ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 299, No. 2, December, pp. 350-355, 1992

Crystal Structure of Peroxynitrite-Modified Bovine Cu,Zn Superoxide Dismutase Craig D. Smith,**’ Mike Carson,* Mark van der Woerd,* Jun Chen,? Harry Ischiropoulos,t and Joseph S. Beckman? *Center for Macromolecular Crystallography The University of Alabama at Birmingham,

and tDepartment of Anesthesiology, Birmingham, Alabama 35294

School of Medicine,

Received June 17, 1992, and in revised form August 6, 1992

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing threedimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160,181-217,1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr108 of a second dimer. o 1992 Academic PWSS, IUC.

utes to the cytotoxicity of macrophages and possibly of neutrophils (9, 10). Peroxynitrite (ONOO-) is a powerful oxidant produced by the rapid spontaneous reaction between nitric oxide and superoxide (11). One mechanism whereby superoxide contributes to tissue injury may involve formation of the intermediate peroxynitrite anion (11). Pathological conditions such as ischemia followed by reperfusion, inflammation, and sepsis can give rise to increased production of superoxide and nitric oxide (12,13), thereby increasing peroxynitrite formation. Cu,Zn superoxide dismutase has been shown to reduce injury to reperfused ischemic heart, brain, and other tissues (14,15). Superoxide dismutase may be protective by scavenging superoxide before it reacts with nitric oxide to form peroxynitrite (16). Cu,Zn superoxide dismutase (SOD)’ efficiently reduces intracellular concentration of Og- by way of the following two-step reaction (17): O;- + SOD(Cu’+)

+ H+ --* SOD’(Cu+) + O2

O;- + SOD’(Cu+) + H+ --* SOD(Cu2+) + H202. Superoxide (Oi-) is formed during oxidative metabolism (1) and has been implicated in several pathological processes, including ischemia/reperfusion, inflammation, and hyperbaric oxygen toxicity. Superoxide also contributes to the microbicidal action of phagocytes (2). A major target of superoxide in viva may be nitric oxide, recently identified as the endogenous factor mimicked by nitrovasodilators such as nitroglycerin and nitroprusside (35). Endothelium and neurons produce nitric oxide by a calmodulin-activated enzyme, nitric oxide synthase. This enzyme oxidizes free arginine and requires biopterin, NADPH, and oxygen (6-8). Production of nitric oxide by a calmodulin-independent nitric oxide synthase contrib1 To whom correspondence

should be addressed. Fax: (205) 934-0480.

The reaction of bovine Cu,Zn superoxide dismutase with superoxide is characterized by an unusually high rate constant of 2 X 10’ M-’ s-l over a broad pH range (18). The twofold symmetry of the beta-barreled dimer positions the two active sites on opposite sides of the molecule and facing away from each other (19). An N-acetylated monomer consists of 151 amino acid residues which tightly bind one copper ion and one zinc ion. A twofold symmetric electrostatic field is created around the dimer by charged surface groups. The clustering of positive charges around the active site, coupled with the broad distribution of negative charges elsewhere, attracts the negatively-charged superoxide anion to the active site * Abbreviation

used: SOD, superoxide dismutase.

350 All

0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.

PEROXYNITRITE-MODIFIED

BOVINE

copper and may be responsible for the nearly diffusionlimited reaction rate constant (20,21). A catalytically required copper is buried at the bottom of the active site hydrophobic pocket, shaped to accommodate superoxide (17). We have found that peroxynitrite is also a substrate for Cu,Zn superoxide dismutase. The catalytic action of bovine Cu,Zn superoxide dismutase permanently modifies Tyr-108 of the enzyme to yield 3-nitrotyrosine on the superoxide dismutase (22). The data given here provide evidence for the self-nitration and suggest a mechanism for the transfer of a nitrating species generated at the active site to the nitration site 18 A away. MATERIALS

