Superoxide Dismutase from Escherichia coli B

Vol.245,

THE JOURNAL OP BIOLOCWAL CHEMISTRY No. 22, Issue of November 25, pp. 6175-6181, 1970 Printed

Superoxide A NEW

in

U.S.A.

Dismutase

MANGANESE-CONTAINING

from

Escherichia

coli B

ENZYME* (Received for publication,

BERNARD

From

JR.,$ J. M. MCCORD,~ AND

B. KEELE, the Department

of Biochemistry,

Duke

I. FRIDOVICH

University

Medical

SUMMARY

Durham,

AND

North

Carolina

27706

METHODS

Cytochrome c, type III, and xanthine were obtained from Sigma. E. coli B harvested in late log phase were purchased from Miles Laboratories, Elkhart, Indiana. Microgranular diethylaminoethyl cellulose (DE-32) and microgranular carboxymethyl cellulose (CM-52) were obtained from Reeve Angel Company, New York, New York. Ammonium sulfate, enzyme Xanthine oxidase, purified from grade, was a product of Mann. raw cream by a procedure which avoided exposure to proteolytic agents (7), was generously provided by Drs. F. Brady and K. V. All other materials were obtained from commerRajagopalan. cial sources at the highest available states of purity. Superoxide dismutase was assayed, and units were defined as previously described (3). Ultracentrifugal analyses were performed at pH 7.3 in 0.10 M KCl, 0.05 M potassium phosphate, on a Beckman model E analytical ultracentrifuge by the method of Yphantis (8). Assessments of the purity of the enzyme by disc gel electrophoresis were performed essentially as described by Davis (9). The molecular weight of the subunits of the enzyme was estimated by disc gel electrophoresis on 10% acrylamide gels, in the presence of sodium dodecyl sulfate, as described by Weber and Osborn (10). The following molecular weight standards were used to calibrate the gels: phosphorylase a, 94,000; human transferrin, 77,000; bovine serum albumin, 68,000; catalase, 60,000; ovalbumin, 43,000; pepsin, 35,000; chymotrypsinogen A, 26,000; and ribonuclease, 13,600. Electron paramagnetic resonance spectroscopy was performed on a Varian model E-9 HF EPRl spectrometer, with a 9.5 GHz microwave bridge assembly, which was operated at a modulation frequency of 100 kHz. Manganese was determined quantitatively on the basis of the intensity of its EPR signal and by the calorimetric method of Sri?astava, Pandya, and Zaidi (11). Zinc assays were performed as described by Malmstrom (12), and copper assays were performed by the method of Felsenfeld (13). RESULTS

Puri$cation of Enzyme-Frozen E. coli, 500 g, was thawed overnight at 5” and then suspended in 2500 ml of 0.05 M potassium phosphate, 1 X 10m4 M EDTA, at pH 7.8. The suspended cells were disrupted by sonication of 400-ml batches at 0” in a Rosett cell for 3 min. A Branson sonifier was used at a power setting of 125 watts. The suspension was clarified by centrifugation for 60 min at 13,200 X g, and after the addition of KC1 to a final concentration of 0.10 M the supernatant solution was 1 The nance

6176

abbreviation

used

is: EPR,

electron

paramagnetic

reso-

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The ability of certain enzyme preparations specifically to inhibit the oxygen-dependent reduction of cytochrome c by xanthine oxidase (1) led to the discovery of an enzymic activity responsible for catalyzing the dismutation of superoxide free radicals (2) and to the isolation of this enzyme from erythrocytes (3). The superoxide dismutase from erythrocytes was found to be identical with the copper-containing proteins hemocuprein (4) and erythrocuprein (5, 6), which had been isolated, without knowledge of their function, from bovine and human erythroVarious tissues from a wide assortment of cytes, respectively. mammalian organisms were surveyed, and all were found to contain high levels of superoxide dismutase. If we suppose that superoxide dismutase serves to protect cells against the deleterious effects of the superoxide free radical, then we should expect to find it in all aerobic cells, including the simplest microorganisms. The present report describes the purification and properties of superoxide dismutase from Escherichia coli. * This work was supported in full by Grant GM-10237 from the National Institutes of Health, Bethesda, Maryland. $ Postdoctoral Fellow of the National Institutes of Health. Present address, Institute of Dental Research, University of Alabama Medical Center, Birmingham, Alabama 35233. $ Postdoctoral Fellow of the National Institutes of Health.

