An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase: RATES ENHANCED VIA PROPOSED ENZYME-ASSOCIATED PEROXYCARBONATE INTERMEDIATE

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 23, Issue of June 6, pp. 21032–21039, 2003 Printed in U.S.A.

An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase RATES ENHANCED VIA PROPOSED ENZYME-ASSOCIATED PEROXYCARBONATE INTERMEDIATE* Received for publication, January 16, 2003, and in revised form, March 14, 2003 Published, JBC Papers in Press, March 20, 2003, DOI 10.1074/jbc.M300484200

Jennifer Stine Elam‡§, Kevin Malek§¶, Jorge A. Rodriguez¶, Peter A. Doucette¶, Alexander B. Taylor‡, Lawrence J. Hayward储, Diane E. Cabelli**, Joan Selverstone Valentine¶‡‡, and P. John Hart‡ ‡‡ From the ‡Department of Biochemistry and the X-ray Crystallography Core Laboratory, University of Texas Health Science Center, San Antonio, Texas 78229-3900, ¶Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, 储Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and **Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973

* This work was supported by the NINDS, National Institutes of Health Grant NS39112 (to P. J. H.), NIGMS, National Institutes of Health Grant GM28222 (to J. S. V.), NINDS, National Institutes of Health Grant NS44170 (to L. J. H.), the Robert A. Welch Foundation (to P. J. H.), and the Amyotrophic Lateral Sclerosis Association (to P. J. H., J. S. V., and L. J. H.). Pulse radiolysis studies were carried out at the Center for Radiation Chemical Research, Brookhaven National Laboratory, which is supported under contract DE-AC02-98CH10886 with the U. S. Department of Energy and supported by its Division of Chemical Sciences, Office of Basic Energy Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 1P1V) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http:// www.rcsb.org/). § Both authors contributed equally to this work. ‡‡ To whom correspondence may be addressed: Dept. of Biochemistry, X-ray Crystallography Core Laboratory, University of Texas Health Science Center San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-0751; Fax: 210-567-6595; E-mail: [email protected]. (P. J. H.) or Dept. of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095. Tel.: 310825-9835; Fax: 310-206-7197; E-mail [email protected] (J. S. V.).

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 (2O2. ⫹ 2H⫹ 3 O2 ⫹ H2O2) 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 O2. 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⫺) and azide (N3⫺) 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 (HPO4⫺2) 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 O2. disproportionation activity, the active site of CuZn-SOD can interact with H2O2 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 (11–14). 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, SOD-Cu(II) ⫹ H2O2 3 SOD-Cu(I) ⫹ 2H⫹ ⫹ O2. REACTION 1 SOD-Cu(I) ⫹ H2O2 3 SOD-Cu(II)(䡠OH) ⫹ OH⫺ REACTION 2 SOD-Cu(II)(䡠OH) ⫹ XH 3 SOD-Cu(II) ⫹ H2O ⫹ X 䡠 REACTION 3

where XH represents amino acids at the active site or an exogenous substrate (8, 17). Analogous to the Fenton reaction, 1 The abbreviations used are: SOD1, superoxide dismutase 1; CuZnSOD, copper-zinc superoxide dismutase; HO䡠, hydroxyl radical; DMPO, 5,5-dimethyl-1-pyroline N-oxide; DCFH, dichlorodihydrofluorescein; FALS, familial amyotrophic lateral sclerosis; SCN, thiocyanate; Mes, 4-morpholineethanesulfonic acid.

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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.

Peroxycarbonate-mediated Oxidation by CuZn-SOD Reaction 2 generates a highly reactive hydroxyl radical (HO䡠). The observation that this HO䡠 does not readily react with scavengers of free HO䡠 such as ethanol led to the proposal that the HO䡠 was “bound” to the catalytic copper ion. This hypothesis was supported by the observation that small anions such as formate (HCO2⫺) and N3⫺ 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.

