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.

Recently a ferroxidase activity has also been reported for the amyloid precursor protein (APP) [21] ( Figure 1C ). APP is a transmembrane protein which consists of two extracellular domains known as the E1 and E2 domains, a short transmembrane section containing part of the Aβ peptide, and a small interacellular domain (AICD) [22] . Alternative splicing of exon regions of the APP mRNA creates APP- isoforms with different amino acid lengths [23] , [24] . APP is of special interest because of its possible role in Alzheimer’s disease [25] . Duce et al. [21] have recently reported that the E2 domain of APP has a putative ferroxidase site, which behaves like the ferroxidase center of ferritins. It was observed that the E2 domain in the presence of the E1 domain has ferroxidase activity equal to that of the full APP-ectodomain and comparable to that of ceruloplasmin. The ferroxidase activity of the E2 domain was inhibited by Zn(II) like that of ferritin [26] – [28] . Based on these findings it has been proposed that the ferroxidase activity of APP in Alzheimer’s disease has the same function as the ferroxidase activity of ceruloplasmin coupled to iron-export activity of ferroportin [14] : the Fe(II) ion that is exiting ferroportin binds to the ferroxidase site of APP, it is oxidized by molecular oxygen, and the resulting Fe(III) product is then scavenged by the ferric binding protein, transferrin. In individuals with Alzheimer’s disease Zn(II) would bind to the ferroxidase site in the E2 domain of APP and inhibits its ferroxidase activity, resulting in accumulation of intracellular Fe(II) and subsequent oxidative damage of the cells. Duce et al. [21] used an unfitting structural assignment as discussed previously [29] , and they applied the Fe(III)-transferrin colorimetric assay to measure the ferroxidase activity of APP, its E2 domain, and of the E2-domain derived synthetic 22-residue peptide FD1. Previously we tested the ferroxidase activity of the FD1 peptide by following the production of Fe(III) with the transferrin assay, and the consumption of molecular oxygen amperometrically. We found that the FD1 peptide does not have any ferroxidase activity and that Zn(II) interferes with the transferrin assay [29] . In the present study we re-evaluate the described ferroxidase activity of the E2 domain of APP and the effect of the E1 domain on this activity. We show that consistent with our previous results for the synthetic FD1 peptide [29] the E2 domain of APP does not bind Fe(II) and does not have a ferroxidase activity either in the presence or in the absence of the E1 domain.

In ferritin the ferroxidase reaction occurs in a diiron binding site, the ferroxidase center, with a highly conserved tyrosine in the vicinity of this site essential for the catalytic activity [16] ( Figure 1A ). The Fe(II) binds to this center and reacts with molecular oxygen under formation of either hydrogen peroxide or water [16] . The metastable Fe(III) product leaves the ferroxidase center and enters the protein cavity upon arrival of incoming Fe(II) ions [17] , [18] . In some multicopper oxidases such as ceruloplasmin and haphaestin the ferroxidase reaction appears to occur via outer-sphere electron transfer [4] . It is proposed that electrons are transferred from the Fe(II) ions bound to the protein to a type I copper center and then to a trinuclear copper center where molecular oxygen is reduced to water ( Figure 1B ) [4] . Possible Fe(II) binding sites have been identified in ceruloplasmin [19] , [20] . The resulting Fe(III) product in these ferroxidases is proposed to be scavenged by an Fe(III)-binding protein such as transferrin to prevent precipitation of Fe(III) products.

(A) Quaternary structure of 24-meric ferritin (HuHF, PDB code 2FHA) showing the position of the diiron binding site where the ferroxidase reaction occurs. The two iron binding sites are marked with A and B. (B) Structure of the multicopper oxidase ceruloplasmin (PDB code 1KCW). Ceruloplasmin contains of three type I copper centers (blue sphere), one type II copper center (green sphere), and one type III copper center (orange sphere). Type II and III centers together form a trinuclear copper center which is responsible for four electron oxidation of molecular oxygen to water. Red spheres in the structure show other possible metal binding sites. (C) A schematic representation of the APP and X-ray structure of the E2 domain of APP 695 (PDB 3UMH). The structure shows the specific Cu(II) (red sphere) binding site (M1 site) with four histidines (His313, His382, His432, and His436) as coordinating residues. Glu337 and Glu340 are the putative ligands of the previously defined ferroxidase site in the E2 domain of APP [21] . The numbering of the residues is based on APP 695 .