Center,

MATERIALS

Superoxide dismutase, which catalyzes the disproportionation of univalently reduced oxygen (0~: + 02; + 2H+ -+ O2 + HzOz) and which has previously been demonstrated in a variety of mammalian sources, has now been purified from Escherichia coli. Whereas the mammalian enzyme is blue and contains copper and zinc, the bacterial superoxide dismutaseis red-purple and was found to contain manganese. The molecular weight of the E. coli enzyme was found to be 39,500 by sedimentation equilibrium and was shown, by gel electrophoresis in the presenceof sodium dodecyl sulfate, to be composed of two subunits of equal size. Electron paramagnetic resonance spectrometry and chemical analysis demonstrated between 1.6 and 1.8 atoms of manganeseper molecule of enzyme. The enzyme contains no signiticant amounts of copper or zinc. The ultraviolet and visible absorption spectra of the enzyme are presented, as are the results of amino acid analysis.

June 25, 1970)

Issue of November

25, 1970

B. B. Keeb, Jr., J. M. MeCord, and I. Fridovich TABLE

1

2.4 2.2 2.0

Purification I

Pooled

1.8

Purification

‘rot& T L Volconcen- I “IlIe 2.tion’

stage

w/ml

0.6 0.4 0.2

c...

I 0

Iz

24

48

36

60

72

84

96

108

120

I32

1

144

Jo

FWCtlOfl

cated. I

I

ml

dismutnse

protein

Total

Specific activity

mg

U?zilSjmg

Total units

p 72 * -%

Purification

-fold

I

95 76 76

2,950 224,200

1.8516,700100 2.2491,700 95

1.2

3,300 250,800

2.2491,700

95

1.2

47 30

*,ooo 188,000 127 3,800

2.7500,OOO 83 317,500

97 62

1.5 46

58

56

20

3 ) 100 294,500

150

3,000

100

300,000

After chromat.ography CM-52 DE-32

on

0.6 1.9: 3 I (1 Protein concentration on all fractions, other than those obtained as column effluents, was measured in terms of absorbance at 280 nm assuming that E:?m = 10.0. The concentration of protein in column effluents was determined by the method of Murphy and Kies (14).

Pooled I

I

l-

-6

E 9

0.4 0.2 -

0

2 -2.0

-4

4

8

I2

16

20

24

28

32

36

40

?k .c k

44

s -3.0 ,-

Fraction

2. Chromatography on DE-32. The superoxide dismutase obtained by chromatography on CM-52 was concentrated and then dialyzed against 5 mM potassium phosphate, pH 7.8, as described under “Purification of Enzyme.” It was then applied to a column, 2.5 X 25 cm, of DE-32, which had been equilibrated at 4’ with 5 mM potassium phosphate, pH 7.8, and was eluted with this buffer. O--O, absorbance at 280 nm; l -- -0, superoxide dismutase activity of the fractions collected. FIG.

heatedin 200-mlbatchesto 60” for 3 min and then chilled. Denatured proteins were removed by centrifugation. Streptomycin sulfatewasaddedto the supernatantto a final concentration of 2.5% and, after incubation at room temperature for 30 min,

the suspension

was clarified

by centrifugation.

Solid

ammoniumsulfate wasaddedto bring the supernatantto 50% saturation and, after stirring for 1 hour at room temperature,the mixture was centrifuged.

