ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 360-364, 1979

Manganese-Containing Reversible DENNIS Department

of Biochemistry,

Superoxide Dismutase from Escherichia Resolution and Metal Replacement+ E. OSE AND IRWIN Duke

University

Medical

co/i:

FRIDOVICH Center,

Durham,

North

Carolina

27710

Received September 11, 19’78; revised December 11, 1978 Exposure of the manganese-containing superoxide dismutase ofE.scherichia coli to pH 3.2, in the presence of 0.7 M guanidinium chloride, causes a rapid loss of manganese and of activity. The apoenzyme so produced can be reconstituted by addition of MnCl, followed by neutralization. In contrast, manganese cannot be restored to the apoenzyme by adding MnCl, after neutralization. The reconstituted enzyme is indistinguishable from the native enzyme in terms of its catalytic activity or electrophoretic behavior on polyacrylamide gels. Co(U), Ni(II), Zn(II), Fe(U), or Cu(I1) could compete with Mn(I1) during reconstitution ofthe apoenzyme. In the cases of Co(II), Ni(II), and Zn(II), it was shown that, in preventing reconstitution by Mn(II), they were themselves bound to the enzyme in stoichiometric amounts, in place of Mn(I1). The binding of Fe(I1) was also explored and was distinct in that the enzyme could bind more than stoichiometric amounts of this metal. None of the derivatives, in which Mn(I1) had been replaced by another metal, were catalytically active. Nevertheless, these derivatives could be again resolved by exposure to acid guanidinium chloride and could then be converted back into the active holoenzyme by neutralization after addition of MnCl,. It appears that the active site of this enzyme can accommodate and can tightly bind several metals other than manganese, but exhibits activity only with manganese. It also appears that movement of metal out of or into this site is only feasible at low pH and in the presence of a chaotropic agent. A substantial amount of the cobalt-substituted enzyme was prepared and its optical properties were recorded.

The manganese-containing superoxide dismutase (MnSOD)* of Escherichia coli is not synthesized in the absence of molecular oxygen and, in its presence, its rate of synthesis is elevated by any circumstances which call forth increased intracellular production of 0, (4-7). This is an adaptive response and exposure to oxygen, under conditions which prevent MnSOD synthesis, results in loss of viability (4-7) and in structural damage which can be visualized under the electron microscope (4). The MnSOD thus appears to provide an essential defense against the potential cytotoxicity of 0;. 1 This work was supported by research grants from the National Institutes of Health, Be+.hesda, Md., the United States Army Research Office, Research Triangle Park, N. C., Merck, Sharp and Dohme, Rahway, N. J., and the Glenn Foundation, Manhassett, N. Y. 2 Abbreviations used: MnSOD, Manganese-containing superoxide dismutase. 0003-9861/79/060360-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

The manganese in this enzyme participates in the catalytic mechanism by undergoing a univalent redox cycle (8-10). This manganese is tightly bound and resists removal by chelating agents at neutrality, but it can be removed by exposure to guanidinium chloride or to urea(ll-14) at low PH.” The apoenzyme, so produced, can be reconstituted by addition of MnCl,, under the conditions which labilized the manganese, followed by a return to neutrality and a removal of the chaotropic agent. The apoenzyme cannot, however, be reconstituted by addition of MnCl, at neutral pH. In the case of the E. coli enzyme Co(H), Zn(II), or Ni(I1) was shown to prevent reconstitution by Mn(I1) and in the case of Co(H) this effect was associated with stoichiometric binding 3 Our first report of the reversible resolution of the MnSOD (7) erroneously indicated that the pH was 7.8 during removal of the metal. This was corrected in J. Biol. Chem. 251, 4796 (1976). 360 E. coli

REPLACEMENT

OF MN IN E. co/i SUPEROXIDE

of cobalt in place of manganese (11). In the case of the enzyme from B. stearothermophilus (15) Co(H), Fe(II), Ni(II), and Cu(I1)

were all found able to bind the apoenzyme, in place of manganese. In no case did any metal, save manganese, restore activity to the apoenzyme. We now present a full account of the reversible resolution of the E. coli MnSOD and of replacements of the active site manganese with a variety of other metals. MATERIALS

