J. Mol. Biol. (1976) 105, 327-332

Manganese Superoxide Dismutases from Escherichia coli and from Yeast Mitochondria: Preliminary X-ray Crystallographic Studies The Mn superoxide dismutase from Escherichia coli has been obtained in three crystal forms: (I) from 68% saturated (NH&SO,, space group I’222 or P222,, a :: 47 11, b = 103 A, c = 47-5 A, with one subunit per asymmetric unit; (II) from 50% polyethylene glycol 6000, space group C222, (with approx. P4,2,2 symmetry), (I = 101 A, b = 108 A, c = 180 A, with four subunits (2 molecules) per &symmetric unit; (III) from 52% polyethylene glycol with a different method of preparing the enzyme solution, space group P2,2,2, a :- 47 A, b = 51 A. c = 188 A, with two subunits per asymmetric unit. The yeast mitochondrial Mn superoxide dismutase has yielded the same crystal form both from 30% 2-methyl-2,4-pentane diol and from 2376 polyethylene glycol 6000: space group P2,2,2,, a = 63 A, b = 115 A, c = 125 A, with four subunits (one molecule) per asymmetric unit. A full X-ray crystallographic study of at least one of these enzymes is planned. The superoxide dismutases are very widely distributed enzymes which protect against the damaging effects of the superoxide (0;) radical by catalyzing its dismutation to hydrogen peroxide and molecular oxygen (Fridovich, 1974,1975). These enzymes appear to fall into two major classes: (I) Cu,Zn superoxide dismutases-copper at the active site; activity is cyanidesensitive but protein is stable to chloroform/ethanol (McCord & Fridovich, 1969) ; two identical subunits of molecular weight 16,000 (Fridovich, 1975; Steinman et al., 1974), each with one Cu and one Zn; found in the cytoplasm of an extremely wide variety of eukaryotic organisms (Fridovich, 19741, but is apparently absent in some eukaryotic algae (Asada et al., 1975), fungi (Lavelle & Michelson, 1975), and flagellates (Lindmark & Miiller, 1974); a rather similar Cu,Zn superoxide dismutase has been found in a symbiotic marine photobacterium (Puget & Michelson, 1974). (II) Mn or Fe superoxide dismutases-either manganese or iron at the active site ; activity is insensitive to cyanide but protein is usually not stable to chloroform/ ethanol ; either two or four subunits of molecular weight 19 to 24,000 ; most prokaryotes (Fridovich, 1975) and the mitochondria of eukaryotes (Weisiger & Fridovich, 1973) have Mn superoxide dismutases; Fe superoxide dismutases have been found in t.he periplasmic space of E. co& (Yost t Fridovich, 1973) and as the major superoxide dismutase in several blue-green algae (Asada at al., 1975: Lumsden & Hall, 1974; Misra & Keele, 1975). There is strong homology among the partial sequences of E. coli Mn superoxide dismutase, chicken liver mitochondrial Mn superoxide dismut’ase, and E. coli Fe superoxide dismutase (Steinman & Hill, 1973); it seems probable that all of the 1Mn and Fe enzymes are related and would have similar three-dimensional structures. However, the lack of sequence homology and the great difference in physical properties between those enzymes and the Cu,Zn superoxide dismutases make it likely that the two main classes are unrelated to each other and could he expect,ed to have basically different. t,hree-dimensional struct,ures. 327







We have previously reported (Richardson et al., 1975aJ) the structure of the bovine erythrocyte Cu,Zn superoxide dismutase at 3 A resolution, showing that the subunit conta,ins an eight-stranded barrel of ant’iparallel p sheet), that bhe two active sites in t,he dimer are widely separated, that, the Cu is accessible to solvent and has four histidine ligands, that the Zn has three histidine and one aspartate ligand, and that one of the histidines bridges bet(ween the Cu and the Zn. This letter reports t’hree crystal forms of the Mn superoxide dismutase from E. coli and one crystal form of the mitochondrial Mn superoxide dismutase from yeast (Saccharomyces cerevisiae). We propose to solve the three-dimensional structure of at’ least one of these forms, in order to obtain a definitive answer as to the unrelatedness of t,he two main classes of superoxide dismutase, and in order t,o determine the geometry of an active site which has very simila,r functional properties hut utilizes a different. single, metal.


