THE JOURNAL OP BUXOGICAL CHEMISTRY Vol. 253, No. 14. Issue of duly 25. pp. 5220-5223, 1978 Printed in U.S.A.

A New Iron-containing Superoxide Dismutase from Escherichia coZi*

(Received for publication, July 14, 1977, and in revised form, April 4, 1978)

Harry W. Dougherty, Sharon J. Sadowski, and Edward E. Baker From the Merck Institute for Therapeutic Research, Rahway, New Jersey 07065

Escherichia coli B, grown under aerobic conditions, contains at least three distinct superoxide dismutases, which can be visualized on polyacrylamide gel electro- pherograms of crude soluble extracts of the sonically disrupted cells. Of these, the slowest migrating and the fastest migrating, respectively, have previously been isolated and characterized as manganese-containing and iron-containing enzymes. The enzyme form with medium electrophoretic mobility has now been purified to homogeneity. Its molecular weight is approximately 37,000 and it contains 0.8 atoms of iron/molecule and only negligible amounts of manganese. Like other iron- containing superoxide dismutases and unlike the cor- responding manganienzymes, it is inactivated by EDTA plus HzOz. Its specific activity is comparable to that of the other superoxide dismutases of E. coli.

Two types of subunits could be distinguished upon electrophoresis in the presence of sodium dodecyl sul- fate. One of these migrated identically with the subunit obtained from the manganisuperoxide dismutase, while the other similarly appeared identical with the subunit from the ferrisuperoxide dismutase. This newly iso- lated enzyme thus appears to be a hybrid of the other two forms. In support of this conclusion, we observed that ultrafiltration or storage of the new superoxide dismutase gave rise to the mangani- and ferrienzymes on disc gel electrophoresis or isoelectric focussing.

Escherichia coli B, when grown in the presence of oxygen, contains several forms of superoxide dismutase. Only two of these have heretofore been explicitly recognized, isolated, and characterized. One of these contains manganese (1) and mi- grates rather slowly during electrophoresis on polyacrylamide gels, at pH 8.9 (2). The other contains iron and migrates more rapidly (3). These enzymes each contain 1 atom of metal (manganese or iron) per molecule and have comparable cat- alytic activities, molecular weights, and subunit compositions, all of which, along with their physiological functions, has repeatedly been reviewed (4-9). Another superoxide dismu- tase of E. coli, which has an electrophoretic mobility midway between that of the manganisuperoxide dismutase and the ferrisuperoxide dismutase, was first noticed in this laboratory and has been the subject of extensive investigation. This report describes the purification and characterization of this new form, which we shall call hybrid ferrisuperoxide dismu- tase.

MATERIALS AND METHODS

E. coli B was grown to late log phase on a glucose/salts medium, under vigorous aeration (3). Cells were collected by centrifugation

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

and the cell paste was either used immediately or stored at -20°C until needed. Protein was estimated by the Lowry procedure (IO). Superoxide dismutase was assayed on the basis of its ability to inhibit the autooxidation of pyrogallol (11). An enzyme activity unit, defined as the quantity of enzyme required for 50% inhibition of the initial rate of pyrogallol oxidation, was found to be equivalent to 1.7 pg of purified enzyme. Electrophoresis on pH 7.5% polyacrylamide gels was performed at pH 9.0 on a Buchler Instrument Co. apparatus, as described in the Ortec Co. manual. Isoelectric focussing on polyacryl- amide gels was performed at 400 V for 16 h on the same apparatus (12). Gel electropherograms were stained for protein (13) or for superoxide dismutase activity (2). Because the activity stain depends on the ability of the enzyme to maintain a zone of achromaticity, maximum sensitivity was obtained by prolonging the gel development time to increase the contrast between the bands and the background. Under these conditions, conclusions relative to the specific activity of bands on the gels were not feasible. Electrophoresis in the presence of sodium dodecyl sulfate was performed according to Weber and Osborn (14). Slab gel isoelectric focussing was performed with an LKB Instrument Co. Multiphor Apparatus, as described in the LKB manual. Iron and manganese were estimated by atomic absorption spectrophotometry with a Perkin-Elmer model 303.