Solid ammonium

&fate

was added to

this supernatant,to bring it to 75% saturation with respectto this salt, and after stirring at room temperature for 1 hour, the precipitate was collected by centrifugation, suspendedin 2 mM

-4.0

(

r2 FIG. 3. Equilibrium sedimentation of E. coli superoxide dismutase. Superoxide dismutase at a concentration of 0.3 mg per ml in 0.05 M potassium phosphate, 0.1 mM EDTA, 0.10 M potassium chloride, pH 7.3, was equilibrated at a rotor speed of 35,609 rpm. The ultracentrifuge was equipped with interference optics, and In fringe displacement is here plotted as a function of the square of the distance from the center of rotation.

potassiumacetate buffer, pH 5.5, and then dialyzed at 4” for 96 hours against 20 volumesof this buffer. During this dialysis, the buffer was replaced every 12 hours. The precipitate that formed during dialysis was removed by centrifugation, and the supernatant wasadsorbedonto a column, 2.5 x 28 cm, of CM-


.~ 1. Chromatography on CM-52. The E. coli preparation after dialysis for 96 hours, as described in the text, was applied to a column, 2.5 X 28 cm, of CM-52 equilibrated at 4” with 2 rnM potassium acetate, pH 5.5, and was then eluted with this buffer until G5 fractions (7 ml) had been collected. At this point, 1000 ml of a gradient (0.002 to 0.20 M) in this buffer were applied. O--O, absorbance at 280 nm; -----9 conductivity; l - - -0, superoxide dismutase activity of the fractions collected. The fractions that were pooled for further manipulation are so indiFIG.

Supernatant fron Sonicate Heat step Streptomycin sulfate 50% (NH&S04 75% NL)zS04 precipitate redissolved Dialysis and centrifugation

I

of E. coli super-oxide

1.6

0

6177

6178

Superoxide Dismutase from E. coli B Molecular

weight

(x 10m3) acid

composition

Amino

6 8 centimeters

IO

12

14

nunometers 260 I

I

280 I

I

I

320 I

I

340 I

.08

- 0.8

.07

- 0.7

7.06

g .05 B P .04 9 .03

nanometers FIG. 5. Absorption spectrum The spectrum in the ultraviolet solution containing 0.7 mg per

(....) of E. coli superoxide dismutase. (solid line) was obtained with a ml of the enzyme in 0.05 M potas-

sium phosphate at pH 7.8, while the spectrum in the visible region (segmented line) was obtained from a solution containing 7.0 mg in the same

buffer.

52 which had been equilibrated with the dialysis buffer. The column was washed with 2 mM potassium acetate, pH 5.5, until a large peak of protein had been eluted, and 1000 ml of a gradient (0.002 to 0.20 M) of this buffer were then applied. Fig. 1 prethe

results

..

of this

elution.

The

fractions

that

contained

..

.

Phenylalanine Total residues.

o Duplicate

..

.

samples were hydrolyzed

dismutase” Residues per mole of enzyme (nearest integer)

29

12.3 10.4 41.9 18.5c 21.6c 36.7 15.4 26.4 47.1 20.4d 3.1 13.7d 37.5d 11.8 18.0

Tyrosine

12 10 42 19 22 37 15 26 47 20 3 14 38 12 18 364

for 24, 48, and 72 hours.

b All calculations were based on a molecular weight of 39,500. 0 Values were extrapolated to zero time of hydrolysis. d Samples of 72 hours were used for calculations. the

- 0.9

.I.

sents

acid contentb

29.2

Glutamic acid. Proline........................ Glycine........................ Alanine........................ Valine. Methionine.................... Isoleucine . Leucine