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SOD-Cu(II)(䡠OH) ⫹ HCO3⫺ 3 SOD-Cu(II) ⫹ H2O ⫹ CO3䡠⫺ (free) REACTION 6 CO3䡠⫺ (free) ⫹ XH (exogenous) 3 HCO3⫺ ⫹ X䡠 (exogenous) REACTION 7

or SOD-Cu(II)(䡠OH) ⫹ HCO3⫺ 3 SOD-Cu(II)(CO3䡠⫺) ⫹ H2O

SOD-Cu(II)(䡠OH) ⫹ HCO2⫺ 3 SOD-Cu(II) ⫹ H2O ⫹ CO2䡠⫺

REACTION 8

REACTION 4

SOD-Cu(II)(CO3䡠⫺) ⫹ XH (self) 3 SOD-Cu(II) ⫹ HCO3⫺ ⫹ X䡠 (self)

SOD-Cu(II)(䡠OH) ⫹ N3⫺ 3 SOD-Cu(II) ⫹ OH⫺ ⫹ N3䡠

REACTION 9

REACTION 5

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 H2SO4 (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 ⫽ 61 M⫺1 cm⫺1). 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 ⫻ 104 ⫺1 M cm⫺1 for the purified enzyme. Purity was estimated using SDSPAGE 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⫺ is generated with a G value of 6.13 and has a molar absorptivity of 7950 M⫺1 cm⫺1 at 472 nm. Irradiation of

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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 H2O2 (18). The peroxidase activity of SOD1 is not strictly limited to small substrates that can gain direct access to the copper ion. In the presence of bicarbonate anion (HCO3⫺), larger reporter molecules such as DMPO (5,5-dimethyl-1-pyroline N-oxide), ABTS (2,2⬘-azino-bis-[3-ethylbenzothiazoline-6-sulfonate]), PBN (N-tert-butyl-␣-phenylnitrone), azulenyl nitrone, tyrosine, and DCFH (dichlorodihydrofluorescein) are also oxidized (10, 15–17, 19 –23). Several studies (24 –27) have implicated this expanded peroxidative activity of SOD1 in the toxic gain-offunction of SOD1 mutants associated with the progressive, fatal, neurodegenerative disorder, familial amyotrophic lateral sclerosis (FALS). This expanded FALS SOD1 peroxidase activity exerted either on substrates critical for motor neuron viability or on the SOD1 molecule itself could play a role in FALS etiology (see “Discussion”). It is important to note that the oxidation of substrates too large to traverse the active site channel constriction can occur only in the presence of HCO3⫺ or structurally similar anions such as HSeO3⫺ and HSO3⫺. Other anions such as N3⫺, HCO2⫺, HPO4⫺2, thiocyanate (SCN⫺), nitrate (NO3⫺), and Cl⫺ do not appear to support the oxidation of these larger substrates (16). The relevance of this activity is underscored by the significant concentration of HCO3⫺ found in vivo (⬃25 mM) (28) and by recent studies showing that at physiological pH values (7.4) and low H2O2 concentrations (1 ␮M), HCO3⫺ dramatically enhances DCFH oxidation in a SOD1/ H2O2/DCFH system (23). Several laboratories have sought to delineate the mechanistic role of HCO3⫺ in the external oxidative pathway of SOD1. Sankarapandi and Zweier (16) propose that HCO3⫺ bound to the SOD1 anion-binding site creates a hydrogen-bonding template for H2O2 near the copper ion that facilitates its partitioning into 䡠OH and OH⫺ (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 HCO3⫺. 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 HCO3⫺ was added. On this basis, they suggested that HCO3⫺ does not facilitate H2O2 binding, but rather, HCO3⫺ can itself be oxidized by the copper-bound HO䡠 to carbonate radical anion (CO3䡠⫺), 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” CO3䡠⫺ is indeed formed in the active site channel, the presence of HCO3⫺ 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 HCO3⫺ 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 HCO3⫺ 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 (HCO4⫺) 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).