AND

METHODS

Crystallization. Pharmaceutical grade bovine erythrocyte Cu,Zn superoxide dismutase was provided by Griinenthal, Inc. (Aachen, Germany). Peroxynitrite (11) and peroxynitrite-modified superoxide dismutase (22) were prepared as described previously. Native superoxide dismutase and peroxynitrite-modified superoxide dismutase were crystallized by vapor diffusion under conditions based on the previously used batch method (23). Droplets 4 ~1 in volume, containing 2-7 mg/ ml of protein, 28% 2-methyl-2,4-pentanediol (Eastman Kodak), and 50 mM potassium phosphate at pH 7.4, were suspended from a siliconized glass coverslip. They were sealed with silicone grease (DOW Chemical Co.) over a tissue culture plate well (Linbro) containing 2-methyl-2,4pentanediol diluted to 55% (v/v) with the same phosphate buffer. Both types of crystals grew to 0.3 X 0.6 X 1.0 mm3 in 2 weeks at 5°C. The crystals were mounted directly from the droplets into glass capillary tubes (Charles Supper Co.). They tended to melt unless allowed to slowly warm to room temperature over a period of 10 to 15 h. X-ray diffraction data were collected at room temperature using a Siemens multiwire area detector mounted on a Rigaku RU200 rotating anode X-ray generator. CuKcr radiation was generated at 40 kV and 100 mA from a 0.3 X 3-mm fine focal point, monochromated with a graphite monochromator, and collimated to 0.3 mm. The detector distance was 12 cm and the swing angle was -15”. The time per frame of data varied between 150 and 500 s and the oscillation angle was 0.25”. XENGEN programs were used to reduce the data to structure factors (24). Native and modified superoxide dismutase crystals had the same space group and lattice parameters as published previously (lS), space group C2, a = 93.65 A, b = SO.33 A, c = 71.65 A, and 0 = 95.10”. Area detector data from two native crystals produced 17,989 reflections after scaling and outlier rejection for 89% of unique 2.5 A data. There was at least twofold redundancy in the data up to 2.5 A resolution and a corresponding R.,, of 3.7% (R,,, = C,,(Z,, - (Z),g,jI/Ct,Z,,, whereg,, is the scale factor applied to the ith observed reflection intensity, Z, of the jth reflection). Two crystals of the peroxynitrite-modified superoxide dismutase produced 19,797 unique reflections or 98% of 2.5 A data after similar scaling and outlier rejection. There was at least twofold redundancy to 2.5 A resolution and a corresponding R,,, of 3.3%. The native and peroxynitrite-modified crystal data were scaled together using relative Wilson scaling to produce scale factors of the form A exp(-B sin20/X2), where 0 is the Bragg angle, X is the wavelength, and A and B are constants determined from plotting the natural logarithm of ratios of native to peroxynitrite-modified diffraction intensities as a function of sin2B/h2 and fitting a least-squares line to it. The mean fractional isomorphous difference c llFph 1 - 1FJ/C I Fp I was 12.6% for 16,202 reflections having nonzero structure factors for native and peroxynitritemodified data to 2.5 A resolution. Initial model coordinates for bovine Cu,Zn superoxide dismutase were obtained from the Brookhaven Protein Data Bank (25), entry 2SOD. This structure was determined and partially refined by Tamer et al. (19) to a nominal resolution of 2.0 A. The asymmetric unit of the C2