RESULTS

Varieties of Superoxide Dismutase in E. coli B-Crude soluble extracts of E. coli exhibited several distinct bands of activity on polyacrylamide gel electropherograms, as shown in Fig. 1. The slowest migrating or least anionic of these corresponds to the manganisuperoxide dismutase of this or- ganism (1) and the most strongly anionic corresponds to ferrisuperoxide dismutase. Our interest was roused by the very evident middle band, which had not yet been studied.

Purification of Hybrid Ferrisuperoxide Dismutase-Cell paste (200 g) was suspended in 1.0 liter of 50 mM potassium phosphate, pH 7.8. The suspended cells were disrupted by ultrasound, in 200-ml aliquots applied with a Branson Sonifier operated at a power setting of 150 watts. The temperature was kept at 10°C or lower by cooling in an ice/salt bath and by sonication in two 6-min bursts. Cell debris was removed by centrifugation at 13,000 X g for 20 min and the sediment was extracted with 400 ml of buffer. The pooled extracts were treated with 2.5 g/100 ml of streptomycin sulfate. After stir- ring at 25°C for 30 min, the precipitate was removed by centrifugation at 13,000 x g for 15 min. Solid (NH&SO4 was added to 50% saturation at 25°C and after stirring and chilling in ice for 1 h, the precipitate was removed at 13,000 x g for 20 min. (NH&SO4 was added to the supernatant solution to 85% saturation at 25°C and after 16 h at 4°C the precipitate was collected by centrifugation and was dissolved in 300 ml of 5 mu potassium acetate at pH 5.5. Residual ammonium sulfate was removed by dialysis against. four 30-liter changes of this dilute acetate buffer, over a period of 48 h. The solution was clarified by centrifugation and was run into a column (4.7 x 18 cm) of CM32 (Whatman Co.) which had been equilibrated with the 5 mu acetate at pH 5.5. The column was then washed with 500 ml bf 2 mM potassium acetate at pH 5.5 and protein- containing fractions were pooled and were found to contain

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A New Iron-containing Superoxide Dismutase from E. coli 5221

ferrisuperoxide dismutase and hybrid ferrisuperoxide dismu- tase. Manganisuperoxide dismutase did adhere to the CM32 column and it could be eluted with a linear gradient of potassium acetate from 2 to 200 mu at pH 5.5.

The fractions containing ferrisuperoxide dismutase and hy- brid ferrisuperoxide dismutase were dialyzed in the cold against 30 liters of 5 ells potassium phosphate, pH 7.8 and were applied to a column (3.5 X 17.5 cm) of DE32 which had been equilibrated with 2 II~M potassium phosphate at pH 7.8. The column was then washed with 100 ml of the equilibrating buffer and linear gradients from 2 to 20 mu and then from 20 to 200 mu potassium phosphate at pH 7.8 were applied. The first of these gradients eluted hybrid ferrisuperoxide dismu- tase and the second gradient eluted ferrisuperoxide dismutase.

Fractions containing hybrid ferrisuperoxide dismutase were pooled and the enzyme was precipitated by adding solid (NH&SO, to 90% saturation. The precipitate was collected by centrifugation, was dissolved in 2 mru potassium phosphate, pH 7.8 and was dialyzed against two 2.0-liter changes of this buffer over a period of 18 h in the cold. The protein solution was absorbed into a column (3.5 X 16.5 cm) of DE32 equili- brated with this buffer and was eluted with a linear gradient, from 2 to 20 II~M, of this buffer. Fig. 2 presents the appearance of acrylamide gel electropherograms of the fractions generated during this procedure. It is apparent that excellent separations were achieved. Fig. 3 shows the profile of elution of protein and hybrid ferrisuperoxide dismutase from the DE32 column. Fractions containing hybrid ferrisuperoxide dismutase were pooled (Fractions 66 to 82) and the enzyme was collected by centrifugation after adding (NH&S04 to 90% saturation and was stored under 90% (NH&S04 at -2O’C. It evidenced no loss of activity over a period of 6 months under these condi- tions.