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Peroxycarbonate-mediated Oxidation by CuZn-SOD process where suitable 3␴ difference electron density and reasonable hydrogen bond geometry were indicated. Modeling of Carbonate into the D125H SOD1 Structure—HCO3⫺ was modeled into the SOD1 active site channel based on the position of the observed HSO4⫺ 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 HSO4⫺ 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 HCO3⫺ 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 ⬃0.1 and ⬃0.4 equivalents of copper and zinc, respectively, per dimer (wild type ⫽ 2.0 equivalents) (30, 32). The D125H FALS protein crystallizes from a solution containing 10 mM ZnSO4 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 His120, all at distances of ⬃2.0 Å. A sulfate anion (HSO4⫺) is observed in the active site channel with its OX1 atom acting as a fourth ligand to the zinc ion at a distance of ⬃1.9 Å. The zinc coordination geometry is best described as pseudo-trigonal planar with the zinc ion displaced ⬃0.4 Å from a plane formed by the nitrogen atoms of the three histidine ligands. In addition to its role as a metal ligand, the HSO4⫺ OX1 atom receives a nearly ideal hydrogen bond donated by the NE2 atom of His-63, the bridging imidazolate. The HSO4⫺ 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 HCO3⫺ modeled into the SOD1 active site channel based on the position of the observed HSO4⫺, such that two of its oxygen atoms occupy the same positions as the OX1 and OX2 atoms of HSO4⫺ in the 2

L. Esser, personal communication.

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⫺ water by an electron beam generates the primary radicals, 䡠OH, eaq , and 䡠 H. These radicals are efficiently converted into O2. in the presence of 䡠 ethanol and oxygen by the following reactions: OH ⫹ H3CCH2OH 3 H2O ⫹ H3CC䡠HOH followed by H3C䡠HOH ⫹ O2 3 H3CCO䡠HOH ⫹ ⫺ O2. and eaq ⫹ O2. 3 O2. , H ⫹ O2 3 HO2, where HO2 3 H⫹ ⫹ O2. . The decay of O2. was monitored at 250 –270 nm. An observed first order rate for the catalytic dismutation of O2. in the presence of SOD was extracted from the observed change in absorbance (at 260 nm) with respect to time. The reported rate constants for the studies were calculated by dividing the observed rate by the total concentration of copper bound to the enzyme in solution for CuZn-SOD. A set of self-inactivation experiments were carried out in the presence and absence of 100 mM phosphate at pH 7.2 and 25 °C to facilitate comparison with the studies of Liochev and Fridovich (17) and Sankarapandi and Zweier (16). All of the other self-inactivation experiments were carried out in the absence of phosphate at pH 8.0 and 37 °C to stabilize the concentration of bicarbonate and to mimic in vivo temperatures. Previous work (9, 31) has demonstrated that SOD1 reacts almost exclusively with the peroxide anion and that the self-inactivation reaction has a large activation energy (data not shown). To account for both the increase in effective peroxide anion concentration at pH 8.0 and the increase in reaction rate because of the elevated temperature of 37 °C, a lower concentration of peroxide (relative to that in the self-inactivation at pH 7.2) was used. Conditions of the experiments performed to monitor the effect of bicarbonate anion in the presence of phosphate at 25 °C were as follows: 0.5 ␮M copper-bound CuZn-SOD, 100 mM sodium phosphate, pH 7.2, 10 ␮M EDTA, 20 mM H2O2, with and without 10 mM sodium bicarbonate. Conditions of the experiments performed to monitor the effect of bicarbonate in the absence of phosphate at 37 °C were as follows: 0.4 ␮M copper-bound CuZn-SOD, 0.5 mM Tris, pH 8.0, 100 mM NaCl, 10 ␮M EDTA, either 4 or 8 mM H2O2, with and without 25 mM sodium bicarbonate. 1-ml aliquots were withdrawn at timed intervals, and a drop of EtOH was added just before pulsing to yield an approximate concentration of 0.25 M EtOH in solution. 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 (sod1⫺), 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 C2221 at 4 °C in 1–2 weeks with unit cell parameters a ⫽ 70.5 Å, b ⫽ 101.1 Å, c ⫽ 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% (Vm ⫽ 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. Rfree was monitored in both refinement programs using identical test sets (34). Upon implementing refinement of anisotropic thermal parameters in SHELX-97, both R and Rfree dropped (R from 19.6 to 14.6%, Rfree from 24.8 to 21.2%). Water molecules were introduced late in the refinement