AND

METHODS

Ultrapure guanidinium chloride was obtained from Heico or from Schwarz/Mann. Reagent grade Trizma Base and %hydroxyquinoline hemisulfate were purchased from Sigma. Metal salts were used as reagent grade chlorides. Polypropylene tubes were used for most manipulations. Protein was assayed with ninhydrin after alkaline hydrolysis (I) or by the Lowry method (2) with bovine serum albumin as a standard. Superoxide dismutase activity was measured in terms of the inhibition of the superoxide-dependent reduction of cytochrome c by xanthine plus xanthine oxidase (31. Metal analyses were done by atomic absorption spectrophotometry with a Perkin-Elmer Model 107. Fisher atomic absorption standard solutions were used to calibrate the instrument prior to each use. All assays were repeated from three to six times and agreed within +5%. The enzyme was prepared from Escherichia coli as described previously (11) and was homogeneous by polyacrylamide gel electrophoresis (3a). RESULTS

AND

DISCUSSION

Effect of Low pH and of Guanidinium Chloride

When MnSOD at 0.1 + 0.2 mg/ml was exposed to 0.7 M guanidinium chloride, 5 mM Tris-HCl at pH 3.2 and at 4”C, it lost all activity within 1 min. If MnCl, was then added to 1 x 10e5 M and the pH was raised to 7.8, by cautious titration with NaOH, followed by lo-fold dilution with 50 mM TrisHCl, pH 7.8, essentially full activity was restored. Both the 0.7 M guanidinium chloride and the low pH were needed to affect rapid release of manganese from the enzyme. The apoenzyme could be dialyzed against 0.1 M guanidinium chloride, 5 mM Tris-HCl, pH 3.2, prior to reconstitution by addition of MnCl, and elevation of pH, without substantially changing the amount of activity recovered. There was some time-dependent

DISMUTASE

361

irreversible loss of protein and of reconstitutable activity during this dialysis. When apoenzyme was applied to a Sephadex G-75 column equilibrated with 0.7 M guanidinium chloride, 5 mM Tris-HCl, pH 3.2, it eluted in two peaks, one of which was an aggregate, as evidenced by elution in the excluded volume; while the other eluted as did the native enzyme in the presence of 0.7 M guanidinium chloride, but at pH 7.8. Both the polymeric and the dimeric forms of the apoenzyme were able to regenerate active, native, enzyme when treated with MnCl, at pH 3.2 and adjusted back to pH 7.8. When native, apo and reconstituted enzymes were subjected to electrophoresis on polyacrylamide gels, they exhibited identical mobilities. It is likely that the polymeric form of the apoenzyme, seen at pH 3.2, dissociated to the dimeric form when exposed to the slightly alkaline buffer used for this electrophoresis. CompetitioTz between Manganese and Other Metals

Aliquots of MnSOD were converted to the apoprotein as described above. MnCl, at 1 x lo-” M, in the absence or in the presence of other metal chlorides, was then added. The pH was readjusted to pH 7.8 and the samples were assayed after lo-fold dilution with Tris buffer at pH 7.8. As shown in Table I Co(II), Ni(II), Zn(II), Fe(H), and Cu(I1) completely prevented reconstitution by Mn(II), whereas La(II1) and Pb(I1) were weakly effective competitors. Li(I), Co(II), Mg(II), and Tl(1) were without effect. It appeared likely that metals which prevented reconstitution by Mn(I1) did so by competing for the active site. It would also follow that occupancy of that site by any of the metals, which prevented reconstitution by Mn(II), yielded a catalytically inactive product. The possibility that Co(H), Ni(II), or Zn(I1) prevented reconstitution by Mn(II), by irreversibly inactivating the protein, was explored. Thus, as shown in Table II apoenzyme reconstituted with MnCl, in the presence of a loo-fold excess of Co(H), Ni(II), or Zn(I1) and inactive because of the effect of these competing metals, could subsequently be converted back into apoenzyme and reac-

362

OSE

AND

FRIDOVICH

TABLE

I

COMPETITION BETWEENMETALCATIONS

Sample

Mn(I1)

(M x 105)

Maximum recoverable activity (o/o)

(M X 10")

Competition

Native enzyme Apoenzyme”

690

Manganese-containing superoxide dismutase from Escherichia coli: reversible resolution and metal replacements.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 360-364, 1979 Manganese-Containing Reversible DENNIS Department of Biochemistry,...
405KB Sizes 0 Downloads 0 Views