When the purified enzyme was again examined by disc gel electrophoresis after several months storage, the superoxide dismutase activity was no longer found in a single band as seen in Fig. 2. The preparation exhibited superoxide dismutase

FIG. 1. Disc gel electropherogram (7.5% acrylamide, pH 9) of ap- proximately 400 pg of soluble protein from freshly sonicated E. cob stained for superoxide dismutase activity (anode to the right of figure).

FIG. 2. Disc gel electropherograms, 7.5% acrylamide, pH 9, of fractions eluted from the second DE32 column purification of hybrid ferrisuperoxide dismutase stained for protein and superoxide dismu- tase activity. See Fig. 3. Fraction 52 stained for superoxide dismutase (A, 20 pg) and protein (B, 20 pg); Fraction 67 stained for superoxide dismutase (C, 4 pg) and protein (D, 10 gg); Fraction 70 stained for superoxide dismutase (E, 4 pg) and protein (F, 20 pg).

activity bands (Fig. 4) which appeared identical with those of ferri- and manganisuperoxide dismutase. This observation suggested that the hybrid ferrisuperoxide dismutase might be a hybrid, containing one subunit from ferrisuperoxide dismu- tase and one from manganisuperoxide dismutase. The follow- ing studies, unless indicated otherwise, were performed on the freshly eluted, homogeneous enzyme. The results of the pu- rification are summarized in Table I. In four repetitions, the final recoveries, expressed as the percentage of the total initial activity f the relative error of the mean, were: ferrisuperoxide dismutase, 29 f 10%; hybrid ferrisuperoxide dismutase, 7 & 2%; and manganisuperoxide dismutase, 9 f 1%. In total, ap- proximately 45% of the initial activity was recovered.

FIG. 3. DE32 column fractions, hybrid ferrisuperoxide dismutase. The graph depicts the absorbance at 280 nm (M) and the units superoxide dismutase per ml (p) for the fractions eluted by a 2 mM potassium phosphate buffer (pH 7.8), linear gradient after absorbing the hybrid ferrisuperoxide dismutase combined fraction from the first DE32 column. Fractions 66 to 82 were pooled.

A

!3

FIG. 4. Disc gel electropherograms of the pooled hybrid ferrisu- peroxide dismutase fractions eluted from the second DE32 column (Fractions 66 to 82) after storage for 6 months. A: 18 pg, stained for superoxide dismutase activity. B: 18 pg. stained for protein.

TABLE I Purification of E. coli suDeroxide dismutases

SkP Activity Yield Protein Specific activity

Sonicate + wash (235 g cells)

Streptomycin superna- tant

50 to 85% ammonium sul- fate cut (dialyzed uer- sus 5 mM UC)

CM32 column washout CM32 column gradient

(manganisuperoxide dismutase)

Dialyzed CM32 washout DE32 column (2 to 20 mM

KP, gradient) (hybrid ferrisuperoxide dismu- tase)

DE32 column (20 to 200 mu KP, gradient) (fer- risuperoxide dismu- tase)

Rechromatography of pooled DE32 fractions (2 to 20 mM KP,) (hy- brid ferrisuperoxide dismutase)

total units 266,900

214,200

187,200

158,600 59 3.7 61.0 26,300 10 0.54 190.0

143,000 54 3.5 59.0 20,800 8 0.53 137.0

105,000

10,600 4 0.21 350.0

% m&?/ml 21.0

units/mg 8.0

80

70 8.0 52.0

39 0.55 483.0

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5222 A New Iron-containing Superoxide Dismutase from E. coli

Purity and Molecular Weight-The hybrid ferrisuperoxide dismutase obtained by the procedure described above mi- grated as a single component during polyacrylamide gel elec- trophoresis at pH 4.0, 7.0, and 9.0 (pH 9.0 gels, Fig. 2; pH 4.0 and 7.0, not shown). Isoelectric focussing of the hybrid ferri- superoxide dismutase did demonstrate a minor contamination with manganisuperoxide dismutase but not with ferrisuperox- ide dismutase (Fig. 5).