(-) 300 I

.09

per ml of the enzyme

fhino

superoxide

superoxide

dismutase

activity

were

pooled,

as indicated

in

Fig. 1, and were concentrated by ultrafiltration over a PM-10 membrane (Amicon Corporation, Cambridge, Massachusetts). The resultant red-purple solution, which was dialyzed against several changes of 5 mM potassium phosphate, pH 7.8, for 60 hours at 4”, was placed on a column, 2.5 X 25 cm, of DE-32 which had been equilibrated with this buffer. The enzyme was eluted with 5 mM potassium phosphate, pH 7.8, with the results shown in Fig. 2. The fractions containing the highest specific activity were pooled, as indicated in Fig. 2, and concentrated by ultrafiltration. The results of this purification procedure are summarized in Table I. The yield was 16 mg of superoxide dismutase with a specific activity of 3800 units per mg. This may be compared with the specific activity of 3300 units per mg observed with the enzyme from bovine erythrocytes (3). Moleculur Weight-E. co& superoxide dismutase at a concentration of 0.3 mg per ml was equilibrated in a centrifugal field, and the resultant distribution of protein was analyzed by the method of Yphantis (8). When In fringe displacement, was plotted as a function of the square of the distance from the center of rotation, a straight line was obtained. This is shown in Fig. 3. The slope of this line and an assumed partial specific volume of 0.720 gave a molecular weight of 39,500. The linearity of the data in Fig. 3 indicates a high degree of homogeneity with respect to sedimentation properties. Polyacrylamide disc gel electrophoresis at pH 8.9 in Tris-HCl buffer did, however, expose the presence of an impurity which was estimated to represent approximately 5% of the total protein. Subunit Weight-E. coli superoxide dismutase was dissociated into its component subunits by exposure to sodium dodecyl sulfate with and without fl-mercaptoethanol, and the molecular weight of these subunits was estimated by disc gel electrophoresis in the presence of these agents (10). Proteins of known subunit


FIG. 4. Determination of subunit molecular weight. E. coli superoxide dismutase was subjected to electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate f -mercaptoethanol as described by Weber and Osborn (10). The gels were calibrated in terms of molecular weight by using the protein standards listed in the text under identical experimental conditions. The position of the stained protein bands on the gels was recorded with a Gilford model 2000 spectrophotometer equipped with a gel scanner. It is apparent that sodium dodecyl sulfate was able to cause the dissociation of the enzyme in the absence of fi-mercaptoebhanol (E + SH).

I

coli

. Serine......................... 4

; IO

E.

of

acid

Lysine......................... Histidine...................... Arginine . Aspartic acid. Threonine .

GOIf, &+I 2

II

TABLE Amino

0

Vol. 245, No. 22

Issue of November

25, 1970

B. B. Keele, Jr., J. M. McCord, and I. Fridovich

2 B. B.,. Keele, 0Dservauons.

Jr., J. M. McCord,

and I. Fridovich,

unpublished

I

C

I

I

I

3000

I

3400

I

I

3800

Gauss

of manganese in E. coli superoxide dismutase by EPR spectroscopy. Curve A was obtained by mixing 0.45ml of E. coli superoxide dismutase (1.5 mg per ml) in water with 0.05 ml of 1.0 M HCI and then heating to 100” for 3 min. This acid solution of denatured enzyme was then placed in a flat cell assembly and examined for EPR signals under the following conditions: microwave frequency, 9.552 GHz ; microwave power, 24 mwatts; modulation amplitude, 4 gauss; scan rate, 250 gauss per min; time constant, 1.0 set; receiver gain, 5000; and temperature, 23”. Curve B was obtained when the native enzyme was examined at a concentration of 4.5 mg per ml in water under otherwise identical condiFIG.

tions. solved

6. Detection

Curve

in 0.1

C was M

similarly

obtained

from

0.1 mM

MnClz

dis-

HCI.

detected by the 2,2’-biquinoline method (13), and only insignificant amounts of zinc were found by the dithizone method (12). DISCUSSION

The demonstrated ability of milk xanthine oxidase (2, 3, 18-21), liver aldehyde oxidase (19, 22), bacterial dihydroorotic dehydrogenase (19, 23), and diamine oxidase (24) to generate superoxide radicals indicates the likelihood that other oxidative enzymes can also do so. The ability of reduced ferredoxin (25) and of reduced flavins, quinones, and dyes (26) to carry out the univalent reduction of oxygen to this radical poses the possibility that superoxide radicals may also be generated within cells by nonenzymic oxidations. Indeed, although the quantitatively important sources of superoxide radical within living cells remain to be identified, it appears unavoidable that this radical will be found to be generated, to a greater or lesser extent, in all aerobic cells. In this case, the superoxide dismutase, which we suppose to be a defense against the damaging reactivities of this free radical, should be ubiquitous in aerobic cells. As one means of exploring the validity of these lines of thought, we have sought to isolate the superoxide dismutase of a common microorganism.