Peroxycarbonate-mediated Oxidation by CuZn-SOD

copper-bound HO䡠 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 HCO3⫺ 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 HCO3⫺ 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) HPO4⫺2 anions present compete with HCO3⫺ for binding to the anion-binding site. In support of this finding, previous studies have shown that at a fixed HCO3⫺ 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 HCO3⫺ 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 HCO3⫺ (Fig. 1C). The pathogenic human SOD1 mutant exhibits overall increased rates of self-inactivation compared with wild type. However, HCO3⫺ does not increase inactivation of L38V to any greater extent than it does the wild type, suggesting a common mechanistic pathway of HCO3⫺ enhanced self-inactivation for both proteins. Insight into the mechanism of the HCO3⫺ 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 (HSO4⫺) is positioned at the anion-binding site between Arg-143 and Thr-137. The mode of HSO4⫺ binding to this site provides an excellent template upon which to model the binding of both bicarbonate and phosphate anions. When HCO3⫺ is modeled in the position of the enzymeassociated HSO4⫺, 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 HCO3⫺-mediated enhancement in the rates of both the self-oxidative and external oxidative pathways but does not require that CO3䡠⫺ 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 O2. as part of the normal disproportionation reaction as shown in Reaction 10,

D125H FALS mutant SOD1 structure. The space-filling model in Fig. 2B shows how the SOD1 active site with bound HCO3⫺ would appear looking into the active site from the bulk solvent.

SOD-Cu(II) ⫹ O2. 3 SOD-Cu(I) ⫹ O2

DISCUSSION

or via H2O2 as shown in Reaction 1. In step ii, HCO3⫺ binds to the anion-binding site in the mode predicted by the D125H SOD1 crystal structure. In step iii, HO2⫺ is guided into the active site channel where it reacts with HCO3⫺ to form peroxy-

Because Reaction 2 is the rate-limiting step in the self-oxidative pathway in the absence of HCO3⫺ (8, 9, 12), any diffusible CO3䡠⫺ formed in the active site channel by reacting HCO3⫺ with

REACTION 10

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FIG. 1. Effect of bicarbonate on hydrogen peroxide-mediated SOD1 self-inactivation. A, Tris-HCl-buffered system. Reaction mixture in 0.5 mM Tris-HCl buffer, pH 8.0, contained wild type CuZn-SOD (0.4 ␮M), NaCl (100 mM), EDTA (10 ␮M), H2O2 (8 mM), and either 0 or 25 mM NaHCO3 at 37 °C. Wild type CuZn-SOD had t1⁄2 ⫽ 380 s in the absence of NaHCO3 and t1⁄2 ⫽ 128 s in 25 mM NaHCO3. B, phosphatebuffered system. Reaction mixture in 100 mM sodium phosphate, pH 7.2, contained wild type CuZn-SOD (0.5 ␮M), EDTA (10 ␮M), H2O2 (20 mM), and either 0 or 10 mM NaHCO3 at 25 °C. C, FALS SOD1 mutant L38V demonstrates enhanced self-inactivation rates relative to wild type with and without bicarbonate. Conditions were the same as in A with the exception that the concentration of hydrogen peroxide was 4 mM. Self-inactivation of pathogenic L38V CuZn-SOD is represented in lines designated with open squares. Wild type CuZn-SOD had t1⁄2 ⫽ 510 s in the absence of NaHCO3 and t1⁄2 ⫽ 190 s in 25 mM NaHCO3. L38V CuZnSOD had t1⁄2 ⫽ 424 s in the absence of NaHCO3 and t1⁄2 ⫽ 156 s in 25 mM NaHCO3.

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Peroxycarbonate-mediated Oxidation by CuZn-SOD TABLE I Crystallographic data, phasing, and refinement of human FALS mutant SOD1 D125H

X-ray data ˚) ␭ (A No. of observations No. of unique reflections ˚) Resolution range (A (Last shell) Completeness (%) (Last shell) Rsym (on L) (%)a (Last shell) Phasing No. of sites ˚) Resolution range (A Overall phasing powerb Overall figure of meritc Figure of merit after density modificationd Refinement