0.8 atoms of iron and only 0.07 atoms manganese per molecule of enzyme. Since these preparations were detectably contam- inated with manganisuperoxide dismutase, but not with fer- risuneroxide dismutase. it is clear that iron is a comnonent of

Hybrid ferrisuperoxide dismutase was dialyzed against 50 mu potassium phosphate at pH 7.8 and was then brought to sedimentation equilibrium at 23,950 rpm in a Beckman model E analytical ultracentrifuge. When the logarithm of protein concentration was graphed as a function of the square of the distance from the center of rotation a straight line was ob- tained. Hybrid ferrisuperoxide dismutase was thus homoge- neous with respect to those properties affecting sedimentation such as size, shape, and density. The molecular weight, cal- culated according to Yphantis (15), on the basis of an assumed partial specific volume of 0.725, was 37,000.

Characterization as an Iron-containing Superoxide Dis- mutase-Fig. 6 presents the optical spectra of hybrid ferrisu- peroxide dismutase. Its ultraviolet absorption spectrum was that of a typical protein with a peak at 280 nm. The molar extinction coefficient at 280 nm was 5.1 X lo4 Me1 cm-‘. The absorption in the visible range was similar to that previously reported for the ferrisuperoxide dismutase from E. coli (3). Metal analysis by atomic absorption spectrometry revealed

FIG. 5. Isoelectric focus gels with ampholyte pH gradient 2.5 to 7.25 (right to left). A: 20 pg of hybrid ferrisuperoxide dismutase, stained for protein. B: 4 pg of hybrid ferrisuperoxide dismutase, stained for superoxide dismutase activity. C: 6 pg of ferrisuperoxide dismutase, stained for superoxide dismutase activity. D: 1 pg of manganisuperoxide dismutase; stained for superoxide clismutase ac- tivity.

0.6 -

0.7 -

0.6 -

8 c 0.5 - x b 9 0.4 -

0.3 -

0.2 -

0.1 -

0.0 ’ 250 300 350 400 450 500

Nonometrrt FIG. 6. Absorption spectrum of E. coli hybrid ferrisuperoxide dis-

mutsse at 25’C. The concentrations of protein in the ultraviolet and visible ranges were 0.43 and 8.6 mg/ml (0.012 and 0.23 mu), respec- tively, in 50 ITIM potassium phosphate, pH 7.8, with a l-cm light path.

hybrid ferrisuperoxide dismutase and manganese is’ not. Hy- brid ferrisuperoxide dismutase, like both iron- or manganese- containing superoxide dismutase, was insensitive to 1 mu cyanide.

Iron-containing superoxide dismutases, unlike the man- ganese-containing enzymes, have been found to be inactivated by 0.5 mu H202 plus 0.1 mM EDTA (16). This criterion was applied to hybrid ferrisuperoxide dismutase and the results are shown in Fig. 7. Ferrisuperoxide dismutase was inactivated with tl12 = 12.3 min and hybrid ferrisuperoxide dismutase was similarly inactivated with t1/2 = 24.6 min. In contrast, man- ganisuperoxide dismutase was very resistant and 85% of its activity was retained after 24 h of exposure to the H202 + EDTA mixture.

Disc gel electrophoresis of the soluble proteins from E. coli sonicate after treatment with Hz02 plus EDTA for 90 min revealed that ferrisuperoxide dismutase and hybrid ferrisu- peroxide dismutase were specifically inactivated.

Hybrid Ferrisuperoxide Dismutase as a Hybrid- Electrophoresis of hybrid ferrisuperoxide dismutase, mangan- isuperoxide dismutase, and ferrisuperoxide dismutase in the presence of sodium dodecyl sulfate demonstrated that hybrid ferrisuperoxide dismutase contained two distinct subunits. Both mangani- and ferrisuperoxide dismutase display a single subunit band on sodium dodecyl sulfate gel electrophoresis. When aldolase was used as an internal standard, to allow precise alignment of the gels, it was clear that one of the subunits of the hybrid ferrisuperoxide dismutase migrated identically with the subunits of manganisuperoxide dismutase and the other with the subunits of ferrisuperoxide dismutase. This is shown in Fig. 8. It appeared possible that hybrid ferrisuperoxide dismutase was a hybrid composed of one sub- unit of manganisuperoxide dismutase and one subunit of ferrisuperoxide dismutase.

