An alternative mechanism of bicarbonate-mediated peroxidation by copper-zinc superoxide dismutase: rates enhanced via proposed enzyme-associated peroxycarbonate intermediate.

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 A resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Selfinactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 Å resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.
Copper-zinc superoxide dismutase (SOD1, 1 CuZn-SOD) is a 32-kDa homodimeric protein that catalyzes the disproportionation of superoxide anion into dioxygen and hydrogen peroxide (2O 2 . ϩ 2H ϩ 3 O 2 ϩ H 2 O 2 ) through redox cycling of its catalytic copper ion (1,2). Each subunit of the enzyme contains a progressively narrowing channel lined with charged residues that guide O 2 . toward the active site (3,4). Immediately adjacent to the copper ion, the channel constricts and the guanidinium group of Arg-143 and the side chain of Thr-137 together act to exclude large nonsubstrate anions (5). Small anions such as cyanide (CN Ϫ ) and azide (N 3 Ϫ ) can proceed past this channel constriction and competitively inhibit the enzyme by binding directly to the copper ion (6). Certain larger anions such as hydrogen phosphate (HPO 4 Ϫ2 ) are also pulled into the active site channel but do not bind tightly to the copper. Instead, they remain associated with Arg-143 in the "anion-binding site" approximately 5 Å away (7).
In addition to its well known O 2 . disproportionation activity, the active site of CuZn-SOD can interact with H 2 O 2 to generate a powerful oxidant (8 -10). Once formed, this oxidant can participate in one of two reaction pathways. In the first, designated herein as the self-oxidative pathway, it can inactivate CuZn-SOD by damaging nearby active site histidine copper ligands, resulting in copper loss (11)(12)(13)(14). In the second, designated as the external oxidative pathway, the oxidant instead reacts with exogenous substrates, protecting the enzyme from inactivation (8,10,15,16). The following reaction scheme has been proposed for these pathways as shown in Reactions 1-3, Reaction 2 generates a highly reactive hydroxyl radical (HO ⅐ ). The observation that this HO ⅐ does not readily react with scavengers of free HO ⅐ such as ethanol led to the proposal that the HO ⅐ was "bound" to the catalytic copper ion. This hypothesis was supported by the observation that small anions such as formate (HCO 2 Ϫ ) and N 3 Ϫ that can traverse the active site channel constriction and gain close approach to the copper ion are able to protect the enzyme from inactivation by serving as sacrificial substrates (8 -10) as shown in Reactions 4 and 5.
This single electron oxidation of substrates is referred to as the peroxidase function of SOD1 because of its similarity to the one-electron oxidation by horseradish peroxidase and H 2 O 2 (18).
The peroxidase activity of SOD1 is not strictly limited to small substrates that can gain direct access to the copper ion.
Several laboratories have sought to delineate the mechanistic role of HCO 3 Ϫ in the external oxidative pathway of SOD1. Sankarapandi and Zweier (16) propose that HCO 3 Ϫ bound to the SOD1 anion-binding site creates a hydrogen-bonding template for H 2 O 2 near the copper ion that facilitates its partitioning into ⅐ OH and OH Ϫ (see Reaction 2). Liochev and Fridovich (17) suggest that if this were true, then both the rate of endogenous SOD1 self-inactivation and the rate of oxidation of larger exogenous substrates in Reaction 3 should be enhanced by the presence of HCO 3 Ϫ . To test this hypothesis, they (17) monitored the rate of self-inactivation of SOD1 in 100 mM phosphate buffer and observed no significant rate enhancement when 10 mM HCO 3 Ϫ was added. On this basis, they suggested that HCO 3 Ϫ does not facilitate H 2 O 2 binding, but rather, HCO 3 Ϫ can itself be oxidized by the copper-bound HO ⅐ to carbonate radical anion (CO 3 ⅐Ϫ ), which in turn can diffuse from the active site channel to oxidize larger, bulky, exogenous substrates (Reactions 6 and 7) or remain associated with the anion-binding site to oxidize histidine copper ligands (Reactions 8 and 9) (17, 20, 22, 23).
