The oxidation of sperm whale, horse, and pig oxymyoglobins, catalyzed by ferrocyanide ions: kinetics and mechanism

The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.     

Controlling the electron transfer rate between oxymyoglobin and ferricytochrome C by alterations in the myoglobin contact site. The role of histidines and electrostatic and steric interactions in the mechanism of the reaction

The kinetics of the redox reaction of sperm whale and pig oxymyoglobins (MbO₂) with ferricytochrome C (CytC) from pig heart has been studied in the pH range 5–8. Also, the effects of histidine (His) modification and of the complexing of both myoglobins with Zn²⁺, on the electron transfer rate, has been investigated. It has been shown that pig MbO₂ reduces Cyt C much more effectively than sperm whale MbO₂. The pH dependence of the reaction rate is shown to result from the influence of two histidines, His 12(A10) and His 119(GH1), in the case of sperm whale myoglobin and only of His GH1 in the case of pig MbO₂. The protonation of His A10 at pH<7.5 decreases the rate of the reaction with Cyt C whereas the ionization of His GH1, on the contrary, increases the electron transfer rate 10–30 times (atI=0.03). The His residues of Cyt C are shown to have no effect on the reaction. Complexing of His GH1 with a zinc ion strongly inhibits the reaction of both sperm whale and pig MbO₂ with Cyt C. The reaction of the zinc-MbO₂ complexes, as distinct from the intact oxymyoglobins, becomes independent of pH and ionic strength. Unlike His A10, His GH1 plays a very important role in the formation of the electron transfer complexes, and is probably directly involved in the charge transfer step. Based on the data obtained, the reactive site of the Mb surface has been identified in the A-GH region. The spatial arrangement of the charged groups in the reactive sites of the two myoglobins has been obtained. The solvent accessibilities of all amino acid residues situated there have been calculated, according to Lee and Richards. In order to explain the different reactivities of sperm whale and pig myoglobins, their electrostatic properties and the steric features of the contact sites have been compared.     

Ferrocyanide - a novel catalyst for oxymyoglobin oxidation by molecular oxygen

A comparative study of the rates of ferrocyanide-catalyzed oxidation of several oxymyoglobins by molecular oxygen is reported. Oxidation of the native oxymyoglobins from sperm whale, horse and pig, as well as the chemically modified (MbO(2)) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM-MbO(2)), and the mutant sperm whale oxymyoglobin [MbO(2)(His119-->Asp)], was studied. The effect of pH, ionic strength and the concentration of anionic catalyst ferrocyanide, [Fe(CN)(6)](4-), on the oxidation rate is investigated, as well as the effect of MbO(2) complexing with redox-inactive Zn(2+), which forms the stable chelate complex with functional groups of His119, Lys16 and Asp122, all located nearby. The catalytic mechanism was demonstrated to involve specific [Fe(CN)(6)](4-) binding to the protein in the His119 region, which agrees with a high local positive electrostatic potential and the presence of a cavity large enough to accommodate [Fe(CN)(6)](4-) in that region. The protonation of the nearby His113 and especially His116 plays a very important role in the catalysis, accelerating the oxidation rate of bound [Fe(CN)(6)](4-) by dissolved oxygen. The simultaneous occurrence of both these factors (i.e. specific binding of [Fe(CN)(6)](4-) to the protein and its fast reoxidation by oxygen) is necessary for the efficient ferrocyanide-catalyzed oxidation of oxymyoglobin.     

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.     

Redox reactivity of the heme Fe<Superscript>3+</Superscript>/Fe<Superscript>2+</Superscript> couple in native myoglobins and mutants with peroxidase-like activity

The reaction enthalpy and entropy for the one-electron reduction of the ferric heme in horse heart and sperm whale aquometmyoglobins (Mb) have been determined exploiting a spectroelectrochemical approach. Also investigated were the T67R, T67K, T67R/S92D and T67R/S92D Mb-H variants (the latter containing a protoheme-l-histidine methyl ester) of sperm whale Mb, which feature peroxidase-like activity. The reduction potential (E°′) in all species consists of an enthalpic term which disfavors Fe³⁺ reduction and a larger entropic contribution which instead selectively stabilizes the reduced form. This behavior differs from that of the heme redox enzymes and electron transport proteins investigated so far. The reduction thermodynamics in the series of sperm whale Mb variants show an almost perfect enthalpy–entropy compensation, indicating that the mutation-induced changes in are dominated by reduction-induced solvent reorganization effects. The modest changes in E°′ originate from the enthalpic effects of the electrostatic interactions of the heme with the engineered charged residues. The small influence that the mutations exert on the reduction potential of myoglobin suggests that the increased peroxidase activity of the variants is not related to changes in the redox reactivity of the heme iron, but are likely related to a more favored substrate orientation within the distal heme cavity.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

Near-heme histidine residues of deoxy- and oxymyoglobins.