Oxidation of Fe(II) to Fe(III) is an essential reaction in cellular iron homeostasis. Under physiological conditions this reaction can occur spontaneously in the presence of oxidants such as molecular oxygen or hydrogen peroxide. Spontaneous (i.e. not biologically catalyzed) oxidation of Fe(II) produces Fe(III) and reactive oxygen species (ROS). Fe(III) is essentially insoluble under physiological conditions, with a solubility of 10 −10 M [1] , and will therefore precipitate, whereas ROS such as the hydroxyl radical will react uncontrollably with many components of the cell. To prevent formation of these toxic products and to keep the iron in a soluble form for cellular usage, proteins evolved to carry out controlled catalytic oxidation of Fe(II) to Fe(III) in the ferroxidase reaction. The proteins for which ferroxidase activity has been established can be divided into two main groups: (i) members of the ferritin superfamily [2] , [3] including ferritin ( Figure 1A ), bacterioferritin, and Dps (DNA binding protein from starved cells), and (ii) multicopper oxidases [4] such as ceruloplasmin [5] , [6] ( Figure 1B ) or hephaestin [7] , [8] . The ferroxidase activity of proteins in the ferritin superfamily is essential for controlling the intracellular concentration of Fe(II) or for protection of DNA from reactive oxygen species. For example ferritin and bacterioferritin oxidize excess Fe(II) and store the resulting Fe(III) product in a non-toxic form [9] , [10] . The ferroxidase activity of multicopper oxidases such as ceruloplasmin appears to be essential for transport of iron across cellular membranes [11] – [15] .

Results and Discussion

The E2 Domain Binds Cu(II) Before measuring ferroxidase activity and Fe(II) binding of the E2 domain of APP, we measured binding of Cu(II) to the E2 domain. Cu(II) binding was used to test the correct folding state of the protein because the APP and its E2 or E1 domains do not have any established catalytic activity. Protein crystallography and a number of biochemical and biophysical studies were used before to ascertain the functional fold of the used recombinant protein [28]. Its purification was also assessed by SDS-PAGE analysis (Figure S1). As four histidines must come together from sequentially distant places in primary structure to form the M1-site of the E2 (Figure 1), binding of Cu(II) to the E2 domain is probably one of the best measurements to analyze its correct three-dimensional fold. Using isothermal titration calorimetry (ITC) it has been previously reported that the E2 domain of APP binds Cu(II) with a stoichiometry of circa 0.7 Cu(II) per E2 domain and a dissociation constant of 0.013±0.005 µM [30]. Those measurements were performed in Tris buffer at pH 7.3 (Tris is a competing ligand) to eliminate any low-affinity binding event and the results were corrected for the Cu(II) binding to Tris. We measured binding of Cu(II) to the E2 domain of APP using ITC in Mops buffer pH 7.0 (Figure 2). A model with two independent binding sites was required to obtain a fit to the data of integrated heat of binding. It resulted in two binding events (Figure 2): one binding event with a stoichiometry of 0.77±0.15 Cu(II) per E2 domain and a dissociation constant of 0.08±0.03 µM, and a second low affinity binding event. The stoichiometry of the second binding event could not be determined with precision due to its low affinity; however, this binding event was required to obtain a fit to the experimental data. The thermodynamic parameters of the first binding event in Mops buffer (Figure 2) are within experimental error identical to the previously published results when corrected for Tris binding [30]. The second low affinity binding event is possibly due to non-specific binding of Cu(II) to the E2 domain of APP or Cu(II) induced intermolecular interactions between the E2 domains. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Cu(II) binding to the E2 domain of APP measured by ITC. We measured binding of Cu(II) to the E2 domain in non-coordinating buffer using isothermal titration calorimetry (ITC). Concentration of E2 domain in the cell was 26.5 µM and that of Cu(II) in the syringe was 1.27 mM. Measurements were performed at 25°C in 100 mM Mops 150 mM NaCl, pH 7.0. A model with two independent binding sites was required to obtain a fit to the data of integrated heat of binding. *The stoichiometry of the second binding event could not be determined with statistical significance. The data represent the average of two experiments ± standard deviation. https://doi.org/10.1371/journal.pone.0072177.g002 To check the results of ITC measurements, we monitored binding of Cu(II) to the E2 domain using electron paramagnetic resonance (EPR) spectroscopy. Upon addition of Cu(II) to the E2 domain of APP an EPR signal with four lines centered at g value of 2.2528 appeared (Figure 3A). These peaks arise because of hyperfine coupling to the I = 3/2 Cu(II) nucleus. The EPR spectrum of the Cu(II) binding site of the E2 domain could be simulated assuming superhyperfine splitting (just barely resolved) in the perpendicular direction from four nitrogen ligands (Figure 3A). Thus, this binding event is associated to a specific Cu(II) binding site that is observed in the E2 domain using X-ray crystallography with four histidines as coordinating residues (His313, His382, His432, and His436 based on APP 695 numbering) [30] (Figure 1C). As the amount of Cu(II) increased from 1.2 Cu(II) per E2 domain to 2.4 Cu(II) per E2 domain, the hyperfine pattern of the Cu(II) became more complex (Figure 3A) suggesting the presence of other Cu(II) binding sites with overlapping hyperfine structure except for the low-field peak around 2650 gauss. A plot of EPR intensity at this field strength versus the amount of Cu(II) added to E2 domain showed a stoichiometry of 0.9±0.1 for the first binding site (Figure 3B). Thus, the results of EPR spectroscopy confirmed that Cu(II) binds to the E2 domain at a specific site whose coordination sphere consists of four histidines. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Cu(II) binding to the E2 domain of APP measured by EPR spectroscopy. (A) Binding of Cu(II) to the E2 domain of APP was recorded using electron paramagnetic resonance spectroscopy (EPR). Simulation of the EPR spectrum of Cu(II) bound to the E2 domain was performed using 4 nitrogen atoms as coordinating ligands for the 0.9 Cu(II)/E2 sample. The simulation parameters were: g ⊥ = 2.053 and g ∥ = 2.2528; line widths (gauss) were 11, 11, and 15; copper hyperfine splittings (gauss) were: 24, 24, and 176 gauss; nitrogen hyperfine splittings (gauss) were: 18, 18, and 11. The inserts show enlargements of part of the spectra which are marked by red lines. (B) The EPR intensity at 2650 gauss is plotted as a function of the amount of Cu(II) added to E2 domain. A 159 mM solution of E2 domain in 100 mM Mops 150 mM NaCl pH 7.0 was used. https://doi.org/10.1371/journal.pone.0072177.g003