Cu,Zn SUPEROXIDE

DISMUTASE

351

unit cell of bovine Cu,Zn superoxide dismutase contains four monomers constituting two dimers. Each monomer has a single water oxygen located at the superoxide binding pocket next to the copper ion. The Protein Data Bank file for bovine Cu,Zn superoxide dismutase uses the colors orange, yellow, blue, and green to refer to the four monomers of the asymmetric unit. The orange and yellow monomers constitute one dimer and the blue and green monomers the other. The noncrystallographic symmetry relating these dimers consists of a rotation and translation. The Protein Data Bank coordinates were deposited with respect to the a*bc orthogonal frame of reference. The graphics program PSFRODO (26, 27) and refinement program XPLOR (28) align X with the a axis for an abc* orthogonal reference frame. Therefore the starting Protein Data Bank coordinates were rotated p - 90.0 = 5.1” about the Y axis. The crystallographic R value (C IIF,,,, 1 - I F,.J/C I Fobs I ) for the native data to 2.5 A resolution was 0.323 as calculated by XPLOR before refinement. The native structure was refined in three stages by XPLOR using the “slow-cool” simulated annealing protocol (29) with individual atomic temperature factors. Noncrystallographic symmetry was not imposed during refinement. The molecular structure file used by XPLOR was modified to place hydrogens on the histidines involved with metal binding as follows: His 46 and 118 have a hydrogen on the delta nitrogen; His 44, 69, and 78 have a hydrogen on the epsilon nitrogen; and His 61 is negatively charged with neither hydrogen. These changes eliminated the worst contacts initially found in the structure when all allowed hydrogens were added. The first stage of refinement used native diffraction data between 6 and 3 A. Those atomic coordinates were used for the second round of refinement with 6 to 2.5 A native data. Before the final stage of refinement, the monomers were superimposed to find what structural differences there were among them. Matrices required to transform the three monomers onto the orange monomer were determined by a least-squares fit of the Co atoms using XPLOR. The matrices were applied to the atomic models before and after refinement, as well as to the difference maps to superimpose all four monomers in the same volume. Overall, the four independent monomers were very similar in terms of sidechain conformation before and after the first two rounds of refinement. Only the green monomer Glu-107 and Tyr-108 residues had large conformational differences from the three other monomers. A manual rebuilding of the native orange monomer was made considering only problem residues. Problem residues were identified by five criteria: deviation from normal 9, $ and o angles, unusual sidechain x values compared to those in the rotamer library of Ponders and Richards (30), large real space density residual factors of mainchain and sidechain atoms as calculated by the graphics program “0” (31), large root-meansquare atomic shifts, and large structural strain energies as calculated by XPLOR. Of the 151 residues in the monomer, 43 were deemed to have potential problems by these criteria, and 34 residues were actually adjusted after visual graphics evaluation. The regions adjusted were almost exclusively in the coil and turn regions of the molecule. The extensive sheet region required only minor adjustments for 5 residues. All mainchain conformations were set to allowed 4, $ values. All sidechain conformations were set to the best library rotamer that fit the density, followed by small adjustments of individual dihedral angles. The yellow, blue, and green monomers of the asymmetric unit for the native structure were then generated by applying the previously determined noncrystallographic transformation matrices to the new orange monomer coordinates. This placed the green monomer Glu-107 and Tyr108 sidechains in the same conformation as for the other monomers, No additional water oxygens were included in the model. The rebuilt native model was refined once more using 6-2.5 A data. The R factor for the native structure was 0.328 after refitting the model, 0.209 after energy minimization by XPLOR, and 0.190 after final XPLOR refinement. The rms deviations from ideal values before and after XPLOR refinement were 0.038 and 0.017 A for bonds, 7.2” and 3.9’ for bond angles, and 30.3’ and 28.8” for dihedral angles. The rms shift in coordinates was 0.73 A for Ca atoms and 1.22 A for all atoms.

352

SMITH

The peroxynitrite-modified superoxide dismutase model was created from the refined native model. Tyr-108 on the orange monomer was replaced by an c-nitro-tyrosine residue. The side chain of the adjacent Glu-107 on the orange monomer was rotated about the C-C@ bond (Xl) to match the density of the difference map. Noncrystallographic transformations generated these two residues for the other three monomers. Diffraction data from the peroxynitrite-modified Cu,Zn superoxide dismutase crystals in the 6-2.5 A range were used for this refinement. The R factor was 0.242 for the initial modified model, 0.207 after energy minimization, and 0.187 after refinement.

ET AL.



Ramachandran plots comparing the initial 2SOD native model and the refined native model are presented in Fig. 1. The average root-mean-square shifts for main-chain and side-chain atoms before and after refinement of the native model are summarized in Fig. 2. The final refined native and peroxynitrite-modified superoxide dismutase models were practically identical. The difference Fourier electron density maps showed strong positive electron density next to only one of the t carbons of Tyr-108 on the orange (Fig. 3), yellow, and blue monomers of the asymmetric unit. The peaks were clearly above background with maximum peak values greater than six times sigma of the overall map density. Weaker positive difference density was seen near Tyr-108 of the green monomer. PSFRODO was used to model a nitro (NO,) group attached to the c carbon of Tyr-108 of the orange monomer. The nitro group fitted well into the elongated positive electron density and was coplanar with the tyrosine ring. The N to 0 distance used was 1.20 A, the N to C distance was 1.45 A, and the bond angles were 120” (32). The carboxylate group of Glu-107 in the native model was situated

iI8 3.0

6.0 7.0 1

220 1

*



240 1

260 1.1

a

280

4

300 11

320 I t

340 1

L

Residue number

7.0 4

5.0 d)mc>



4.0 3.0 2.0 1 .o l?o 2.0 3.0 4.0 5.0 6.0 4 7.0





420

I

440

460

480

I

500

1’

520

540

Residue number

7.0 6.0

d)mc> 28 a

+R

+I

Y

Y 0

b

0

5.0 : 1 6.0 7.0

I 620

640

660 ,

.

680 1

‘1 700 1 .

720 I

.

