Conservative and Atypical Ferritins of Sponges

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

Direct Detection of Iron Clusters in L Ferritins through ESI MS Experiments

Human cytoplasmic ferritins are heteropolymers of H and L subunits containing a catalytic ferroxidase center and a nucleation site for iron biomineralization, respectively. Here, ESI-MS successfully detected labile metal-protein interactions...

Disclosing the Molecular Mechanism of Iron Incorporation in Listeria innocua Dps by EPR Spectroscopy

Bacteria overexpress, under condition of starvation or oxidative stress, Dps (DNA-binding proteins from starved cells), hollow sphere formed by 12 identical subunits endowed with ferritin-like activity. The iron oxidation and incorporation in Dps take place using H2O2 produced under starvation as preferred iron oxidant, thereby protecting bacteria from oxidative damage. Even if the role of Dps is well known, the mechanism of iron oxidation and incorporation remain to be elucidated. Here, we have used the EPR technique to shed light on the Fe(II) binding and oxidation mechanism at the ferroxidase center using both the wild-type (wt) protein and mutants of the iron ligands (H31G, H43G and H31G-H43G-D58A). The EPR titration of wt Dps and the H31G mutant with Fe(II) upon H2O2 addition shows that Fe(II) is oxidized with the increase of the signal at g = 4.3, reaching a maximum for 12 Fe(II)/subunit. The EPR signal becomes negligible when the titration is carried out on the triple mutant. These experiments indicate that the iron firstly occupied the A site at the ferroxidase center and confirm that the residues H31, H43 and D58 have a key role in the iron oxidation and incorporation process. Moreover, the data indicate that the ferroxidase center, upon mutation of H31 or H43 to Gly, changes the mode of iron binding. Finally, we demonstrate here that, when the iron micelle forms, the EPR signal at g = 4.3 disappears indicating that iron leaves the ferroxidase center to reach the inner cavity.

Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this proposed iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site.

Reaction of O2with a diiron protein generates a mixed-valent Fe2+/Fe3+center and peroxide

The gene encoding the cyanobacterial ferritinSynFtn is up-regulated in response to copper stress. Here, we show that, whileSynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2with the di-Fe2+center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+form. Iron–O2chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O2chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

Serendipitous crystallization and structure determination of bacterioferritin from Achromobacter

Bacterioferritins (Bfrs) are ferritin-like molecules with a hollow spherical 24-mer complex design that are unique to bacterial and archaeal species. They play a critical role in storing iron(III) within the complex at concentrations much higher than the feasible solubility limits of iron(III), thus maintaining iron homeostasis within cells. Here, the crystal structure of bacterioferritin from Achromobacter (Ach Bfr) that crystallized serendipitously during a crystallization attempt of an unrelated mycobacterial protein is reported at 1.95 Å resolution. Notably, Fe atoms were bound to the structure along with a porphyrin ring sandwiched between the subunits of a dimer. Furthermore, the dinuclear ferroxidase center of Ach Bfr has only a single iron bound, in contrast to the two Fe atoms in other Bfrs. The structure of Ach Bfr clearly demonstrates the substitution of a glutamate residue, which is involved in the interaction with the second Fe atom, by a threonine and the consequent absence of another Fe atom there. The iron at the dinuclear center has a tetravalent coordination, while a second iron with a hexavalent coordination was found within the porphyrin ring, generating a heme moiety. Achromobacter spp. are known opportunistic pathogens; this structure enhances the current understanding of their iron metabolism and regulation, and importantly will be useful in the design of small-molecule inhibitors against this protein through a structure-guided approach.

Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.

Modulating the permeability of ferritin channels

Electric field gradients across the C3 and C4 ferritin channels controls the directional Fe2+fluxes towards the catalytic ferroxidase center.