Eur. .I. Biochcm. 64, 465-470 (1976)

Cobalt Bovine Superoxide Dismutase Reactivity of the Cobalt Chromophore in the Copper-Containing and in the Copper-Free Enzyme Lilia CALABRESE, Dina COCCO, Laura MORPURGO, Bruno MONDOVI, and Giuseppe ROTILIO Institute of Biological Chemistry and Institute of Applied Biochemistry, University of Rome, and Center for Molecular Biology of Consiglio Nazionale delle Ricerche, Rome (Received August 11, 1975/February 3, 1976)

1. The reactivity of the zinc site of bovine superoxide dismutase has been probed by observing optical and electron paramagnetic resonance changes, under several conditions, of the Co(I1)-substituted protein. 2. Only in the absence of copper are the optical and electron paramagnetic resonance spectra of the cobalt chromophore appreciably affected by alkaline pH or by cyanide. With both reagents the reaction with the copper-containing protein appears to involve the water molecule bound to the copper and does not affect the magnetic coupling between copper and cobalt. 3. The reaction of cyanide with the copper-free Co(I1) protein leads to a slow detachment of cobalt from the protein as pentacyanocobalt. An oxygen adduct forms in air, analogous to that described in Co(I1)carbonic anhydrase (Haffner, P. H. and Coleman, J. E. (1975) J . Biol. Chem. 250,996- 1005.) 4. Acid titration modifies the Co(1I) spectra in the same way in the Cu-containing and in the Cu-free protein and brings about uncoupling of the Co(I1) - Cu(I1) system. Protonation of histidine-61 on the zinc facing nitrogen is suggested. 5. H202 modifies the cobalt chromophore only in the presence of copper. Magnetic coupling between Cu(I1) and Co(I1) seems to be still present after H202 inactivation of the enzyme. The Co(I1) derivatives of copper-zinc bovine superoxide dismutase have proved very useful in the study of the zinc site of the enzyme. They have been obtained either by exchange dialysis [l], which results in variable extents (always lower than loo./,) of Co(I1) substitution, or by readdition of 2 Co(I1) per protein dimer ( M , z 32000) to the fully metal-depleted enzyme [2]. Both procedures give active molecules: 100% [l] and 60%) [2] of the activity of the native zinc enzyme respectively. Both types of Co(1I) derivatives show magnetic coupling between the cobalt and copper sites [l-3], such as would be expected if the two metals were bridged by a common ligand [3]. Reduction or absence of the copper gives derivatives with identical EPR [2] and optical [2,3] Co(I1) spectra, which are typical of distorted tetrahedral configuration. Moreover, evidence has been obtained that the Co(I1) site is less exposed to the solvent than the Cu(I1) site, as indicated by EPR [3] and pulsed nuclear magnetic resonance [4] measurements. All results are in line Ahhrrviution. EPR, electron paramagnetic resonance. Enzyme. Superoxide dismutase or superoxide: superoxide oxido-

reductase (EC 1.15.1.1).

with recent X-ray crystallography data, obtained with the native zinc enzyme [5], thus confirming that Co(I1) is a reliable probe for the zinc site. In the present paper are reported new data concerning the reactivity of the Co(I1) chromophore and some aspects of the interdependence of the Co(I1) and Cu(I1) sites.

MATERIALS AND METHODS All materials were of reagent grade and were used without further purification. The enzyme was purified according to McCord and Fridovich [6]. Co(I1)Cu(II) superoxide dismutase was prepared according to Calabrese et al. [I]. The Cu(I1)-free cobalt derivative was prepared by the same procedure using copper-free protein [7] as starting material. Metal analyses were performed with a Hilger & Watts Atomspek Model H 1170 atomic absorption spectrometer equipped with a Varian Techtron heated-graphite atomizer Model 61. pH measurements were obtained with a type PHM4c Radiometer pH-meter. Optical spectra were recorded on a Beckman DK-2A spectrophotometer. EPR spectra were recorded at approxi-

Reactivity of Cobalt in Cobalt Superoxide Dismutase

466

mately 9.24 GHz with a Varian Model V-4502-14 spectrometer. Temperatures between 4 K and 100 K were obtained with an Air Products and Chemicals LT-3-110 liquid-transfer Cryo-Typ refrigerator with automatic temperature controller. RESULTS