and Space-group Determination

Purified enzyme materials were the gift of Irwin Fridovich, Frederick Yost, S. D. Ravindranath and Dennis Ose of the Duke University Biochemistry Department. The E. coli Mn superoxide dismutase was received as a slurry in saturated ammonium sulfate; usually it was centrifuged and the pellet dissolved in 0.05 M-pOtaSSiUm phosphate buffer at pH 78 with 0.1 mM-EDTA; the protein concentration was adjusted to 3 to 4 mg/ml (E. coli enzyme st’ock solution). All work on the E. coli enzyme was done at normal room temperature. The yeast mitochondrial Mn superoxide dismutase was received as a slurry of crystals in deionized water (Ravindranath & Fridovich, 1975) ; for later crystallization trials, the enzyme mixture was dialyzed against 5 mMTris*HCl at pH 8.5 and reconcentrated to between 1 and 3 mg/ml (yeast enzyme stock solution). All manipulations of the mitochondrial enzyme were done at 4°C unless otherwise noted. The polyethylene glycol 6000 stock was 25 g of Union Carbide flakes dissolved in 50 ml water. E. coli crystal form, I: to the E. di enzyme stock, saturated ammonium sulfate was added to make SS%, of the total volume; very small crystals grew in from one to seven months. The space group is either P222 or P222,, with unit cell dimensions of a = 47 A, b = 103 8. c = 47.5 A. F or a molecular weight of 39.500 (Keele et al., 1970) I’, = 2.9 A3 per dalton for one subunit (3 molecule) per asymmetric unit ; this is a midrange V, value for protein crystals, while a F’, either twice or half as large would be outside the normal observed range (Matthews. 1968). E. coli crystal fornt, II: equal amounts of E. c&i enzyme stock and polyethylene glycol6000 stock were mixed; long. square. pinkish needles grew in several days to a few weeks. The needles could be grown to almost any length but were always less t,han 0.1 mm wide. The same crystal form was also grown from polyethylene glycol 20000. The space group is C222,, wit,h unit’ cell dimensions of a. = 101 A, b -= 108 A. c 5 180 A. V, = 3.06 for four subunim (2 molecules) per asymmetric unit. The diffraction pattern shows an approximat’e P4,2,2 symmetry: the h01 and Ok1 zones show essentially identical intensity patterns at least out to 15 A resolution and the c axis shows 1 # 4n systematic absences broken by only two faint spots. Since the 180 A spacing is along t)he needle axis, prevention of spot, overlap necessitated either cleavage of the cry&al or use of a small collimator. This crystal form is extremely stable in the X-ray beam : a still photograph taken aftor 17 days of exposure showed






dismutase, crystal form 111 (P2,2,2; from polyethylene FIG:. 1. Crystal of E. coli Mn superoxide glycol), approximately 1 mm long. The 001 crystal face is in front, the 011 and Oli faces are at thfa top, and the 101 and 1Oi faces form the termination at, the right-hand end of the crystal.

intense reflections out to the edges of the film (taken on a precession camera with 6 cm crystal-to-film distance). E. c*oli crystal form 111: in this case the initia,l slurry was spun through a Millipore (:S filter: the trapped gelatinous protein deposit (still quite wet with ammonium sulfat*e solution) was scraped from the filter and dissolved in O*Oti M-phosphate buffer at pH 7%; the solution was then filtered through very coarse filter paper. Polyethylene glycol6000 stock was added to make 52% of the tot’al volume. and in one to two weeks crystals grew as clusters, plates. or blocks. Figure 1 is a photograph of one of these crystals which is approximately 1 mm long. They are deep pink when viewed down the CLaxis, it clear yellowish brown down the b axis, and almost colorless down the c axis. Figuna 2 shows a precession photogra,ph of the h,O8zone. The space group is 51 A. c =- 188 8. V, = 2.8 for P%,212. with unit cell dimensions of a = 47 A, h two subunits (1 molecule) per a’symmetric unit. Yenst mitochondrial crystals: Ravindranath & Fridovich (1975) initially grew crysta,ls by dialysis against 0.1 mM-EDTA and then against water. We have obtained more sbable crystals, probably of the same crystal form, by adding to the enzyme stock either 30yo 2-methyl-2,4-p entane diol or 23% polyethylene glycol 6000 stock, mixed at room t’emperature and then returned to 4°C. The crystals are rhombic in cross-