The E2 Domain does not Show Ferroxidase Activity in the Transferrin Assay We showed that the results of Cu(II) binding to the E2 domain of APP were reproducible, which suggested to us that our E2 preparation was in a properly folded state. Subsequently, we checked if we can reproduce the ferroxidase activity of the E2 domain reported by Duce et al [21]. We measured the kinetics of Fe(II) oxidation by recording incorporation of the Fe(III) product into apo-transferrin and formation of the Fe(III)-transferrin complex at 460 nm (Figure 4A). We compared this activity with the ferroxidase activity of two ferritins, i.e. eukaryotic human H ferritin (HuHF) and archaeal Pyrococcus furiosus ferritin (PfFtn) as measured by following the formation of an Fe(III)-mineral core inside the cavity of these proteins in the absence of transferrin. Fe(III)-mineral core formation in HuHF was followed at 310 nm using a molar extinction coefficient of 2.47 mM−1cm−1 [31], [32], and for PfFtn at 315 nm using a molar extinction coefficient of 2.5 mM−1cm−1 [33] (Figure 4A). The transferrin assay was not used to measure the ferroxidase activity of ferritin because ferritin binds and stores the Fe(III) and therefore, the rate of transferrin-Fe(III) complex formation does not represents the actual rate of the ferroxidase activity of ferritin [29]. Both in the absence of the E2 domain and in its presence the rates of Fe(III)-transferrin complex formation were within experimental error identical; thus the E2 domain of APP (in the absence of the E1 domain) does not show ferroxidase activity in the transferrin assay (Figure 4A). These rates were within experimental error identical to those we observed previously in the presence or absence of the FD1 peptide [29]. In contrast, the ferroxidase activities of HuHF and PfFtn were significantly higher than the background oxidation of Fe(II) that was measured by the transferrin assay. The lower activity of PfFtn (Figure 4A) in comparison to that of HuHF at 37°C is because PfFtn is a hyperthermophilic protein which has its optimal activity at temperatures around 100°C. The E2 domain can bind to Cu(II) and this binding induces a large conformational change [30]. Therefore, we measured if binding of Cu(II) to the E2 domain can induce ferroxidase activity. We incubated the E2 domain with one Cu(II) per E2 domain and we looked for ferroxidase activity by measuring incorporation of the Fe(III) product into transferrin (Figure 4A). The results show that binding of Cu(II) does not induce ferroxidase activity in the E2 domain of APP. The presence of Cu(II) slightly increased the background oxidation of Fe(II) by molecular oxygen and incorporation of the Fe(III) product into transferrin in the presence or absence of the E2 domain (Figure 4A). Finally, we tested the effect of pH on the ferroxidase activity of the E2 domain (Figure 4B). The initial rate of background oxidation of Fe(II) and incorporation of the resultant Fe(III) product into transferrin increases hyperbolically as the pH increases from 6 to 8.5 consistent with our previous results [29]. Moreover, at none of the tested pH values the presence of the E2 domain increased the rate of Fe(II) oxidation above the background reaction (Figure 4B). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. The E2 domain of APP does not have ferroxidase activity in transferrin assay. (A) The initial rate (µM Fe(III) formed per min) of Fe(III) formation was measured in the presence and absence of the E2 domain of APP using the transferrin assay. The effect of Cu(II) was tested on the ferroxidase activity of the E2 domain. The results were compared with the ferroxidase activity of HuHF and PfFtn. The initial rate of ferroxidase activity of ferritin was obtained from the initial slope of the progress curves at 310 nm for HuHF or at 315 nm for PfFtn. Concentrations of the E2 domain, HuHF (monomer) or PfFtn (monomer) were 1.6 µM. Measurements were performed at 37°C in 100 mM Mops, 100 mM NaCl pH 7.0. The concentrations of Fe(II) and of transferrin were 80 µM and 100 µM respectively. (B) The effect of pH on the ferroxidase activity of the E2 domain was measured and was compared with that of background oxidation of Fe(II) and incorporation of the Fe(III) product into transferrin. The concentrations of E2 domain, of transferrin, and of Fe(II) were 1.6 µM, 100 µM, and 80 µM, respectively. Measurements were performed at 37°C. https://doi.org/10.1371/journal.pone.0072177.g004