weight were used as standards, as described under “Materials and Methods.” The superoxide dismutase was found to be composed of subunits having a molecular weight of approximately 21,600. As shown in Fig. 4, sodium dodecyl sulfate, in the presence or absence of P-mercaptoethanol, was able to dissociate superoxide dismutase. We may conclude that E. coli superoxide dismutase is composed of two subunits of equal size, the association of which does not involve disulfide bridges. Spectral Properties-The color of the E. coli superoxide dismutase was obviously different from the color of the erythrocyte enzyme. Its absorption spectrum in the visible region confirmed Thus, as shown in Fig. 5, the E. coli enzyme these differences. had an absorption maximum at 473 nm with a molar extinction coefficient of 400. The erythrocyte enzyme, on the other hand, absorbed at 680 nm (3). The differences in spectral properties between the superoxide dismutases from E. co& and from erythrocytes extended to the ultraviolet region as well. Thus, as shown in Fig. 5, the E. coli enzyme exhibited the usual protein absorption maximum at 283 nm, whereas the erythrocyte enzyme had previously been seen to absorb maximally at 258 nm (3). Amino Acid AnalysisE. coli superoxide dismutase was dialyzed at 4” against changes of de-ionized water for 1 week, and its concentration was then estimated on the basis of absorbance in the short ultraviolet. Aliquots were hydrolyzed under reduced pressure by heating to 110” in the presence of 6 N HCI for 24, 48, and 72 hours. After removal of the .HCl under reduced pressure, the samples were assayed for their amino acids on a Beckman model 120 C amino acid analyzer. The results of this analysis are presented in Table II. When the amino acid composition of the E. co& enzyme is compared with that of the human The erythrocyte enzyme (15), striking differences are apparent. erythrocyte enzyme contains no tyrosine and no methionine, whereas the E. co&i enzyme contains 3 residues of methionine and 12 of tyrosine. Less pronounced differences occur in virtually all of the amino acids in these proteins. Manganese-EPR spectroscopy of native E. coli superoxide dismutase failed to show the characteristic copper(H) signal observed with the bovine erythrocyte and heart superoxide dismutase2 or with human cytocuprein (16) which is, in fact, superoxide dismutase. Indeed, no EPR signal was observed with the native enzyme. Upon denaturation by boiling in 0.1 N HCl, however, the very characteristic signal of manganese(I1) appeared. This is shown in Fig. 6. Spectrum A was obtained from 1.35 mg per ml of E. coli superoxide in 0.10 N HCl which had been exposed to 100” for 3 min; Spectrum B was obtained from the native enzyme at a concentration of 4.5 mg per ml in water; while Spectrum C was that obtained from 0.1 mM MnClz in 0.10 N HCl. These spectra, which were obtained with a flat cell assembly at room temperature, were repeated three times. Each repetition was preceded by careful checking of the alignment of the flat cell in the magnet,ic field. The results obtained, which were perfectly reproducible, indicated that the E. coli superoxide dismutase contained 1.6 atoms of manganese per molecule of enzyme. Quantitative calorimetric analysis for manganese after wet-ashing of the enzyme by the method of Sribastava et al. (11) demonstrated 1.8 atoms of manganese per molecule of enzyme. Because erythrocyte superoxide dismutase contains both copper (4, 5, 16) and zinc (17), the E. COG enzyme was assayed for both of these metals. Copper could not be

6 179

&peroxide

Dismutase from E. coli B

3 J. M.

McCord

and

I. Fridovich,

unpublished

observations.