Building on this model, we reasoned that if "diffusible" CO 3 ⅐Ϫ is indeed formed in the active site channel, the presence of HCO 3 Ϫ in the reaction mixture must partially protect the enzyme from self-inactivation as is observed with formate or azide in Reactions 4 and 5 (8 -10). Here, we test the effect of HCO 3 Ϫ on the rate of self-inactivation in the absence of other oxyanions that might compete for binding to the anion-binding site (e.g. phosphate). We find that the rate of self-inactivation of wild type SOD1 is significantly enhanced under these conditions rather than diminished. Thus, the strong oxidant produced in this experiment arises from a pathway that is mechanistically distinct from Reactions 2 and 6. We also show that the human Leu-38 to Val (L38V) FALS SOD1 protein demonstrates increased rates of self-inactivation relative to the wild type protein whether HCO 3 Ϫ is present or not. Finally, x-ray crystallographic analysis of the human Asp-125 to His (D125H) FALS SOD1 protein suggests a mechanism for both the selfoxidative and external oxidative pathways that proceeds through an enzyme-associated peroxycarbonate (HCO 4 Ϫ ) intermediate. This chemistry has direct relevance to the understanding of SOD1-mediated oxidative cellular damage and how members of the "wild type-like" and "metal-binding region" mutant classes of FALS SOD1 proteins can be fused into a single class of molecules that are toxic to motor neurons (for review see Ref. 29).

EXPERIMENTAL PROCEDURES
Materials-All of the solutions were prepared using distilled water passed through a Millipore ultrapurification system. EDTA was purchased from Sigma. pH was adjusted by the addition of H 2 SO 4 (double distilled from Vycor, GFC Chemical Co.) and NaOH (Puratronic, Baker Chemical Co.). Monobasic phosphate buffer (Ultrex, JT Baker Co.) at a concentration of 100 mM was used in all of the measurements requiring phosphate. Sodium bicarbonate (EM Science) at a concentration of either 10 or 25 mM was used in all of the measurements that required bicarbonate anion. Solutions buffered using 0.5 mM Tris were adjusted with 100 mM sodium chloride to negate the effect of shifts in ionic strength between experiments with and without bicarbonate anion. Hydrogen peroxide was of the highest purity (The Olin Corporation). The concentration of hydrogen peroxide was measured by the titration against iodate and by its absorbance at 230 nm (extinction coefficient ϭ 61 M Ϫ1 cm Ϫ1 ). Ethanol was purchased from Quantum Chemical Co.
Expression and Purification of Wild type and L38V SOD1-Human wild type and L38V SOD1 proteins were expressed in insect cells and purified as described previously (30). The metallation states of protein samples were not altered following purification. SOD1 protein concentrations were determined using an extinction coefficient of 1.08 ϫ 10 4 M Ϫ1 cm Ϫ1 for the purified enzyme. Purity was estimated using SDS-PAGE and electrospray mass spectrometry. Metal content analyses were performed using inductively coupled plasma mass spectrometry techniques.
Pulse Radiolysis Experiments-Pulse radiolysis experiments were performed using the 2 MeV Van de Graaff accelerator at Brookhaven National Laboratory. Dosimetry was established using the KSCN dosimeter, assuming that (SCN) 2 Ϫ is generated with a G value of 6.13 and has a molar absorptivity of 7950 M Ϫ1 cm Ϫ1 at 472 nm. Irradiation of water by an electron beam generates the primary radicals, ⅐ OH, e aq Ϫ , and ⅐ H. The solutions were immediately pulse-irradiated, and their SOD activity was determined. SOD activity is known to be ionic strength-dependent, and the pK of ethanol is well above 9; therefore, variation in the final EtOH concentration would not alter the ionic strength of the solution. The indicated reaction temperatures were maintained in a thermostated water bath for the duration of the experiments. The pulse radiolysis cell was thermostated to the same temperature as the water bath. D125H SOD1 Purification, Crystallization, and Structure Determination-Recombinant human D125H CuZn-SOD was obtained as described previously through Saccharomyces cerevisiae expression under control of the ySOD1 promoter in the strain EG118 (sod1Ϫ), which lacks the gene encoding the yeast CuZn-SOD polypeptide (30,32). D125H SOD1 at 20 mg/ml in 2.25 mM sodium phosphate buffer, pH 7.0, 60 mM NaCl, crystallized as thick rectangular blocks in space group C222 1 at 4°C in 1-2 weeks with unit cell parameters a ϭ 70.5 Å, b ϭ 101.1 Å, c ϭ 143.1 Å from hanging drops containing equal volumes (1-2 l) of protein solution and reservoir solution (10 mM zinc sulfate, 25% v/v polyethylene glycol monomethyl ether 550, 100 mM MES, pH 6.5). All of the crystals were quickly swept through a cryoprotecting solution containing 50% sorbitol in reservoir solution and flash-cooled in liquid nitrogen prior to x-ray data collection. The wavelength for optimal copper and zinc anomalous signal was determined by scanning x-ray fluorescence of the crystals prior to x-ray data collection near regions corresponding to the absorption maximum of each metal. Copper exhibited no significant absorption, whereas zinc exhibited strong absorption at 1.2811 Å. X-ray diffraction data were obtained at the NSLS beamlines X12B (native data set) and X8C (zinc anomalous data set). For both data sets, the crystal-to-detector distance was 150 mm and the oscillation angle was 0.7°.