The E2 Domain does not Consume Molecular Oxygen to Catalyze Oxidation of Fe(II) To further test the proposed ferroxidase activity of the E2 domain of APP we recorded consumption of molecular oxygen which is the second substrate in the ferroxidase reaction. We compared the results of the E2 domain with those of HuHF and of BSA. HuHF consumes molecular oxygen to catalyze oxidation of Fe(II) and thus is used as a positive control. BSA is not able to catalyze oxidation of Fe(II) and is used as a negative control. HuHF shows significant consumption of dioxygen upon addition of 50 Fe(II) per subunit of protein (1200 Fe(II) per 24-mer) (Figure 5A). We found a stoichiometry of circa 3.5 Fe(II) per molecular oxygen consistent with the literature for Fe(II) added to HuHF in a ratio greater than 150 Fe(II) per 24-meric ferritin [34]. The activities of the E2 domain of APP and of BSA were zero the same as the FD1 peptide which we have tested previously [29]. Thus, consistent with the results obtained from UV-visible spectroscopy, we conclude that the E2 domain in the absence of the E1 domain does not catalyze oxidation of Fe(II) as measured on molecular-oxygen consumption. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. The E1 domain does not induce ferroxidase activity in the E2 domain of APP. (A) Consumption of molecular oxygen upon addition of Fe(II) was measured for HuHF (2.7 µM monomer), E2 domain (2.7 µM), E1 domain (2.7 µM), E2 domain (2.7 µM) in the presence of E1 domain (2.7 µM), and BSA (2.7 µM). The concentration of Fe(II) was 270 µM. Measurements were performed in 100 mM Mops, 100 mM NaCl pH 7.0. Temperature was 22°C. (B) The transferrin assay was used to measure the ferroxidase activity of the E2 domain (1.6 µM) in the presence of different amounts of E1 domain (gray circles), that of E1 domain alone (1.6 µM, blue rectangle), or that of background oxidation of Fe(II) and incorporation of the Fe(III)-product into transferrin in the absence of the E1 and E2 domain (purple triangle). In all experiments concentration of transferrin was 100 µM and that of Fe(II) was 80 µM. Temperature was 37°C. https://doi.org/10.1371/journal.pone.0072177.g005