Proton NMR titration curves of the histidine Cepsilon-H resonances of the deoxy and oxy forms of human, horse, and sperm whale myoglobins (Mb) were determined and compared with the results for the met and azide forms. One extra titrating resonance (H-8) was observed for each deoxy-Mb compared with the corresponding met-Mb, and a further extra resonance (H-9) was observed for the oxy-Mb form. These resonances correspond to the two additional resonances previously described for azide-Mb [Hayes, M., Hagenmaier, H., & Cohen, J. S. (1975) J. Biol. Chem. 250, 7461--7472]. This new evidence prompts us to reassign these resonances to the near-heme histidine residues.     

1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.     

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu²⁺ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in β-sheet content. Cu²⁺ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu⁺ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu²⁺ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure, allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu²⁺.

The mechanism of oxymyoglobin oxidation catalyzed by ferrocyanide ions: chemically modified and mutant sperm whale myoglobins

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.     

Structural insights into a low-spin myoglobin variant with bis-histidine coordination from molecular modeling

Rational design of functional enzymes is a powerful strategy to gain deep insights into more complex native enzymes, such as nitric oxide reductase (NOR). Recently, we engineered a functional model of NOR by creating a two His and one Glu (2‐His‐1‐Glu) non‐heme iron center in sperm whale myoglobin (swMb L29E, F43H, H64, called Fe_BMb(‐His)). It was found that Fe_BMb(‐His) adopts a low‐spin state with bis‐His coordination in the absence of metal ions binding to the designed metal center. However, no structural information was available for the variant in this special spin state. We herein performed molecular modeling of Fe_BMb(‐His) and compared with the X‐ray structure of its copper bound derivative, Cu(II)‐CN^?‐Fe_BMb(‐His), resolved recently at a high resolution (1.65 Å) (PDB entry 3MN0). The simulated structure shows that mutation of Leu to Glu at position 29 in the hydrophobic heme pocket alters the folding behavior of Mb. The hydrogen bond between Glu29 and His64 further plays a role in stabilizing the bis‐His (His64/His93) coordination structure. This study offers an excellent example of using molecular modeling to gain insights in rational design of both structural and functional proteins. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Peroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation

Peroxynitrite-mediated oxidation of ferrous nitrosylated myoglobin (Mb(II)-NO) involves the transient ferric nitrosylated species (Mb(III)-NO), followed by NO dissociation and formation of ferric myoglobin (Mb(III)). In contrast, peroxynitrite-mediated oxidation of ferrous oxygenated myoglobin (Mb(II)-O₂) involves the transient ferrous deoxygenated and ferryl derivatives (Mb(II) and Mb(IV)O, respectively), followed by Mb(III) formation. Here, kinetics of peroxynitrite-mediated oxidation of ferrous carbonylated horse heart myoglobin (Mb(II)-CO) is reported. Values of the first-order rate constant for peroxynitrite-mediated oxidation of Mb(II)-CO (i.e., for Mb(III) formation) and of the first-order rate constant for CO dissociation from Mb(II)-CO (i.e., for Mb(II) formation) are h = (1.2 ± 0.2) × 10⁻² s⁻¹ and k_off(CO) = (1.4 ± 0.2) × 10⁻² s⁻¹, respectively, at pH 7.2 and 20.0 °C. The coincidence of values of h and k_off(CO) indicates that CO dissociation represents the rate limiting step of peroxynitrite-mediated oxidation of Mb(II)-CO.     

Neurotoxic effects of copper: Inhibition of glycolysis and glycolytic enzymes

The effects of Cu²⁺ on glycolysis and several glycolytic enzymes were studied in rat brain extracts in vitro. At concentrations reportedly found in Wilson's disease, Cu²⁺ significantly inhibited lactate production from glucose or glucose-6-phosphate in rat brain postnuclear supernatant with an IC₅₀ of about 3 M. Cu²⁺ also inhibited several glycolytic enzymes. Amongst the latter, Cu²⁺ was most effective in inhibiting hexokinase (IC₅₀ for Cu²⁺=7 M), moderately effective in inhibiting pyruvate kinase (IC₅₀ for Cu²⁺=56 M), but least effective in inhibiting lactate dehydrogenase (IC₅₀ for Cu²⁺=300 M). These results suggest that inhibition of brain glycolysis may have pathophysiological importance in copper poisoning and in Wilson's disease.     

