are endowed with ferroxidase activity and can harbour up to 4500 iron atoms as oxy-hydroxide micelles. The release of the metal upon reduction can alter the microorganism-host iron balance and hence permit bacteria to overcome iron limitation. In bacteria, the relevance of the Dps (DNA-binding proteins from starved cells) family in iron storage-detoxification has been recognized recently. The seminal studies on the protein from Listeria innocua demonstrated that Dps proteins have ferritin-like activity and most importantly have the capacity to attenuate the production of ROS. This latter function allows bacterial pathogens that lack catalase, e.g. Porphyromonas gingivalis, to survive in an aerobic environment and resist to peroxide stress.

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.

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.

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.

Incorporation of iron by the unusual dodecameric ferritin from Listeria innocua

The polypeptide chain that assembles into the unusual dodecameric shell of Listeria innocua apoferritin lacks the ferroxidase centre characteristic of H-type mammalian chains, but is able to catalyse both Fe(II) oxidation and nucleation of the iron core. A cluster of five carboxylate residues, which correspond in part to the site of iron core nucleation typical of L-type mammalian ferritins, has been proposed to be involved in both functions. The features of the iron uptake kinetics and of Fe(II) autoxidation in the presence of citrate followed spectrophotometrically confirm this assignment. In Listeria the kinetics of iron uptake is hyperbolic at low Fe(II)-to-dodecamer ratios and becomes sigmoidal when iron exceeds 150 Fe(II) atoms per dodecamer, namely when a fast crystal growth phase follows a slow initial nucleation step. Iron autoxidation in the presence of citrate displays a similar behaviour. Thus the time course is sigmoidal at low citrate-to-Fe ratios at which Fe(III) polymerization is predominant, but is hyperbolic at ligand concentrations high enough to prevent polymerization. The marked inhibitory effect of Tb(III) on the kinetics of iron incorporation confirms that carboxylates provide the iron ligands in L. innocua apoferritin. Iron uptake followed in steady-state fluorescence experiments allows one to distinguish Fe(II) binding and oxidation from the subsequent movement of Fe(III) into the apoferritin cavity as in mammalian ferritins despite the different localization of the tryptophan residues.

Transcription of the Listeria monocytogenes fri gene is growth-phase dependent and is repressed directly by Fur, the ferric uptake regulator.

The Listeria monocytogenes fri gene encodes the only ferritin-like protein of this pathogen, a Dps protein (DNA binding protein from starved cells). Listeria Dps is endowed with the capacity to detoxify concurrently free iron and H(2)O(2), is essential for virulence and is required for efficient bacterial growth at early stages of the infection process. The transcription of fri is known to depend on sigma(A) and sigma(B) factors, to be affected by growth conditions and to be derepressed in a perR (peroxide-inducible stress response regulator) mutant background. The present work shows that fri transcription is restricted to the exponential phase of growth, whereas the Dps protein has a long half-life and is detected in significant amounts also in stationary phase cells. Expression of fri is downregulated under iron-rich conditions and is controlled directly by Fur, the ferric uptake regulator, which binds within the DNA region encompassing nucleotides from position -23 to position +90 relative to the proximal sigma(A) transcription startpoint. The putative Fur-box is proposed to coincide with the putative Per-box both in sequence and position. The primary structure of L. monocytogenes Fur has a high degree of similarity with homologues of known X-ray crystal structure. The molecular model of L. monocytogenes Fur built on this basis shows that the ligands of the structural Zn(II) and of the regulatory Fe(II) are conserved and are located in positions fully compatible with their respective roles.

Stoichiometry of iron oxidation by apoferritin

Thus, in vitro studies show that, in the presence of air, the protein catalyses the oxidation of Fe’*, which is then incorporated into the shell. In a previous paper [3] we have shown that in anaerobiosis the protein binds some Fe2+ and con- verts it to Fe3+ on admission of oxygen. In this respect the protein acts as an oxidoreductase; there- fore, it seemed of interest to measure the stoichiom- etry of the reaction between oxygen and iron as catalysed by apoferritin. The results indicate that 4 FeV are oxidized per oxygen molecule and that the product of oxygen reduction is water. The presence of catalase does not affect the time course nor the stoichiometry of the reaction. STOICHIOMETRY OF IRON OXIDATION BY APOFERRITIN

Iron entry route in horse spleen apoferritin: Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4-maleimido-tempo

Apoferritin has been selectively labeled with a maleimide nitroxide derivative at Cys‐126, located in the hydrophilic 3‐fold channels. Titration of this derivative with Fe(II), which gives rise to the initial Fe(III)‐apoferritin complex, produces, at low metal‐to‐protein ratios, a decrease of the intensity of the label EPR signal due to the occurrence of a magnetic dipolar interaction. A label‐metal distance ranging between 8–12 Å can be estimated from titrations performed with VO(IV), which is known to bind in the 3‐fold channels, and likewise produces a decrease in the label EPR signal. The present findings indicate that iron binds in the hydrophilic channels in its higher oxidation slate and that these channels represent the metal entry route at least at low metal‐to‐protein ratios.