Reaction of Co(II) Superoxide Dismutase with Cyunide Azide does not react with Co(I1) either in the Cu(1I)- Co(11) or in the copper-free, Co(I1) protein, even at very high excesses. Cyanide has already been reported to bind preferentially to copper in the Co(11)-Cu(I1) protein [ 3 ] , and also to bind to the Co(I1) protein only at rather high molar ratios [2]. The reactions with cyanide were reinvestigated in greater detail. In the Co(I1)-Cu(I1) protein the Co(I1) optical spectrum is unaffected by cyanide, even at molar ratios as high as 50, and the reaction with the copper does not affect the magnetic coupling between the two sites. On the other hand in the copper-free Co(I1) protein anaerobic addition of 1 equiv. of cyanide at pH 8.9 leads to a slow decrease of the visible absorption, which levels off in about 30 min (Fig. 1, curve b); 4 equiv. of cyanide are required for almost complete bleaching. A larger amount is required at pH 7.0 indicating that CN- is the reacting species, since the Co(I1) chromophore is insensitive of pH in this range. However, removal of cyanide by acidification does not reverse the reaction. The aerobic titration with cyanide follows a similar pattern, but a new shoulder at 380nm is formed in the optical spectrum (Fig. 1, curve c). The reaction is not reversed by dialysis, though the 380-nm band disappears. Atomic absorption controls show that a considerable amount of cobalt is lost after dialysis. In Fig.2 is reported the EPR spectrum of the protein anaerobically reacted with 4 equiv. of cya-

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nide (curve b) and after admission of air (curve c). The signal around g = 2 in curve (b) is reminiscent of that of pentacyano-Co(I1) [8], while the signal of curve (c) resembles that of the oxygen adduct of lowspin Co(I1)-carbonic-anhydrase . cyanide complex [8]. The reaction with oxygen is not reversible as the original signal is not restored by subsequent degassing. In the aerobic reaction with cyanide the EPR signal of the oxygen adduct is only detectable for a short period of time and is not related to the appearance of the 380-nm band.

Injlumce o j p H on Co ( I I ) Superoxide Dismutuse The optical and EPR spectra of copper-free Co(I1)superoxide dismutase were modified either by raising or lowering the pH from neutrality (Fig.3A and B). Full reversibility of the observed changes occurred between pH 3.0 and pH 11.O and was still good, though partial, from pH 12.0. In the Co(I1)-Cu(11) protein no relevant change of either optical or EPR spectra is observed in the pH range 6-12. In the pH range 10- 12 the residual Cu(I1) EPR spectrum, resulting from a Co(1I) content less than stoichiometric to copper [I], undergoes axialization as in the native protein [9] with no change of signal intensity. In the acid range, however, several major modifications occur. The Co(1I) optical absorption band decreases with decreasing pH, as it does in the copper-free Co(I1) protein, while the Cu(I1) absorption near 680 nm is only slightly affected (Fig.4A). The shape of the Cu(I1) EPR signal becomes distinctly axial as in the native protein at about pH 3.0 [7] and its intensity increases with decreasing pH. Integration of the copper signal at pH 3.0 gives a two-fold increase compared to the higher pH signal. The EPR signal of Co(I1) also increases and shows, at pH 3.0 (Fig.4B, curve b), the same shape as in the copper-free protein at the same pH (Fig. 3 B, curve b). No attempt was made to inte-

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Fig. 1. O ~ I ~ C ~ Sof’thr ~ ~ C’oiIl) C I I Zcopprr-jrw superoxide disiriutuse in the prc~.c.em~~ of’cjunide. Curve (a): 0.65 m M protein, 0.7 m M Co(I1) in 0.1 M borate bul€er, pH 8.9. Curve (b): 30 min after addition of I equiv. C N in an anaerobic Thunberg cuvetle. Curve (c): 30 min after the addition of 1 equiv. C N - in the presence of air

L. Calabrese, D. Cocco, L. Morpurgo, B. Mondovi, and G. Rotilio

461

Fig.2. EPR spectrti ofthe reaction ~ f ~ i j ( l l ) ~ s u p e rcfismutasc. o . ~ i ~ ~ with ~ cyanide. Curve (a): the same solution of curve (a) in Fig. I . Curve (b): after addition of 4 equiv. C N - in an anaerobic Thunberg-type EPR tube. Curve (c): after addition of 4 equiv. CN- in the presence of air. EPR conditions: temperature, 10 K ; microwave frequency, 9.24 G H z ; microwave power, 6 mW; modulation amplitude, 10 fi. The inset contains an enlargement of curves (b) and (c) in the 3-kG region to show details

