Emilia Chiancone

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.

Iron Incorporation into Escherichia coli Dps Gives Rise to a Ferritin-like Microcrystalline Core

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d5 configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, witht 1 2 of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core.

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: Structural and functional properties

The bivalve mollusc Scapharca inaequivalvis contains in the coelomic fluid erythrocytes with a dimeric (HbI) and a tetrameric (HbII) hemoglobin like the other members of the arcid family. The tetrameric protein is made up from two types of polypeptide chain, while the dimeric protein is made from a single type of chain which differs from the other two in terms of molecular weight and isoelectric point. The optical and circular dichroism spectra show that the heme environment in HbI and HbII resembles that of vertebrate hemoglobins, although distinctive features are present in the deoxygenated derivative. p]The dimeric HbI in the pH range 6 to 9 does not change its association state upon deoxygenation, while the tetrameric HbII polymerizes as indicated by the appearance of a fast peak in the sedimentation velocity patterns. The dependence of the areas and sedimentation coefficients of the fast and slow peaks on protein concentration is characteristic of a rapidly established association-dissociation equilibrium between tetramers and polymers higher than octamers. The pH, ionic strength and temperature dependence of polymer formation indicate that both hydrophobic and ionic interactions stabilize the polymers. The functional properties of HbI and HbII differ. HbI shows co-operative oxygen binding (h = 1·5) and a constant oxygen affinity (p12 = 7.8 mm Hg) over the pH range 5.5 to 9.5. HbII likewise shows co-operativity in oxygen binding (h = 2·0). Its oxygen affinity at neutral and alkaline pH values is slightly lower (p12 = 9.1 mm Hg) than that of the dimeric protein, but becomes higher at pH values below 6.5 due to the presence of an acid Bohr effect. At high protein concentrations, under conditions of extensive polymerization of the deoxygenated derivative, the oxygen affinity is lowered and co-operativity slightly increased. Both phenomena require that the oxygen affinity of the polymer be lower than that of the tetramer, consistent with the predictions of linkage theory.

Nuclear magnetic resonance quadrupole relaxation studies of chloride binding to human oxy- and deoxyhaemoglobin.

Abstract Chloride binding to oxy-, carbon monoxy- and deoxyhaemoglobin and to myoglobin has been measured directly in quadrupole relaxation experiments on the excess line-width of the nuclear magnetic resonance signals of 35 Cl − associated with its binding to the protein. The measurements, which have been extended over a wide range of conditions, suggest that in haemoglobin there are at least two classes of chloride binding sites. The high affinity sites are oxygen linked and over the range 0.1 to 2.5 m -NaCl deoxyhaemoglobin binds more chloride ions than liganded haemoglobin. In contrast no such difference exists between the corresponding myoglobin derivatives. The difference in chloride binding between oxy- and deoxyhaemoglobin may be correlated with the conformational transitions associated with ligand binding in haemoglobin and is reflected in the effect of chloride on the oxygen equilibrium. Competition experiments with ATP indicate that the high affinity chloride binding sites correspond to those for the organic phosphates.

Studies on erythrocruorin. I. Physicochemical properties of earthworm erythrocruorin.

Abstract Erythrocruorin has been purified by preparative ultracentrifugation from the blood of Lumbricus. The protein is homogeneous in the ultracentrifuge and in gel electrophoresis. The sedimentation coefficient is 59.6, and the molecular weight by light scattering is 3.45 × 106 daltons. The protein has 2.67% heme iron corresponding to a minimum molecular weight of ~23 × 103 daltons. The full protein molecule thus contains 144 hemes arranged in a subunit structure similar to that of other erythrocruorins and of Spirographis chlorocrun.

The properties and interactions of the isolated α and β chains of human haemoglobin: I. Sedimentation and electrophoretic behaviour

The α and β chains of human haemoglobin A 1 may be isolated by starch block electrophoresis or by chromatography after treatment of the protein with p -mercuribenzoate. The chains so isolated do not show any free SH groups; however, after treatment with cysteine or thioglycolate, between 1 and 2 SH groups per β chain and 1 per α chain become titratable. The chains before and after such treatment are designated α PMB† or β PMB and α SH or β SH respectively. Ultracentrifugal analysis shows that the isolated α SH and α PMB chains are both monomeric; in contrast the β PMB chains tend to polymerize. The β SH chains are heterogeneous but the major component appears to be a tetramer and may be identified with haemoglobin H. A mixture of α PMB and β PMB chains in equal amounts is homogeneous and has a sedimentation coefficient in the neighbourhood of 3. A similar mixture of α SH and β SH chains behaves like haemoglobin A both in the ultracentrifuge and in starch gel electrophoresis. Certain systems which are homogeneous in the centrifuge and on Sephadex show multiple bands in electrophoresis, which implies differences beyond those due to molecular weight. These results are discussed in terms of the interactions between the chains under various conditions.

Studies on erythrocruorin: II. Dissociation of earthworm erythrocruorin

Earthworm erythrocruorin (Lumbricus terrestris) dissociates into subunits at alkaline pH values. The dissociation products have been correlated to definite molecular structures on the basis of electron microscope and minimum molecular weight data. The final state in dissociation is approached by complex kinetics as determined in rapid mixing and static light-scattering experiments. On the other hand, the final state reflects only in part an equilibrium situation, since the intact molecules at any given pH are not in equilibrium with the dissociated components. Between the dissociated subunits a reversible equilibrium is established which is sensitive to the presence of ligand. The results give clear evidence of molecular microheterogeneity in the system.

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue.

Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi‐drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium‐dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r‐sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r‐sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non‐ionic detergent Triton X‐114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium‐dependent translocation of sorcin in the specialized functions of sorcin‐expressing cells is discussed.