Fru. 2. X-ray diffraction photograph form III (P2,2,2; from polyethylrne angle.





of thv h0Z zone of E. coli Mn supwoxicie diamutaso crystal glycol). Taken at room temperat,ure, p 15” prncwsion

section, of about 0.2 mm longest dimension, and very pale brown. A photograph of one batch of them is shown in Ravindranath & Fridovich (1975), and Figure 3 shows a 7” precession photograph of the h01 zone. The space group is P2,2,2,, with unit cell dimensions of a = 62 A, 6 = 115 A, c = 125 A. V, = 2.3 for four subunits (1 molecule of iM, 97,000; Weisiger 6 Fridovich. 1973) per asymmetric unit.

Discussion All four of these crystal forms of Mn superoxide dismutase diffract well out to at least 2.5 A resolution. At present the E. c.oZiform III crystals are the most promising, both because of their large size and because they have only two subunits per asymmetric unit. We will proceed with data collection and heavy-atom derivative search on those crystals and also on the yeast mitochondrial crystals, and will continue


toward a. three-dimensional prows most satisfactory.

Departmellt of Hiochemiutr; tDepa,rtment of Anatomy Duke ITniversity. Dm-ham, Keceix rd 5 April “2






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REFERENCES Asada, K., Yoshikawa, K., Takahashi, M., Maeda, Y. & Enmanji. K. (1975). ,1. Biol. Chem. 250, 2801-2807. Fridovich, I. (1974). A&an. Enzymol. 41, 35.-97. Fridovich, I. (1975). Annu. Rev. Biochem. 44, 147- 159. Keele, B. B. Jr, McCord, J. M. & Fridovich, I. (1970). J. Biol. Chem. 245, 61766181. T,avelle, F. & Michelson, A. M. (1975). Biochimie, 57, 375-381. Lindmark, D. G. & Muller, M. (1974). J. Riol. Chem. 249, 4634.-4637. Lumsden, J. & Hall, D. 0. (1974). Riochem. Biophys. Res. Gommun. 58, 35 41. Matthews, B. W. (1968). .I. Mol. Biol. 33, 491-497. McCord, J. M. & Fridovich, I. (1969). ,I. Biol. Chem.. 244, 6049-6055. Misra, H. I’. & Keele, B. B., Jr (1975). Biochim. Biophys. Acta, 379, 416425. Puget, K. & Michelson, A. M. (1974). Rio&em. Biophys. Kes. Common. 58, 830-838. Ravindranath, S. D. & Fridovich, I. (1975). J. Biol. Chem. 250, 610776112. IXichardson, J. S., Thomas, K. A., Ruhin, R. H-. K- Richardson, D. (1. (1975a). Proc. .Vat. Acad. Sci., U.S.A. 72, 1349 -1353. Richardson, 6. S., Thomas, K. A. & Richardson, D. C. (19755). Biochem. Biophya. Res. Commun,. 63, 986.-992. Steinman, H. M. & Hill, R. L. (1973). E’roc. Xat. dead. Sci., U.S.A. 70, 3725-3729. Steinman, H. M., Naik, V. R., Abernethg, J. L. &r. Hill, R. L. (1974). J. Biol. Chem. 249, 732667338. Weisiger, R. A. & Fridovich, I. (1973). ,I. Bid. Chem. 248, 3582-3592. Yost, F. a. Jr & Fridovich, I. (1973). J. Biol. Chem. 248, 4905-4908.

Manganese superoxide dismutases from Escherichia coli and from yeast mitochondria: preliminary x-ray crystallographic studies.

J. Mol. Biol. (1976) 105, 327-332 Manganese Superoxide Dismutases from Escherichia coli and from Yeast Mitochondria: Preliminary X-ray Crystallograph...
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