140 1

c

Residue number

FIG. 1. Ramachandran plots for the orange monomer only. (a) Data from the 2SOD Brookhaven Protein Data Bank entry. (b) After final refinement. Glycine residues are represented as 0, other amino acids as 0. Shading represents observed data base frequencies. The two lighter shades apply to glycine only; the two darker shades apply to all other amino acids. In those categories the darker shade means a higher frequency of occurrance than in the lighter shaded area.

FIG. 2. Root mean square shifts to atomic coordinates in A after refinement for mainchain atoms ((Dmc)) and for sidechain atoms ((Dsc)). The monomers are numbered as 1-151 orange, 201-351 yellow, 401-551 blue, and 601-751 green.

between a positive and a negative difference density peak, suggesting a small shift of the Glu-107 sidechain away from the nitro group on the tyrosine. A nitro group was

PEROXYNITRITE-MODIFIED

BOVINE

Cu,Zn

SUPEROXIDE

DISMUTASE

353

FIG. 3. Stereo pair showing positive (blue) and negative (red) electron difference density, e,e~son) ~ pcson). The model residues are Glu-107 and Tyr-108 of the native orange monomer after the first refinement. The nitro group is built onto the epsilon carbon of Tyr-108 and situated in the strongest positive difference density of the map. The contour level was t5u for the positive density and -3.5~ for the negative density. FIG. 4. Stereo pair showing the superposition of the yellow, blue, and green monomers onto the orange monomer, before final refinement. The green phenol side chain had not been repositioned. Difference electron densities as well as the model residues shown were superimposed by applying noncrystallographic symmetry. The electron density was contoured at +5u.

354

SMITH

modeled similarly into difference density observed for the yellow and blue monomers. The bound nitro group has only a local effect on the structure at residues Tyr-108 and Glu-107. The Tyr-IO8 phenolic group of the native structure orients the flat side of the ring toward a hydrophobic pocket composed of residues Phe-62, Pro-64, Leu-65, Val-79, and Leu-101. The addition of the hydrophilic NO2 group causes the tyrosine ring to twist a small amount, which moves the NO% group from the pocket and toward the outside solvent. The Glu107 sidechain is pushed slightly outward as a result. The appearance of weak difference density at the green Tyr-108 contrasted with the strong and clear difference density found for the other monomers. The green monomer should have been nitrated to the same extent as the others since the reaction took place in solution prior to crystallization. There were indications that the green monomer might have greater disorder for the Glu-I07 and Tyr-108 residues. An alternate conformation differing from the other three monomers had been modeled by Tainer et al. (19) in the Protein Data Bank 2SOD structure (Fig. 4). The alternate conformation for the green monomer has the Glu-107 and Tyr-108 sidechains pointing away from each other, whereas on the other three monomers these residues are oriented closer and approximately parallel to each other. To decide which conformation was supported by elec- FSOD,calc maps were calculated. tron density, ~FsOD,~,,~ Phases for the map were calculated omitting contribution from atoms of Glu- 107, Tyr- 108, and Ser- 109 in the green monomer. Electron density corresponding to both conformations was observed for the green monomer. The magnitude of the electron density indicated that both conformations were occupied to similar extents. Examination of the orange monomer 2FSOD,obs - FsoD,~~~~ map, calculated with complete model phases, did not indicate the presence of the alternate conformation. DISCUSSION The strong positive peaks in the difference electron density map appear to be due to the addition of a nitro group to one of the epsilon carbons of the single tyrosine (Tyr-108) found in each monomer. The tyrosine nitration site is 18 A from the active site copper on the same monomer and 36 A from the copper of the active site on the opposite side of the dimer. These results are consistent with our previous experimental observations (22). Cu,Zn superoxide dismutase reacted with tetranitromethane, commonly used to nitrate tyrosine residues (34), produces a pH-dependent uv-visible spectrum similar to that of Cu,Zn superoxide dismutase modified with peroxynitrite. The Raman spectrum for 3-nitro-L-tyrosine was clearly present in the spectrum of Cu,Zn superoxide dismutase reacted with peroxynitrite. The yellow-colored reaction product has full enzymatic activity compared to native

ET AL.