phore, leads to the suspicion that the manganese in the resting This is based on the observation that enzyme may be Mn(II1). the red-purple color is more characteristic of Mn(II1) complexes than of Mn(I1) complexes (33). The many differences between the mammalian and the E. coli superoxide dismutase are interesting from an evolutionary point of view. It is extremely difficult to imagine that such different proteins evolved from a common ancestral form. One is rather led by these data to speculations concerning the independent development of these enzymes in evolutionary lines which had already separated during the anaerobic phase of life’s history on earth, or alternatively, to the proposal that the original superoxide dismutase was lost in the E. coli line by a long but temAlthough these specuporary restriction to an anaerobic milieu. lations need not be pursued here, they will obviously generate the need to isolate and examine the superoxide dismutase from a wide variety of living things. Aclcnowbdgments-We would like to express sincere thanks to Dr. K. V. Rajagopalan who performed the electron paramagnetic resonance examinations of the E. co& superoxide dismutase and who, by this means, actually discovered the manganese in this enzyme. We are also indebted to Dr. Bolling Sullivan for the amino acid analyses and to Mr. Salvatore Pizzo who determined the molecular weight of the subunits of the enzyme by disc gel electrophoresis in the presence of sodium dodecyl sulfate. The sedimentation equilibrium centrifugation was kindly performed by Mr. Jim Huston. We wish to thank Dr. George H. Reed for his suggestion concerning the probable valence state of the manganese in the resting enzyme. REFERENCES 1. FRIDOVICH, I., J. Biol. Chem., 237, 584 (1962); 242,, 1445 (1967). 2. MCCORD, J. M., AND FRIDOVICH, I., J. Biol. Chem., 243, 5753 (1968). J. M., AND FRIDOVICH, I., J. Biol. Chem., 244, 6049 3. MCCORD, (1969). T., AND KEILIN, D., Proc. Roy. Sot., Ser. B, Biol. Sci., 4. MANN, 126, 303 (1939). 5. MARKOWITZ, H., CARTWRIGHT, G. E., AND WINTROBE, M. M., J. Biol. Chem., 234, 40 (1959). J. R., MARKOWITZ, H., AND BROWN, D. M., J. Biol. 6. KIMMEL, Chem., 234, 46 (1959). 7. BRADY, F. O., Ph.D. Dissertation, Duke University, 1969. D. A., Biochemistry, 3, 297 (1964). 8. YPHANTIS, B. J., Ann. N. Y. Acad. Sci., 121, 404 (1964). 9. DAVIS, Biol. Chem., 244,440s (1969). 10. WERER, K., AND OSBORN, M.,“J. 11. SRI~TASTAVA, S. P., PANDYA, K. P., AND ZAIDI, S. H., Analyst, 94, 823 (1969). B. G., Methods Biochem. Anal., 3, 327 (1956). 12. MALMSTROM, 13. FELSENFELD, G., Arch. Biochem. Biophys., 37, 247 (1960). J. B., AND KIES, M. W., Biochim. Biophus. 14. MURPHY, _ _ Acta, 46. 384 (1960). J. W., AND DEUTSCH, H. F., J. Biol. Chem., 244, 4565 15. HARTZ, (1969). R. J., AND DEUTSCH, H. F., J. Biol. Chem., 244, 6087 16. CARRI&, (1969). 17 CARRICO, R. J., AND DEUTSCH, H. F., J. Biol. Chem., 246, 723 (1970). I., AND HANDLER, P., J. Biol. Chem., 236, 1836 18. FRIDOVICH, (1961). 19. GREENLEE, L., FRIDOVICH, I., AND HANI)LER, P., Biochemistry, 1, 779 (1962). 20. KNOWLES, P. F., GIBSON, J. F., PICK, F. M., AND BRAY, R. C., Biochem. J., 111, 53 (1969). I., Biochim. Biophys. Acta, 21. NAKAMURA, S., AND YAMAZAIZI, 189, 29 (1969).

A..