If hybrid ferrisuperoxide dismutase were a hybrid, then it might be expected to give rise to the other two superoxide dismutases by separation and resegregation of subunits. Long term storage of the enzymes was sufficient to allow the expres- sion of both ferri- and manganisuperoxide dismutase as de-

Time (min)

FIG. 7. Effect of hydrogen peroxide on E. coli ferrisuperoxide dismutase (M) manganisuperoxide dismutase (- - -), and hybrid ferrisuperoxide dismutase (m). The superoxide dismutases (about 100 units) were each incubated in 10 mu potassium phosphate (pH 7.8) with 0.1 mM EDTA and 0.5 mu Hz02 at 25°C. At intervals, aliquota of the treated enzyme were assayed under the standard conditions for activity.

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A

B

A New Iron-containing Superoxide Dismutase from E. coli 5223

wonder why the hybrid ferrisuperoxide dismutase, being a hybrid, contains predominantly iron rather than manganese

, or a mixture of iron and manganese. One answer could lie in the relative availability of iron and manganese in E. coli. Thus, the heterodimers could have nearly equal affinity for iron and manganese, but a relative surplus of iron could nevertheless assure that the completed hybrid ferrisuperoxide dismutase contained predominantly iron.

D i

FIG. 8. Sodium dodecyl sulfate disc gel electrophoresis of E. coli superoxide dismutases. The gels were stained with 1% fast green in a solution with 10% acetic acid and 45% methanol. The cathode end of each gel is at the left and the dye band is indicated by the India ink mark at the anode end. A, 25 pg of aldolase and 15 pg of hybrid ferrisuperoxide dismutase. B, 25 pg of aldolase and 39 pg of ferrisu- peroxide dismutase. C, 25 pg of aldolase and 20 pg of manganisu- peroxide dismutase. D, 20 pg of hybrid ferrisuperoxide dismutase.

scribed above. Generation of active manganisuperoxide dis- mutase by this process would be dependent upon acquisition of manganese from the medium, since hybrid ferrisuperoxide dismutase does not contain maganese. Manganisuperoxide dismutase, hybrid, ferrisuperoxide dismutase, and ferrisuper- oxide dismutase exhibit very different isoelectric points during isoelectric focusing and are thus well separated by this process. Yet hybrid ferrisuperoxide dismutase, excised and eluted as a single band (~15.44) from a shallow pH gradient slab gel after isoelectric focussing, was found to contain both manganisu- peroxide dismutase (p1 6.64) and ferrisuperoxide dismutase (~14.93) after concentration by ultrafiltration and subsequent analysis by polyacrylamide gel electrophoresis or isoelectric focussing.

It has recently been reported (28) that anaerobically grown E. coli K12 contain only ferrisuperoxide dismutase and that exposure to air induced the synthesis of both manganisu- peroxide dismutase and hybrid ferrisuperoxide dismutase. It was furthermore seen that the induction of the hybrid ferri- superoxide dismutase was not observed until manganisu- peroxide dismutase had been induced and, after induction, the hybrid ferrisuperoxide dismutase appeared at levels lower than either the manganisuperoxide dismutase or the ferrisu- peroxide dismutase. These results can now be interpreted in terms of the hybrid structure of hybrid ferrisuperoxide dis- mutase. Thus, there could be no hybrid ferrisuperoxide dis- mutase until the synthesis of manganisuperoxide dismutase was initiated after exposure to oxygen. Furthermore, if hom- oaffinity exceeds heteroaflinity, then the production of hybrid ferrisuperoxide dismutase should not precede that of mangan- isuperoxide dismutase.

Acknowledgment-We are indebted to Dr. Irwin Fridovich for his many invaluable contributions to this work and report.

REFERENCES

1.

2.

3.

4.

5. 6.