Cu,Zn superoxide dismutase as would be expected for a modification distant from the active site. Removal of Cu from Cu,Zn superoxide dismutase by prior dialysis against cyanide prevented nitration by peroxynitrite. Cu ion can be reintroduced to restore full catalytic activity to both 0, and ONOO- (22). This suggests that peroxynitrite does not simply attack the Tyr-108 residue directly but that the active site copper is involved in the nitration reaction. We propose that peroxynitrite reacts with the active site Cu of Cu,Zn superoxide dismutase to form a nitrating agent resembling nitronium ion (NO;) (22). However free nitronium ion is highly reactive and unlikely to survive long enough to diffuse to a Tyr-108 18 A away before reacting with water to form nitrate. A more likely possibility is that a moderately stable intermediate is formed at or near the active site. Peroxynitrite may transiently bind to the copper in the active site or a resulting nitronium ion might form a nitrocarboxylate intermediate with Glu-131 situated at the mouth of the active site 8 A from the copper. A subsequent molecular collision could then allow transfer of a nitro group to the Tyr-108 of another molecule. Modeling experiments using PSFRODO indicate that no steric hindrance prevents the active site region of one dimer from approaching closely Tyr-108 on another dimer. Tyr-108 is in an anionic region of the dimer, while Glu-131 and the active site are in a cationic region. Such a charge arrangement would facilitate the collision of those oppositely charged regions. The transfer process would depend upon the translational and rotational diffusion rates of Cu,Zn superoxide dismutase dimers and thus be a much slower mechanistic step than the initial reaction of peroxynitrite with Cu,Zn superoxide dismutase. Kinetic experiments have shown that the rate of selfnitration of Cu,Zn superoxide dismutase is only 1.0 -+ 0.1 Me’ s-l (22), approximately lOO,OOO-fold slower than nitration of low molecular weight phenolics by Cu,Zn superoxide dismutase (33). Furthermore, the rate of selfnitration varied with the square of superoxide dismutase concentration, consistent with a nitration mechanism requiring collision between two superoxide dismutase molecules (22). The confinement of the nitro group electron density to one side of the phenolic ring was unexpected. Although double nitration of tyrosine is improbable because the first nitrate deactivates the second nitration site, access to either side of the ring in the native model appears unobstructed. Thus both single site nitrations could potentially exist and appear simultaneously in the electron density difference maps as a disordered group. Comparison of the native and modified structures reveals that nitration of the tyrosine caused a small twist of the phenolic ring to position the nitro group into solvent rather than toward the hydrophobic interior. The observed orientation satisfies the placement of the nitro group in a more hydrophilic environment compared to the other ori-

PEROXYNITRITE-MODIFIED

BOVINE

entation. However, the aromatic ring of nitrotyrosine remains stacked against hydrophobic residues. The weak difference density on Tyr-108 of the green monomer may be a result of disorder. An alternate, extended conformation seems possible for the Glu-107 and Tyr-108 sidechains, which makes these groups more solvent accessible. However, crystal packing contacts make the extended conformation more difficult to attain in all monomers except the green one. The nitro group oxygens of the orange, yellow, and blue nitro-Tyr-108 residues are within 4 A of other symmetry related monomers. In contrast the green nitro-Tyr-108 has no symmetry related monomer residues within a 10 A distance. These conditions could allow the green nitro-Tyr-108 sidechain to have greater disorder than the other three monomers and may explain the weak difference density observed at the green monomer for the nitro group. The two possible conformations for nitrotyrosine may explain the observed splitting of several Raman bands assigned to the nitro moiety of peroxynitrite-modified superoxide dismutase (22), since the environments for each conformation are different. The close proximity of Glu-107 to the nitro group may be responsible for the small pKa shift from 7.3 for free 3nitrotyrosine to 7.8 observed for nitrated Cu,Zn superoxide dismutase tyrosine (22). The refined peroxynitrite modified Cu,Zn superoxide dismutase structure shows the Glu-107 carboxylate group interacting with the nitro group rather than the Tyr-108 hydroxyl group. The proximal nitro oxygen can easily hydrogen bond to the Tyr108 hydroxyl hydrogen. The presence of the Glu-107 carboxylate group near the nitro oxygen distal to the tyrosine hydroxy group may repel the partial negative charge there and make the other oxygen more negative. The slight buildup of charge density on the oxygen closer to the hydroxyl group would strengthen the hydrogen bond to the tyrosine hydroxyl group. The pKa of the hydroxyl group would shift upward as a result. REFERENCES

333,664-666.

7. Schmidt, H. H. W., Pollock, J., Nakane, M., and Gorsky, L. (1991) Proc.

Natl.

Acad.

Sci. USA

88,

365-369.