CNS

˚) Resolution range (A Rcryst (%)e Rfree (%)f F/␴F

35.8–1.4 20.3 22.3 ⬎0

Native

Zinc

1.0000 658,344 95,234 50–1.4 1.45–1.4 94.7 85.1 4.8 42.6

1.2811 143,764 57,725 50–2.0 2.07–2.0 85.2 52.3 4.1 6.6 6 37.0–2.0 2.2 0.43 0.80

Shelx-97

10.0–1.4 14.6 21.2 ⬎0

Final model

0.012 2.12 3156 736 6 zinc 3

a Rsym ⫽ ⌺兩I ⫺ 具I典兩/⌺I, where I is the observed intensity and 具I典 is the average intensity of multiple symmetry-related observations of that reflection. b Phasing power ⫽ 公FH2/公E2, where Fh is the heavy-atom structure factor amplitude and E is the residual lack of closure error. c Figure of merit represents the weighted mean of the cosine of phase error. d Density modification using solvent flipping implemented in CNS (34). e Rcryst ⫽ ⌺兩兩Fobs兩 ⫺ 兩Fcalc兩兩/⌺兩Fobs兩. f Rfree ⫽ ⌺兩兩Fobs兩 ⫺ 兩Fcalc兩兩/⌺兩Fobs兩, where 兩Fobs兩 is from a test set not used in the structural refinement (2002 reflections). R.m.s.d., root mean square deviation.

carbonate (HCO4⫺) with the concomitant release of OH⫺ as described in step iv. This peroxo species could form at either of the copper-distal oxygen atoms of the bicarbonate labeled OX2 or OX3 in Fig. 2B as shown in Reaction 11. SOD-Cu(I)(HCO3⫺) ⫹ HO2⫺ 3 SOD-Cu(I)(HCO4⫺) ⫹ OH⫺ REACTION 11

There are subsequently two possible fates for this enzymeassociated HCO4⫺ 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 HCO4⫺, and it partitions into CO3䡠⫺ ⫹ OH⫺ as shown in Reaction 12. SOD-Cu(I)(HCO4⫺) 3 SOD-Cu(II)(CO3䡠⫺) ⫹ OH⫺ REACTION 12

Non-diffusible enzyme-associated CO3䡠⫺ can catalyze the hydroxylation of nearby histidine copper ligands by oxidizing them to their corresponding histidinyl radicals followed by the addition of OH⫺ 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 CO3䡠⫺ 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 HCO4⫺ and it partitions into HCO3⫺ and HO䡠 as shown in Reaction 13. SOD-Cu(I)(HCO4⫺) ⫹ H⫹ 3 SOD-Cu(II)(HCO3⫺) ⫹ 䡠OH REACTION 13

The HO䡠 produced can directly attack histidine copper ligands or oxidize substrates exogenous to the active site channel, leaving HCO3⫺ 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 H2O2-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 HO2⫺ (or H2O2) 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 (⬃25 mM) (28)

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˚) R.m.s.d. bonds (A R.m.s.d. angles (degrees) No. protein atoms No. water molecules No. metal ions No. sulfate anions

Peroxycarbonate-mediated Oxidation by CuZn-SOD

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FIG. 2. X-ray crystal structure of the copper-binding site of FALS SOD1 mutant D125H. A, the active site of one monomer of D125H is superimposed on 1.4 Å electron density with coefficients 2mFo ⫺ DFc contoured at 1.3 ␴. The histidine copper ligands and zinc ions are labeled. 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 (⫹) 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.

and that this activity has been measured at H2O2 concentrations as low at 1 ␮M at neutral pH (23). In pathological conditions of oxidative stress where H2O2 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,

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Peroxycarbonate-mediated Oxidation by CuZn-SOD 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 ⬃85–90% of all ALS cases, might also be triggered by oxidatively damaged wild type SOD1. Acknowledgments—We thank L. Flaks and J. Berendzen for help and support at beamline X8C at the National Synchrotron Light Source, Brookhaven National Laboratory, D. Cascio for valuable discussions, and S. Holloway for assistance with the illustrations. REFERENCES

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 H2O2-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-

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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, HO2. 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.

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Enzyme Catalysis and Regulation: An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase: RATES ENHANCED VIA PROPOSED ENZYME-ASSOCIATED PEROXYCARBONATE INTERMEDIATE

J. Biol. Chem. 2003, 278:21032-21039. doi: 10.1074/jbc.M300484200 originally published online March 20, 2003

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Jennifer Stine Elam, Kevin Malek, Jorge A. Rodriguez, Peter A. Doucette, Alexander B. Taylor, Lawrence J. Hayward, Diane E. Cabelli, Joan Selverstone Valentine and P. John Hart