Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin

Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV–vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

A Synthetic Peptide with the Putative Iron Binding Motif of Amyloid Precursor Protein (APP) Does Not Catalytically Oxidize Iron

The ?-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III) product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II) just as in ferritin. We measured the ferroxidase activity indirectly (i) by the incorporation of the Fe(III) product of the ferroxidase reaction into transferrin and directly (ii) by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II) oxidation by molecular oxygen. Zn(II) binds to transferrin and diminishes its Fe(III) incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and – by implication – of APP should be re-evaluated.BT/BiotechnologyApplied Science

Effect of concentration of Fe(II) on non-enzymatic oxidation of Fe(II) and incorporation of the Fe(III) product into transferrin.

<p>Progress curves were recorded at 460 nm for formation of diferric-transferrin and the initial rates were plotted versus concentration of Fe(II). The error bars represent standard deviation of three independent measurements. Concentration of apo-transferrin was 100 µM. Buffer was 100 mM Hepes, 100 mM NaCl, pH 7.2. Measurements were performed at 37°C.</p

The FD1 peptide does not catalytically oxidize iron as measured by the transferrin assay.

<p>(<b>A</b>) Non-enzymatic oxidation of Fe(II) and incorporation of Fe(III) into transferrin was compared with that of BSA, HuHF, ceruloplasmin (CP) and FD1. 52 µM of apo-transferrin was mixed with 2 µM BSA or 0.18 µM HuHF (24-mer) or 2 µM FD1 peptide, or 0.1 µM ceruloplasmin, and then an aliquot of 5 µl anaerobic solution of ferrous sulphate was added. Measurements were done in 100 mM Hepes, 100 mM NaCl, pH 7.2. Final concentration of Fe(II) was 40 µM. (B) Effect of pH on non-enzymatic oxidation of Fe(II) in the presence of transferrin was checked at three different pH values in the presence or absence of FD1 peptide (2 µM): pH 6.2, 100 mM Mes, 100 mM NaCl; pH 7.2, 100 mM Hepes, 100 mM NaCl, and pH 8.2, 100 mM Tris, 100 mM NaCl. Concentration of apo-transferrin was 52 µM. Final concentration of Fe(II) was 40 µM. Measurements were performed at 37°C in triplicate with two different batches of transferrin.</p

Thermodynamic parameters for Zn(II) binding to transferrin and FD1 peptide.

<p>Binding of Zn(II) to transferrin and FD1 peptide was measured by isothermal titration calorimetry. N is the stoichiometry of Zn(II) binding per protein. Measurements were performed at 25°C. A two independent binding site model or a single binding site model was used to fit the data points for integrated heat of binding. Standard deviations were obtained from triplicate experiments.</p

Fe(II) and Zn(II) binding to transferrin and to FD1 measured by isothermal titration calorimetry.

<p>(A) Anaerobic Fe(II) titration of the FD1 peptide. Concentration of FD1 in the cell was 70 µM and that of Fe(II) in the syringe was 2.23 mM. The Fe(II) solution was prepared in 100 mM Mops, 100 mM NaCl pH 5.8, and FD1 was in 100 mM Mops, 100 mM NaCl, pH 7.0. The data for the integrated heat of binding were corrected for the heat of dilution due to titration of Fe(II) to buffer (control). Measurements were performed at 25°C. (B) Zn(II) binding to transferrin and (C) to the FD1 peptide. For experiments with transferrin concentration of protein in the cell was 126 µM and the concentration of Zn(II) in the syringe was 12 mM. For experiments with the FD1 peptide, concentration of peptide in the cell was 70 µM and that of Zn(II) in the syringe was 3.35 mM. Transferrin, FD1 peptide, and Zn(II) were prepared in 100 mM Mops, 100 mM NaCl, pH 7.0. Measurements were performed at 25°C. The data for the integrated heat of binding were corrected for the heat of dilution due to titration of Zn(II) to buffer (control).</p

Inhibitory effect of Zn(II) on diferric-transferrin complex formation.

<p>(A) Initial rates of diferric-transferrin formation at 460 nm are plotted versus concentration of Zn(II). Concentration of apo-transferrin was 80 µM and that of Fe(II) was 200 µM. (B) Comparison of the inhibitory effect of Zn(II) on the diferric-transferrin complex formation for the non-enzymatic oxidation of Fe(II) by molecular oxygen in the presence of transferrin (Tf 70 µM) and for the ferroxidase activity of ceruloplasmin (CP 0.08 µM) measured with the transferrin assay. Final concentration of Fe(II) was 60 µM and that of Zn(II) was 70 µM. Measurements were done in 100 mM Hepes buffer, 100 mM NaCl, pH 7.2 in triplicate at 37°C.</p

Iron-sulphur clusters as inhibitors and catalysts of viral replication

A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron–sulfur [FeS] clusters, which are among the oldest classes of bio-inorganic cofactors, have barely been considered. Here we discuss how several [FeS] cluster-containing proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biological ‘fuses’ to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorganic virology/immunology and that such studies may reveal new molecular insight into the links between viral infections and diseases like cancer and neurodegeneration