8. Granger, D. L., Hibbs, J. B., Jr., Perfect, J. R., and Durack, (1988) J. Clin. Inuest. 81, 1129-1136.

9. Stuehr, D. J., and Nathan,

C. (1989) J. Exp. Med. 169,1543-1555.

10. Marletta, M. A., Yoon, P. S., Iyengar, R., Leaf, C. D., and Wishnok, 27, 8706-8711. J. S. (1988) Biochemistry 11. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. M., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1621-1624. 12. Tamura, Y., Lingua, C., Driscoll, E. M., Jr., Hoff, P. T., Freeman, B. A., Gallagher, K. P., and Lucchesi, B. R. (1988) Circ. Res. 63, 944-959.

13. Liu, T. H., Beckman, J. S., Freeman, B. A., Hogan, E. L., and Hsu, C. Y. (1989) Am. J. Physiol. 256, H589-H593. 14. Imaizumi, S., Wollworth, V., Fishman, R. A., and Ghan, P. H. (1990) Stroke 21, 1312-1317. 15. Kawamoto, S., Inoue, M., Tashiro, S., Morino, Y., and Miyauchi, Y. (1990) Arch. Biochem. Biophys. 277, 160-165. 16. Beckman, d. S. (1990) Nature 345, 27-28. 17. Tainer, .I. A., Getzoff, E. D., Richardson, cJ. S., and Richardson, D. C. (1983) Nature (London) 306, 284-287. 18. Fridovich, I. (1986) Arch. Biochem. Biophys. 247, I-11. 19. Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Hiol. 160, 181-217. 20. Koppenol, W. H. (1981) in Oxygen and Oxy-radicals in Chemistry and Biology (Rodgers, M. A. J., and Powers, E. L., Eds.), pp. 671674, Academic Press, New York. 21. Getzoff, E. D., Tainer, J. A., Weiner, P. K., Kollman, P. A., Richardson, J. S., and Richardson, D. C. (1983) Nature 306, 287-290. 22. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431-437. 23. Richardson, D. C., Bier, C. J., and Richardson, J. S. (1972) J. Biol. Chem.

247,

6368-6369.

24. Howard, A. H., Gilliland, G. L., Finzel, B. C., Poulos, T. L., Ohlendorf, D. H., and Salemme, F. R. (1987) J. Appl. Cryst. 20, 383387. 25. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr., Brice, M. D., Rogers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) J. Mol. Biol. 112, 535-542. 26. Jones, T. A. (1978) J. Appl. Cryst. 11, 268272. 27. Pflugrath, J. W., Saper, M. A., and Quiocho, F. A. (1984) in Method and Applications in Crystallographic Computing (Hall, S., and Ashida, T. Eds.), pp. 404-407, Clarendon, Oxford. 28. Briinger, A. T., Kuriyan, 458-460. tallogr.

J., and Karplus,

A 46,

M. (1987) Science 235,

A., and Erickson,

J. (1990) Acta Cry+

585-593.

30. Ponder, J. W., and Richards, F. M. (1987) J. Mol. Biol. 193, 775791. 31. Jones, T. A., Bergdoll, M., and Kjeldgaard, M. (1989) in Crystallographic and Modeling Methods in Molecular Design (Bugg, C. E., and Ealick, S. E., Eds.), pp. 189-199, Springer-Verlag, New York. 32. Weast, R. C. Ed., (1973) in CRC Handbook of Chemistry ics, 54th ed., pp. F198-F199, CRC Press, Cleveland.

and Phys-

33. Beckman, J. S., Ischiropoulos, H., Zhu, L., van der Woerd, M., Smith, C. D., Chen, J., Harrison, J., Martin, J. C., Tsai, M. (1992) Arch. Biochem.

D. T.

355

DISMUTASE

29. Brunger, A. T., Krukowski,

1. Cadenas, E. (1989) Annu. Reu. Biochem. 58, 79-110. 2. Fridovich, I. (1989) J. Biol. Chem. 264, 7761-7764. 3. Palmer, F. M. J., Ferrige, A. G., and Moncada, S. (1987) Nature (London) 327, 523-526. Toricol. 30, 535-560. 4. Ignarro, L. J. (1990) Annu. Reu. Pharmacol. 5. Furchgott, R. F., and Vanhoutte, P. M. (1989) FASEB J. 3, 20072018. 6. Palmer, R. M. J., Ashton, D. S., and Moncada, S. (1988) Nature (London)

Cu,Zn SUPEROXIDE

Biophys.

298,

34. Riordan, ,J. F., Sokolovsky, 6, 358-361.

438-445.

M., and Vallee, B. L. (1967) Biochemktry

Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the exis...
11MB Sizes 0 Downloads 0 Views