Diffraction data were processed with the DENZO/SCALEPACK suite (HKL2000) (33). Single wavelength anomalous dispersion phasing to 2.0 Å in CNS (34) yielded an overall figure of merit of 0.43. Density modification using solvent flipping improved the figure of merit to 0.8 and produced readily interpretable electron density maps. The molecular 2-fold axis of one D125H CuZn-SOD dimer is coincident with the crystallographic 2-fold axis parallel to b, and the asymmetric unit thus contains three D125H monomers. The crystals have a solvent content of 53% (V m ϭ 2.7). Model building and manual readjustments were performed in the program O (35). Initial stages of refinement were accomplished in CNS, and in the final stages, SHELX-97 was used. R free was monitored in both refinement programs using identical test sets (34). Upon implementing refinement of anisotropic thermal parameters in SHELX-97, both R and R free dropped (R from 19.6 to 14.6%, R free from 24.8 to 21.2%). Water molecules were introduced late in the refinement process where suitable 3 difference electron density and reasonable hydrogen bond geometry were indicated.
Modeling of Carbonate into the D125H SOD1 Structure-HCO 3 Ϫ was modeled into the SOD1 active site channel based on the position of the observed HSO 4 Ϫ in the D125H FALS mutant SOD1 structure. The carbonate molecule was downloaded from the Hetero-compound Information Center (HIC-Up, Uppsala, Sweden) (website: x-ray.bmc.uu.se/ hicup/) (Release 6.1) (36). The anion was positioned in the molecular graphics program O, such that two of its oxygen atoms occupy the same positions as the OX1 and OX2 atoms of HSO 4 Ϫ in the D125H structure. The figures were created using MOLSCRIPT (37), BOBSCRIPT (38), GL_RENDER, 2 and/or POV-Ray (39).

RESULTS
Pulse Radiolysis (Self-inactivation of SOD1)-The rate of self-inactivation of wild type CuZn-SOD in the presence (25 mM) and absence of bicarbonate anion in Tris buffer (0.5 mM, pH 8.0) is shown in Fig. 1A. Although bicarbonate anion is not necessary to detect the self-inactivation of SOD1 (upper line), if present, it increases the rate of self-inactivation by nearly 3-fold. As shown in Fig. 1B, when the self-inactivation of SOD1 is monitored in 100 mM phosphate buffer, pH 7.2, the addition of 10 mM bicarbonate has little effect. To determine the effect of bicarbonate anion on SOD1 mutant proteins found to cause familial amyotrophic lateral sclerosis, we compared the selfinactivation of wild type SOD1 and the FALS mutant L38V. L38V shows increased self-inactivation rates relative to those of wild type whether or not bicarbonate is present. Fig. 1C shows that the presence of HCO 3 Ϫ increases the rate of selfinactivation of both proteins to approximately the same extent, suggesting a common mechanistic pathway for this effect.