Superoxide dismutase was found in E. coli and has been isolated from that source. This result, although not conclusive in itself, is consistent with the position taken above, The enzyme obtained from E. co& although possessing a specific activity virtually identical with that of the mammalian enzyme, was nevertheless grossly different from it in many important respects. The molecular weight of the mammalian enzyme has been shown to be 32,600 (3), whereas that of the E. co& enzyme is 39,500. The mammalian enzyme is composed of two subunits whose association depends upon disulfide bonds,a while the association of the subunits of the E. co& enzyme deThe amino acid composipends upon noncovalent interactions. tions of these enzymes bear no relation to each other, and their solubility properties reflect these differences. The mammalian enzyme contains no methionine and no tyrosine (15), and its spectrum, which is very atypical for a protein, is strongly influenced by the spectrum of phenylalanine (3). In contrast, the E. coli enzyme contains 3 eq of methionine and 12 of tyrosine and exhibited the usual protein ultraviolet absorption band near 280 nm. The E. coli enzyme was unable to survive the acetone precipitation that was successfully applied during the purification of the erythrocyte superoxide dismutase. Finally, and most strikingly, the mammalian enzyme contains copper and zinc (17), whereas the E. co&i enzyme contains manganese. Only a few proteins and only one other enzyme have thus far The pyruvate been found to contain tightly bound manganese. carboxylase from chicken liver mitochondria has been shown to contain 3.6 atoms of tightly bound manganese per molecule of enzyme (27). It was not possible to dissociate reversibly the manganese of pyruvate carboxylase, and we have also been unable, thus far, to accomplish this with the superoxide dismufrom jack beans, called tase from E. coli. A hemagglutinin concanavalin A, has also been found to contain manganese (2%30), and in this case it was possible to demonstrate that a reversible loss of hemagglutinin activity accompanied the reversible removal of manganese (28). Concanavalin A has been assayed for superoxide dismutase and contains no activity. Another mangano-protein has recently been isolated from peanuts (31). It has been reported that E. coli possesses a specific transport system for the uptake of manganese (32). Since superoxide dismutase probably functions as an oxidoreductase, alternately being oxidized and reduced by its radical substrate, the metal ions associated with these enzymes may oscillate between two valence states during the course of the catalytic action. EPR studies on the mammalian superoxide dismutase have indicated that all of the copper in the resting enzyme can be accounted for as CU(II).~ Similar studies performed on the E. coli enzyme, however, do not lead to conclusive results as to the valence state of the manganese in the resting enzyme. The fact that no manganese signal could be observed in the native enzyme may be explained in terms of the metal being Since Mn(II1) is not a present as either Mn(I1) or Mn(II1). paramagnetic species, it would not be expected to give a signal. It could readily be reduced upon denaturation of the enzyme, however, giving rise to the characteristic Mn(I1) signal. Although Mn(I1) is a paramagnetic species, it need not produce a signal if it is tightly bound to protein and thereby immobilized within its ligand field. Such an effect on the EPR signal of bound Mn(I1) has been observed in the case of concanavalin A (30). One additional consideration, that of the visible chromo-

Vol. 245, No. 22

Issue of November

25, 1970

B. B. Keele, Jr., J. M. McCord,

22. RAJAGOPALAN, K. V., FRIDOVICH, I., AND HANDLER, P., J. Biol. Chem., 237, 922 (1962). 23. ALEMAN, V., AND HANDLER, P., J. Biol. Chem., 242,4087 (1967). 24. ROTILIO, G., CALABRESE, L., FINAZZI-AQRO, A., AND MONDOW, B., B&him. Biophys. AC& 198, 618 (1970). 25. NILSSON, R., PICK, F. M., AND BRAY, R. C., Biochim. Biophys. Acta, 192, 145 (1969). J. M., AND FRIDOVICH, I., J. Biol. Chem., 246, 1374 26. MCCORD, (1970). 27. SCRUTTON, M. C., UTTER, M. F., AND MILDVAN, A. S., J. Biol. Chem., 24l, 3480 (1966).

and I. Fridovich

28. SUMNER, (1936): 29. AGRAWAL,

J. B..

AND

6181 HOWELL.

S. F..

J. Biol. Chem..I 116. I 583

’

B. B. L., AND GOLDSTEIN, I. J., Arch. Bioch,em. Biojohus.. 124, 218 (1968). 30. RI&, 6. II., AN; CO&N, M., J. Biol. Chem., 246, 662 (1970). 31. DIECKERT, J. W., AND ROZACKY, E., Arch. Biochem. Biophys.,

134, 473 (1969). 32. SILVER, S., AND KRALOVIC,

Commun., 34, 640 (1969). 33. SASTRY, G. S., HAMM, R. E.,

M. L., Biochem. AND

POOL,

Biophys.

K. H., And.

Res.

Chem., 4l,

857 (1969).