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study.

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.     

A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin

The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb.     

Effect of copper on inhibition by nitrapyrin of growth ofNitrosomonas europaea

Exponentially growing cultures ofNitrosomonas europaea were inhibited by addition of 0.5 g nitrapyrin ml^–1. This inhibition was increased by simultaneous addition of 0.046 g Cu²⁺ ml^–1 as copper sulfate. This contradicts a previous report that copper relieves inhibition of ammonia oxidation by nitrapyrin, which report has formed the basis for hypotheses regarding the mechanism of action of this inhibitor.     

Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I,II, and IV Leads to Increased ROS Production

Oxidative damage has been implicated in disorders associated with abnormal copper metabolism and also Cu²⁺ overloading states. Besides, mitochondria are one of the most important targets for Cu²⁺, an essential redox transition metal, induced hepatotoxicity. In this study, we aimed to investigate the mitochondrial toxicity mechanisms on isolated rat liver mitochondria. Rat liver mitochondria in both in vivo and in vitro experiments were obtained by differential ultracentrifugation and the isolated liver mitochondria were then incubated with different concentrations of Cu²⁺. Our results showed that Cu²⁺ induced a concentration and time-dependent rise in mitochondrial ROS formation, lipid peroxidation, and mitochondrial membrane potential collapse before mitochondrial swelling ensued. Increased disturbance in oxidative phosphorylation was also shown by decreased ATP concentration and decreased ATP/ADP ratio in Cu²⁺-treated isolated mitochondria. In addition, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling, and release of cytochrome c following of Cu²⁺ treatment were well inhibited by pretreatment of mitochondria with CsA and BHT. Our results showed that Cu²⁺ could interact with respiratory complexes (I, II, and IV). This suggests that Cu²⁺-induced liver toxicity is the result of metal’s disruptive effect on liver hepatocyte mitochondrial respiratory chain that is the obvious cause of Cu²⁺-induced ROS formation, lipid peroxidation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.     

Amicyanin metal-site structure and interaction with MADH: PAC and NMR spectroscopy of Ag-, Cd-, and Cu-amicyanin

To investigate the structural control mechanisms in the metal site of amicyanin when interacting with MADH, redox-inactive Ag⁺- and Cd²⁺-substituted amicyanins were studied with perturbed angular correlations of -rays (PAC) spectroscopy. PAC experiments on ^111mCd-substituted amicyanin revealed two different metal-site structures, which are very likely in dynamic exchange on a ~5 ns timescale. Only one structure binds to MADH. The dissociation constants, K _d, are 9±2 M with MADH^red and 38±11 M with MADH^ox, indicating that the Cd-amicyanin binding affinity is regulated by the MADH redox state. PAC experiments on ¹¹¹Ag-substituted amicyanin also showed two different forms of Ag-amicyanin, probably reflecting relaxation from Ag to Cd geometry. No binding of Ag-amicyanin to MADH could be observed with PAC, suggesting that the K _d is larger than 43 M, based on the 95% confidence limit. NMR revealed large chemical shift differences between native copper amicyanin and both metal-substituted forms. Affected residues are found up to 15 Å away from the metal ion. The Ag⁺- and Cd²⁺-substituted amicyanins demonstrate no change in coordination as a function of pH, contrary to Cu⁺-amicyanin which shows protonation of the copper ligand His96 with pK _a=6.8. It is concluded that, contrary to other blue copper proteins, Ag⁺-amicyanin is not a close mimic of Cu⁺-amicyanin, and that structural changes in the metal site have large effects on the affinity for the redox partner.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AOM angular overlap model - HSQC heteronuclear single-quantum coherence - MADH methylamine dehydrogenase - MADH^ox oxidized MADH - MADH^red reduced MADH - NOESY nuclear Overhauser effect spectroscopy - NQI nuclear quadrupole interaction - PAC perturbed angular correlations of -rays - TOCSY total correlation spectroscopy     

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Background

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.

Methodology/Principal Findings

As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn₂-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu²⁺ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu²⁺: a moderate-affinity site (10⁶ M^-1) and a high-affinity site (10⁸ M^-1). Furthermore, Cu²⁺ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.

Conclusions/Significance

We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu²⁺ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.