Crystal Structure of D125H SOD1-The x-ray crystal structure of the human FALS mutant D125H was determined to 1.4 Å resolution using single wavelength anomalous dispersion phasing methods (Table I). The as-isolated D125H SOD1 protein is nearly devoid of metal ions, binding only ϳ0.1 and ϳ0.4 equivalents of copper and zinc, respectively, per dimer (wild type ϭ 2.0 equivalents) (30,32). The D125H FALS protein crystallizes from a solution containing 10 mM ZnSO 4 at pH 6.5. Zinc is found to occupy both metal binding sites, a fact confirmed through the analysis of fluorescence spectra that precede the x-ray data collection experiments and through single wavelength anomalous dispersion phasing of experimental electron density maps using zinc as the anomalous scatterer. Fig. 2A shows the zinc-occupied copper binding site of a D125H monomer superimposed on 1.4 Å electron density contoured at 1.2 . The Zn(II) ion is coordinated by the three copper ligands, His-46, His-48, and His-120, all at distances of ϳ2.0 Å. A sulfate anion (HSO 4 Ϫ ) is observed in the active site channel with its OX1 atom acting as a fourth ligand to the zinc ion at a distance of ϳ1.9 Å. The zinc coordination geometry is best described as pseudo-trigonal planar with the zinc ion displaced ϳ0.4 Å from a plane formed by the nitrogen atoms of the three histidine ligands. In addition to its role as a metal ligand, the HSO 4 Ϫ OX1 atom receives a nearly ideal hydrogen bond donated by the NE2 atom of His-63, the bridging imidazolate. The HSO 4 Ϫ OX2 atom participates in hydrogen-bonding interactions with the epsilon and guanidinium nitrogens of Arg-143 and with the ND2 atom of the side chain of Asn-26 from a symmetry-related D125H molecule in the crystal lattice. The symmetry-related Asn-26 side chain also donates a hydrogen bond to the backbone oxygen atom of Gly-141, which forms part of the active site rim. Fig. 2A also shows HCO 3 Ϫ modeled into the SOD1 active site channel based on the position of the observed HSO 4 Ϫ , such that two of its oxygen atoms occupy the same positions as the OX1 and OX2 atoms of HSO 4 Ϫ in the D125H FALS mutant SOD1 structure. The space-filling model in Fig. 2B shows how the SOD1 active site with bound HCO 3 Ϫ would appear looking into the active site from the bulk solvent. DISCUSSION Because Reaction 2 is the rate-limiting step in the self-oxidative pathway in the absence of HCO 3 Ϫ (8, 9, 12), any diffusible CO 3 ⅐Ϫ formed in the active site channel by reacting HCO 3 Ϫ with copper-bound HO ⅐ must protect the enzyme from self-inactivation (to some extent) in a way analogous to that observed for formate or azide (Reactions 4 and 5) (8 -10). However, we find that the rate of self-inactivation of wild type SOD1 in 0.5 mM Tris buffer, pH 8.0, is significantly enhanced when 25 mM HCO 3 Ϫ is added (Fig. 1A). The strong oxidant produced in this experiment must therefore arise from a pathway distinct from that described in Reactions 2 and 6. When we repeat the self-inactivation reaction using conditions identical to those used previously (10 mM HCO 3 Ϫ in 100 mM phosphate, pH 7.2) (17), we do not observe this rate enhancement (Fig. 1B). We interpret this to mean that the (excess) HPO 4 Ϫ2 anions present compete with HCO 3 Ϫ for binding to the anion-binding site. In support of this finding, previous studies have shown that at a fixed HCO 3 Ϫ concentration, the rate of oxidation of DMPO in the external oxidative pathway is significantly attenuated by increasing phosphate concentrations (16). Conversely, at a fixed phosphate concentration, the self-inactivation rates are enhanced by increasing HCO 3 Ϫ concentrations (40). We next compared the self-inactivation rate of wild type SOD1 with that of the L38V FALS mutant in the presence and absence of HCO 3 Ϫ (Fig. 1C). The pathogenic human SOD1 mutant exhibits overall increased rates of self-inactivation compared with wild type. However, HCO 3 Ϫ does not increase inactivation of L38V to any greater extent than it does the wild type, suggesting a common mechanistic pathway of HCO 3 Ϫ enhanced self-inactivation for both proteins.