Fig. 3.Optic.aland E P R spectra qf C u ( l I ) supero.~idedismuluseat variouspH values. (A) Optical spectra. Curve (a) : water solution (approximately pH 6) of 0.5 m M protein, 0.7 mM Co(I1). Curve (b): the same as in curve (a) brought to p H 3.5 by small additions of concentrated HCI. Curve (c) : the same as in curve (a) brought to p H 1 1.5 by sinall additions of concentrated NaOH. (B) EPR spectra. Curve (a) : 0.36 mM protein, 0.57 mM Co(II), water solution. Curve (b): the same, pH 3.5. Curve (c): the same, pH 11.5. EPR conditions as in Fig.2

grate the Co(I1) signals; but a very similar Co(I1) signal was obtained when the Co(T1) - Cu(I1)protein was reduced with H 2 0 2at neutral pH and then brought to pH 3.0 (Fig.4B, curve d), indicating a comparable paramagnetism of the cobalt in the two cases. The uncoupling of the two metal sites at low pH is not due to protein denaturation, since good reversibility is observed by raising the pH back to neutrality (Fig. 4B, curve c).

Reaction of Co(I1) Superoxide Dismutase with H 2 0 2 The reaction of H 2 0 2 with the copper of native superoxide dismutase leads to reduction of the metal ion, and then to a slow reoxidation by oxygen which results in an inactive enzyme with altered EPR spectrum of the reoxidized copper [lo, 111. This denaturdtion of the copper site has been related to the distruction of a single histidine residue per site [ll]. In the

Reactivity of Cobalt in Cobalt Superoxide Dismutase

468

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Fig.4. Optical and EPR spectra of C o ( I I ) - C u j I I ) superoxide dismutase at acidpH. (A) Optical spectra of the protein in water, curve (a); and at pH 3.0, curve (b). Protein was 0.23 mM, Co(I1) 0.32 mM. (B) EPR spectra of the same protein in water, curve (a); at pH 3.0 with HCl, curve (b); back to neutrality from pH 3.0 with NaOH, curve (c); and after reduction with 50-fold excess of H,O, and lowering the pH to about 3 with HCI, curve (d). EPR conditions as in Fig. 2. The copper signal was recorded at 100 K to avoid saturation

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0 Fig. 5. EPR spectru r$Co(II) - CujII) superoxide dismutase treated with H,O,. Curve (a): the protein in water solution. Curve (b): immediately after addition of 20-fold excess of H,Oz. Curve (c) : after 150 min incubation in air. EPR conditions as in Fig. 2 and 4. In the inset the corresponding optical spectra are shown

L. Calabrese, D. Cocco, L. Morpurgo, B. Mondovi, and G. Rotilio

copper-free Co(I1) protein no change of optical and EPR spectra is observed after 150 min incubation in the presence of a 20-fold excess of HzOz. A similar treatment of the Co(I1)- Cu(I1) protein (Fig. 5) shows, in the phase of slow reoxidation of copper, a gradual decrease of the Co(I1) EPR signal with a significant shift of the maximum to lower magnetic fields. This change was paralleled by an alteration of the Co(I1) visible spectrum (Fig. 5 inset). The intensity of the final copper EPR signal, as evaluated by double integration, was not significantlyhigher than that seen before adding H,O,. Full reduction of copper with dithionite did not apparently modify the intensity of the cobalt signal. However the cobalt signal after H,O, treatment is broader than that of the untreated protein and quantitative conclusions can not be drawn on simple inspection of the major peak near g = 4.3.

