Kourosh Honarmand Ebrahimi

The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.

A conserved iron-binding site, the ferroxidase center, regulates the vital iron storage role of the ubiquitous protein ferritin in iron metabolism. It is commonly thought that two Fe(II) simultaneously bind the ferroxidase center and that the oxidized Fe(III)-O(H)-Fe(III) product spontaneously enters the cavity of ferritin as a unit. In contrast, in some bacterioferritins and in archaeal ferritins a persistent di-iron prosthetic group in this center is believed to mediate catalysis of core formation. Using a combination of binding experiments and isotopically labeled (57)Fe(II), we studied two systems in comparison: the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus (PfFtn) and the eukaryotic human H ferritin (HuHF). The results do not support either of the two paradigmatic models; instead they suggest a unifying mechanism in which the Fe(III)-O-Fe(III) unit resides in the ferroxidase center until it is sequentially displaced by Fe(II).

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 Conserved Tyrosine in Ferritin Is a Molecular Capacitor

A highly conserved tyrosine residue of unknown function is present in the vicinity of the di‐iron catalytic center of the ubiquitous iron‐storage protein ferritin. The di‐iron center with a gateway FeII/FeIII‐binding site nearby provides the vital iron‐storage mechanism of the protein. It is believed that, in eukaryotic ferritin, this center catalyzes simultaneous oxidation of two FeII ions, whereas in microbial ferritin it catalyzes simultaneous oxidation of three FeII ions. To understand the role of the conserved tyrosine, we studied the intermediates and products that are formed during catalysis of FeII oxidation in the di‐iron catalytic centers of the hyperthermophilic archaeal Pyrococcus furiosus ferritin and of eukaryotic human H ferritin. Based on our spectroscopic studies and modeling, we propose a merger of the models for eukaryotic and bacterial ferritin into a common mechanism of FeII oxidation in which the conserved tyrosine acts as a single‐electron molecular capacitor to facilitate oxidation of FeII.

The Amyloid Precursor Protein (APP) Does Not Have a Ferroxidase Site in Its E2 Domain

The ubiquitous 24-meric iron-storage protein ferritin and multicopper oxidases such as ceruloplasmin or hephaestin catalyze oxidation of Fe(II) to Fe(III), using molecular oxygen as oxidant. The ferroxidase activity of these proteins is essential for cellular iron homeostasis. It has been reported that the amyloid precursor protein (APP) also has ferroxidase activity. The activity is assigned to a ferroxidase site in the E2 domain of APP. A synthetic 22-residue peptide that carries the putative ferroxidase site of E2 domain (FD1 peptide) has been claimed to encompass the same activity. We previously tested the ferroxidase activity of the synthetic FD1 peptide but we did not observe any activity above the background oxidation of Fe(II) by molecular oxygen. Here we used isothermal titration calorimetry to study Zn(II) and Fe(II) binding to the natural E2 domain of APP, and we employed the transferrin assay and oxygen consumption measurements to test the ferroxidase activity of the E2 domain. We found that this domain neither in the presence nor in the absence of the E1 domain binds Fe(II) and it is not able to catalyze the oxidation of Fe(II). Binding of Cu(II) to the E2 domain did not induce ferroxidase activity contrary to the presence of redox active Cu(II) centers in ceruloplasmin or hephaestin. Thus, we conclude that E2 or E1 domains of APP do not have ferroxidase activity and that the potential involvement of APP as a ferroxidase in the pathology of Alzheimer’s disease must be re-evaluated.

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

The β-amyloid precursor protein (APP), which is a key player in Alzheimers 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.

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.

A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of an uncharacterized branch of the ferritin-like superfamily

Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins. In an attempt to identify new iron-storage systems, we have found and characterized a new member of the ferritin-like superfamily of proteins, which unlike the multimeric storage system of ferritin, bacterioferritin, and Dps is monomeric in the absence of iron. Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin. We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.

The radical‐SAM enzyme Viperin catalyzes reductive addition of a 5′‐deoxyadenosyl radical to UDP‐glucose in vitro

Viperin, a radical‐S‐adenosylmethionine (SAM) enzyme conserved from fungi to humans, can restrict replication of many viruses. Neither the molecular mechanism underlying the antiviral activity of Viperin, nor its exact physiological function, is understood: most importantly, no radical‐SAM activity has been discovered for Viperin. Here, using electron paramagnetic resonance (EPR) spectroscopy, mass spectrometry, and NMR spectroscopy, we show that uridine diphosphate glucose (UDP‐glucose) is a substrate of a fungal Viperin (58% pairwise identity with human Viperin at the amino acid level) in vitro. Structural homology modeling and docking experiments reveal a highly conserved binding pocket in which the position of UDP‐glucose is consistent with our experimental data regarding catalytic addition of a 5′‐deoxyadenosyl radical and a hydrogen atom to UDP‐glucose.

Spectroscopic evidence for the role of a site of the di-iron catalytic center of ferritins in tuning the kinetics of Fe(ii) oxidation.

Ferritin is a nanocage protein made of 24 subunits. Its major role is to manage intracellular concentrations of free Fe(ii) and Fe(iii) ions, which is pivotal for iron homeostasis across all domains of life. This function of the protein is regulated by a conserved di-iron catalytic center and has been the subject of extensive studies over the past 50 years. Yet, it has not been fully understood how Fe(ii) is oxidized in the di-iron catalytic center and it is not known why eukaryotic and microbial ferritins oxidize Fe(ii) with different kinetics. In an attempt to obtain a new insight into the mechanism of Fe(ii) oxidation and understand the origin of the observed differences in the catalysis of Fe(ii) oxidation among ferritins we studied and compared the mechanism of Fe(ii) oxidation in the eukaryotic human H-type ferritin (HuHF) and the archaeal ferritin from Pyrococcus furiosus (PfFtn). The results show that the spectroscopic characteristics of the intermediate of Fe(ii) oxidation and the Fe(iii)-products are the same in these two ferritins supporting the proposal of unity in the mechanism of Fe(ii) oxidation among eukaryotic and microbial ferritins. Moreover, we observed that a site in the di-iron catalytic center controls the distribution of Fe(ii) among subunits of HuHF and PfFtn differently. This observation explains the reported differences between HuHF and PfFtn in the kinetics of Fe(ii) oxidation and the amount of O2 consumed per Fe(ii) oxidized. These results provide a fresh understanding of the mechanism of Fe(ii) oxidation by ferritins.

Phosphate accelerates displacement of Fe(III) by Fe(II) in the ferroxidase center of Pyrococcus furiosus ferritin

The iron‐storage protein, ferritin, is widely found in all Domains of life. A conserved diiron center in ferritin catalyzes oxidation of Fe(II) and regulates storage of the resultant Fe(III) oxidation product. When this center is filled with Fe(III), in bacterial or archaeal ferritin the presence of phosphate accelerates the rate of Fe(II) oxidation. The molecular mechanism underlying this stimulatory effect of phosphate is unknown. Using site directed mutagenesis of the residues in the diiron center of the archaeal ferritin from Pyrococcus furiosus we show that phosphate facilitates displacement of Fe(III) by Fe(II) from this site. Therefore, the rate of Fe(II) oxidation increases only when the ferroxidase center is filled with Fe(III).