Insight into the mechanism of the HCO 3 Ϫ effect on both the self-oxidative and external oxidative pathways comes from the x-ray crystal structure of human FALS mutant D125H. Although there is substantial evidence of oxyanion binding to SOD1 in solution (7), the D125H structure presented here is the first high resolution crystal structure to reveal spatial details of how an oxyanion can be bound in the active site channel. A hydrogen sulfate anion (HSO 4 Ϫ ) is positioned at the anion-binding site between Arg-143 and Thr-137. The mode of HSO 4 Ϫ binding to this site provides an excellent template upon which to model the binding of both bicarbonate and phosphate anions. When HCO 3 Ϫ is modeled in the position of the enzymeassociated HSO 4 Ϫ , we see that it is capable of simultaneously interacting with the metal ion, Arg-143, and an asparagine residue (Asn-26) from a symmetry-related SOD1 protein in the crystal lattice ( Fig. 2A). That oxyanions bound at the SOD1 anion-binding site can be in close contact with a metal (in this case, zinc) at a position very nearly corresponding to that of Cu(I) in the wild type protein was unanticipated. The interaction with the side chain of Asn-26 is particularly intriguing, because it demonstrates that such a bound oxyanion can also simultaneously contact much larger molecules (in this case, another SOD1 protein) in the bulk solvent. Based on this structure and our chemical data, we now propose the following novel mechanism that can explain the HCO 3 Ϫ -mediated enhancement in the rates of both the self-oxidative and external oxidative pathways but does not require that CO 3 ⅐Ϫ act as a diffusible oxidant. This mechanism is illustrated schematically in Fig. 3 where the steps are labeled as i-vi in a counterclockwise direction. In step i, the Cu(II) ion is reduced to Cu(I). This can occur via O 2 . as part of the normal disproportionation reaction as shown in Reaction 10, REACTION 10 or via H 2 O 2 as shown in Reaction 1. In step ii, HCO 3 Ϫ binds to the anion-binding site in the mode predicted by the D125H SOD1 crystal structure. In step iii, HO 2 Ϫ is guided into the active site channel where it reacts with HCO 3 Ϫ to form peroxy- There are subsequently two possible fates for this enzymeassociated HCO 4 Ϫ that lead to the formation of a strong oxidant (step v), designated as [O*] in Fig. 3. In the first pathway, the Cu(I) ion donates an electron to HCO 4 Ϫ , and it partitions into CO 3 ⅐Ϫ ϩ OH Ϫ as shown in Reaction 12.

REACTION 12
Non-diffusible enzyme-associated CO 3 ⅐Ϫ can catalyze the hydroxylation of nearby histidine copper ligands by oxidizing them to their corresponding histidinyl radicals followed by the addition of OH Ϫ from the bulk solvent to form 2-oxo-histidine (Fig. 3B) (41). Histidine copper ligands modified in this way result in copper cofactor loss and enzyme inactivation. Alternatively, enzyme-associated CO 3 ⅐Ϫ can catalyze the oxidation of exogenous substrates that can gain close approach, perhaps at the solvent-exposed position near that occupied by the symmetry-related Asn-26 side chain shown in Fig. 2A. Exogenous substrates such as DMPO can be hydroxylated either through a nucleophilic addition of water to a DMPO-carbonate radical intermediate or to a DMPO radical cation intermediate (22,23). In the second pathway, the Cu(I) ion donates an electron to HCO 4 Ϫ and it partitions into HCO 3 Ϫ and HO ⅐ as shown in Reaction 13.
The HO ⅐ produced can directly attack histidine copper ligands or oxidize substrates exogenous to the active site channel, leaving HCO 3 Ϫ in the anion-binding site (vi) and completing the cycle. The salient feature of this mechanism is that a strong oxidant is generated in situ that protrudes into the bulk solvent or reacts with residues in and around the active site.
Investigations of proteolyzed H 2 O 2 -treated SOD1 using mass spectrometry indicate that multiple amino acids in the vicinity of the catalytic copper ion can be oxidatively damaged (13,14). These residues include His-46, His-48, Pro-62, His-63, and His-120 (human numbering). The positions of these residues relative to the enzyme-associated bicarbonate anion are shown in Fig. 2B. Uchida and Kawakishi (13) have reported that His-118 in the bovine enzyme (His-120 in the human) is selectively converted to 2-oxo-histidine at its C⑀1 atom (13). As first proposed by Sankarapandi and Zweier (16), the examination of Fig. 2, A and B, suggests that there does indeed exist a pre-formed hydrogen-bonding template comprised of the OX2 atom of the enzyme-bound bicarbonate anion and the carbonyl oxygen of Gly-141. In the D125H crystal structure, this hydrogen-bonding position is occupied by the ND1 atom of Asn-26 coming from a symmetry-related molecule in the crystal lattice. It is tempting to speculate that the reason for selective self-oxidation at His-118 (His-120) is that HO 2 Ϫ (or H 2 O 2 ) preferentially forms the peroxycarbonate moiety on the OX2 atom of the enzyme-bound bicarbonate anion where it is stabilized by hydrogen bonding interactions with the carbonyl oxygen of Gly-141. In either of the peroxycarbonate-partitioning pathways described above, the strong oxidant subsequently derived would be in close proximity to the C⑀1 atom of His-120.