The E1 Domain does not Induce Ferroxidase Activity in the E2 Domain Duce et al. [21] reported that the E1 domain stimulates the ferroxidase activity of the E2 domain circa two-fold to a level that is identical to that of recombinant soluble APP695α. Therefore, we measured the ferroxidase activity of the E2 domain of APP in the presence of different amounts of E1 domain using both the transferrin assay and dioxygen-consumption measurements. In the transferrin assay (Figure 5B), the presence of different amounts of the E1 domain did not affect the activity. Within experimental error the activity of the E2 domain was always identical to that of background oxidation of Fe(II) and incorporation of Fe(III) product into transferrin. Furthermore, the E1 domain alone also did not show any ferroxidase activity. Consistent with these data, oxygen consumption measurements also showed that in the presence of one E1 domain per E2, the activity is identical to that of the E2 domain alone and to that of BSA, i.e. zero (Figure 5A). Only HuHF as a positive control showed significant ferroxidase activity upon addition of Fe(II). Therefore, we conclude that the E1 domain does not activate the E2 domain for ferroxidase activity.

The E2 Domain does not Bind Fe(II) Because we found that the E2 domain of APP does not catalyze oxidation of Fe(II) using molecular oxygen and that the E1 domain does not induce any ferroxidase activity in the E2 domain, we tested if the E2 domain binds Fe(II) at all. We measured binding of Fe(II) to the E2 domain under anaerobic conditions using isothermal titration calorimetry (ITC) and we compared the results with those of Fe(II) binding to PfFtn as a positive control. Consistent with our previous observation [17], for PfFtn we observed three binding events per subunit (Figure 6A): one high affinity binding event with a stoichiometry of one and association constant of (9.00±0.4)·105 M−1, and two lower affinity binding events each with a stoichiometry of one and an association constants of (3.3±0.2)·104 M−1 and (1.4±0.1)·104 M−1, respectively. These binding events have been assigned to binding of Fe(II) to the ferroxidase center and a gateway site in its vicinity [17]. The thermodynamic parameters of these bindings were within experimental error identical to our previous results [29]. Fe(II) binding to HuHF under anaerobic conditions also shows three binding sites the same as PfFtn [17]. For the E2 domain of APP (Figure 6B) however, within the sensitivity of the ITC experiments we did not observe significant binding of Fe(II) at pH 7.0. We found that the solution after ITC experiments turned milky suggesting aggregation of the E2 domain, which is possibly due to metal ion induced intermolecular interactions between E2 domains. This was possibly the reason for the observation of a small amount of heat consumed during the anaerobic Fe(II) titration (Figure 6B). This is in line with our observation that Fe(II) did not bind to APP-E2 crystals in soaking experiments the same as those reported by Dahms et al. ([30] and S.O.Dahms personal communication). Thus, in contrast to the data reported by Duce et al. [21] we conclude that the E2 domain of APP does not bind Fe(II) and it does not have a ferroxidase site. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. The E2 domain does not bind Fe(II). (A) Anaerobic binding of Fe(II) to Pyrococcus furiosus ferritin (PfFtn) was measured using isothermal titration calorimetry (ITC). Concentration of apo-PfFtn in the cell was 96 µM (subunit) and that of Fe(II) solution in the syringe was 9.18 mM. Measurements were performed at 25°C. A control experiment was performed in the absence of PfFtn to obtain the heat of dilution of Fe(II) titrated in buffer (purple rectangle). The data of integrated heat of binding of Fe(II) to apo-PfFtn (red circles) were corrected for the heat of dilution. The black line shows the fit of an equation with three sequential binding sites. A model of three sequential binding sites was required to obtain a fit to the data of integrated heat of binding. (B) Anaerobic binding of Fe(II) to the E2 domain of APP was measured by ITC. The concentration of the E2 domain in the cell was 40 µM and that of Fe(II) in the syringe was 1.27 mM. Measurements were performed at 25°C. A control experiment was performed in the absence of the E2 domain to obtain the heat of dilution of Fe(II) in buffer (purple rectangle). The data of integrated heat of binding of Fe(II) to the E2 domain (red circles) were corrected for heat of dilution. For all measurements buffer was 200 mM Mops, 150 mM NaCl, pH 6.9. Each experiment was performed at least in duplicate. https://doi.org/10.1371/journal.pone.0072177.g006