Keele, B. B., Jr., McCord, J. M., and Fridovich, I. (1970) J. Biol. Chem. 246,6176-6181

Beauchamp, C. O., and Fridovich, I. (1971) Anal. Biochem. 44, 276-287

Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908

Fridovich, I. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 35-97

HaUiweII, B. (1974) New Phytol. 73, 1075-1086 Bors, W., Saran, M., Lengfelder, E., Sp5tt1, R., and Michel, C.

(1974) Curr. Top. Radiat. Res. Q. 9,247-309 Fridovich, I. (1975) Annu. Rev. Biockem. 44,147-159 Asada, K. (1976) J. Jpn. B&hem. Sot. 48,226-257 Fridovich, I. (1976) Adv. Exp. Med. Biol. 74.530-539 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randah, R. J.

(1951) J. Biol. Chem. 193,265-275 Marklund, S., and Markhrnd, G. (1974) Eur. J. Biochem. 47,

469-474

DISCUSSION

Hybridization of subunits is a well known phenomenon. The best studied case probably being the generation of five isozymes of lactic dehydrogenase from two types of subunits (17). The copper-zinc superoxide dismutases, which are di- merit, readily form hybrids. This was seen in humans heter- ozygous for a rare form of this enzyme (18). Because of the evolutionary conservation of this enzyme, interspecies hybrids have also been generated (19,20). Although the ferrisuperox- ide dismutase and manganisuperoxide dismutase of E. coli appear to be very different because they depend upon different prosthetic metals, they are actually closely related and exhibit a striking degree of sequence homology (21). That being the case, hybridization of subunits derived from these two en- zymes is not surprising. We have now presented data which indicate that hybrid ferrisuperoxide dismutase is such a hy- brid.

The manganisuperoxide dismutase from E. coli (l), Bacil- lus stearothennophilus (22), and Pleurotus olearius (23) all contain only 1 manganese atom per two subunits and the same stoichiometry between metal and subunits has been seen for ferrisuperoxide dismutase from several sources (3, 24-27). Hybrid ferrisuperoxide dismutase also fits this pattern in that it contains one metal per two subunits. Since mangan- isuperoxide dismutase and ferrisuperoxide dismutase appear to be homodimers (21), we must suppose that the metal binding site is created by the association of the subunits. This implies that separated subunits should have much less affinity for metal than the dimeric form. We may then very well

7. 8. 9.

10.

11.

12. Wrigley, C. W. (1971) Methods Enzymol. 22,559-564 13. Davis, B. J. (1964) Ann. N. Y. &ad. Sci. 121,494-427 14. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 15. Yphantis, D. A. (1964) Biochemistry 3.297-317 16. Asada, K., Yoshikawa, K., Takahashi, M., Maeda, Y., and En-

manii. K. (1975) J. Biol. Chem. 260.2801-2807 17.

18. 19. 20. 21.

ChiIson,‘O. P., Costello, L. A., and Kaplan, N. 0. (1965) Biochem- istry4,271-281

Beckman, G. (1973) Hereditas 73,305-310 Khan, P. M. (1971) Arch. Biochem. Biophys. 145,470-483 Tegelstrom, H. (1975) Hereditas 81, 185-198 Steinman, H. M., and HiiI, R. L. (1973) Proc. Natl. Acad. Sci.

U. S. A. 70,3725-3729 22.

23.

24.

25.

26. 27.

28.

Bridgen, J., Harris, J. I., and Northrop, F. (1965) FEBS Lett. 49, 392-395

LaveUe, F., Durosay, P., and MicheIson, A. M. (1974) Biochimie (Paris) 56,451-k%

Misra, H. P., and Keele, B. B., Jr. (1975) Biochim. Biophys. Acta 379,418-425

Lumsden, J., Commack, R., and HaII, D. 0. (1976) Biochim. Biophys. Acta 438, 380-392

Yamakura, F. (1976) Biochim. Biophys. Acta 422,280-294 Bannister, J. H., Bannister, W. H., and Anastasia, A. (1976) Znt.

J. Biochem. 7,541~546 Hassan, H. M., and Fridovich, I. (1977) J. Bacterial. 129,

1574-1583

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H W Dougherty, S J Sadowski and E E BakerA new iron-containing superoxide dismutase from Escherichia coli.

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