The potential relevance of this peroxidative chemistry to FALS is underscored by the fact that bicarbonate is normally present in tissue at relatively high concentration (ϳ25 mM) (28)  and that this activity has been measured at H 2 O 2 concentrations as low at 1 M at neutral pH (23). In pathological conditions of oxidative stress where H 2 O 2 may persist in the cytosol long enough to react with SOD1, the external oxidative path-way could significantly increase tyrosine oxidation and nitration (22,42). Such products are signs of oxidative damage that, in sufficient amounts, could potentially lead to apoptosis. This idea has received support from other studies. For example, A sulfate anion (green and yellow, all of the oxygen atoms with the exception of the one designated with a red asterisk) is found associated with Arg-143 in the anion-binding site and is bound to the zinc ion through its OX1 atom. Bicarbonate anion (yellow, OX1, OX2, and oxygen labeled with the red asterisk) is modeled based on the position of the sulfate anion (see "Experimental Procedures"). The side chain of Asn-26 (green) comes from a symmetry-related molecule in the crystal lattice and hydrogen bonds simultaneously to the OX2 atom of the oxyanion and to the carbonyl oxygen atom of Gly-141. B, space-filling model of the D125H active site with bound bicarbonate when viewed from the solvent. D125H carbon and oxygen atoms are shown in gray, and nitrogen atoms are shown in blue. The carbon atoms of Arg-143 and Thr-137, residues forming the active site channel constriction, are shown in pink. The positive charge on the guanidinium group of Arg-143 is represented by a (ϩ) symbol. Residues known to be oxidatively damaged in the active site through mass spectrometry analyses (13,14) are shown in light green. The C⑀1 position of His-120 is indicated (see "Discussion"). The zinc ion is shown in yellow. The carbonate oxygen atoms are labeled OX2 and OX3 (red), and its carbon atom is shown in black.
human neuroblastoma cells transfected with the G93A SOD1 mutant demonstrate increased DCFH oxidation relative to cells transfected with wild type SOD1 (43). In spinal cord extracts of G93A-expressing transgenic mice, increased oxidation of the spin trap azulenyl nitrone is observed when compared with those of nontransgenic animals or transgenic mice expressing wild type human SOD1 (44,45).
Although pathogenic SOD1 might oxidatively damage neuronal cellular constituents directly through enhanced rates of peroxidation, perhaps the most enticing hypothesis on how the enhanced peroxidase activity in pathogenic SOD1 proteins could cause ALS is that this activity can facilitate SOD1 misfolding and aggregation. High molecular weight-insoluble protein complexes, composed in part of FALS SOD1, are now widely believed to play a role in ALS pathogenesis either by sequestering heat shock proteins (46,47) and/or interfering with the neuronal axonal transport (48,49) and protein degradation (50,51) machineries. The H 2 O 2 -mediated oxidation of histidine residues that bind metals in the SOD1 active site has been shown to stimulate SOD1 aggregation relative to the unoxidized protein in vitro (52). Moreover, recent results from our own laboratory demonstrate that, unlike the holo-wild type protein, two metal-deficient pathogenic SOD1 proteins, H46R and S134N, can form higher order filamentous assemblies through non-native SOD1-SOD1 protein-protein interactions (53). These non-native interactions occur only through sub-units of the SOD1 protein that are devoid of copper, zinc, or both. Thus, any chemistry that could result in an increase in the amount of metal-deficient SOD1 could lead to pathogenesis indirectly through the gradual accumulation of such higher order SOD1 assemblies and aggregates. Finally, if enhanced rates of self-inactivation are related to increased aggregation of SOD1 with itself or with other proteins, it is possible that sporadic ALS, which comprises ϳ85-90% of all ALS cases, might also be triggered by oxidatively damaged wild type SOD1.
FIG. 3. Proposed mechanism for bicarbonate-mediated peroxidation in SOD1 (see "Discussion"). i, Cu(II) is reduced to Cu(I). ii, bicarbonate binds to the anion-binding site in the manner predicted by the D125H SOD1 crystal structure. iii, HO 2 . reacts with bicarbonate to form peroxycarbonate (iv). v, the oxygen radical species formed [O*] may then oxidize endogenous or exogenous substrates, leaving bicarbonate bound to the anion-binding site (vi). B, the attack of an oxygen radical species on one of the histidine copper ligands in SOD1 leads to the formation of a 2-oxo histidine adduct, leading to cofactor loss and enzyme inactivation.