DISCUSSION The data reported in the present paper confirm that in bovine superoxide dismutase Zn(II), or Co(II), is far less exposed to the solvent than Cu(I1). In fact, in some conditions such as at alkaline pH and with CN-, copper is apparently the most reactive species and reaction of cobalt can only occur in the absence of copper. Previous data [12-141 have shown that these reactions involve the water molecule co-ordinated to the copper in a position open to solvent access. The histidine-61 bridging copper and zinc [ 5 ] is likely to be unaffected by these reagents, which do not have uncoupling effect on the Co(I1) - Cu(I1) system. The reaction of Co(I1) with cyanide in the copper-free protein is slow and does not show any spectral evidence for a monocyanide or a dicyanide adduct, as in the case of carbonic anhydrase [8,15]. After addition of equimolar amounts of cyanide the visible absorption spectrum of cobalt decreases, as well as its EPR spectrum, and the resulting species appears to be cobalt already removed from the protein. The EPR spectrum of this species in anaerobiosis is reminiscent of pentacyanocobalt [8], while in the presence of air both the EPR signal at g e 2 and the optical band near 380 nm recall the adduct formed with oxygen in the case of Co(I1) carbonic anhydrase, which is likely to be an oxygen addition product of Co(CN),I3- [15]. The reaction of the native protein with CN- can thus be outlined as the following sequence of events: monocyanide complex of copper(II), tetracyanide complex of copper(II), reduction and removal of the copper, attack and removal of the zinc. This sequence accounts for the complete metal removal obtained when the protein is treated with CN- at pH > 8 in the preparation of the apoprotein [7]. A different pattern of reaction is represented by the acid titration of the protein, which dramatically

469

affects the Co(I1) site, irrespective of the presence of the copper. The strong decrease of the optical spectrum of cobalt following acidification either in the coppercontaining or copper-free protein shows that the distorted tetrahedral geometry of the site [3] is modified giving a new species with a much lower absorption coefficient. Also the features of the EPR spectra (see Fig. 3 B and 4B) with the appearance of a shoulder at lower field, which is probably due to unresolved cobalt hyperfine structure, suggest a different geometry [16]. As uncoupling of the Co(I1)-Cu(I1) antiferromagnetic system also occurs on such a titration, histidine-61 is very likely to be the proton acceptor in the protonation reaction which breaks the coupling. This protonation should occur at the cobalt facing nitrogen because of the higher stability constant of the Cu(I1)- histidine bond. This could explain why the optical spectrum of the copper is relatively unchanged at low pH while that of the cobalt almost disappears. (see Fig. 3 A and 4A). A third type of reaction occurs with H,Oz, which slowly and irreversibly modifies the cobalt chromophore only in the presence of copper. Previous data showed the destruction of one histidine residue during enzyme inactivation by H202 [ l l ] . This residue is not likely to be the bridging histidine-61, in fact when the Co(I1) - Cu(I1) enzyme reoxidizes after H,O, treatment some coupling still occurs even with the altered cobalt site, as shown by the intensity of Cu(I1) EPR signal. As a concluding remark these results show clearly that the more significant aspects of the Zn site reactivity are better described in terms of reaction of the Znimidazolate - Cu system as a whole. More experimental evidence and discussion concerning this point will be presented in a further paper. The skilful technical assistance of Messrs Mario Sanchioni and Amleto Ballini is gratefully acknowledged.

REFERENCES 1 . Calabrese, L., Rotilio, G. & Mondovi, B. (1972) Riochirn. Biophys. Actu, 263, 827 - 829. 2. Fee, J . A. (1973) J . B i d . Chem. 248, 4229-4234. 3. Rotilio, G., Calabrese, L., Mondovi, B. &. Blurnberg, W . E. (1974) J . Biol. Chem. 249, 3157-3160. 4. Rigo, A,, Terenzi, M., Franconi, C., Mondovi, B., Calabrese, L. 81 Rotilio, G. (1974) FEBS Lett. 39, 154- 156. 5. Richardson, J. A,, Thomas, K. A., Rubin, B. H. & Richardson, D. C. (1975) Pmc. NutlAcud.Sci. U.S.A. 72,1349- 1363. 6. McCord, J. M . & Fridovich, I . (1969) J . Biol. Chem. 244, 6056- 6063. 7. Rotilio, G., Calabrese, L., Bossa, F., Barra, D., Finazzi-Agro, A. &. Mondovi, B. (1972) Biochemistry, 11, 2182-2187. 8. Cockle, S. A. (1974) Biochern. J . 137, 587-596. 9. Rotilio, G., Finazzi-Agro, A., Zalabrese, L., Bossa, F., Guerrieri, P. &. Mondovi, 6.(1971) Biochernistvy, 10, 616-621.

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10. Rotilio, G., Morpurgo, L., Calabrese, L. & Mondovi, B. (1973) Biochim. Biophys. Acta, 302, 229- 235. 11. Bray, R. C., Cockle, S. A,, Fielden, E. M., Roberts, P. B., Rotilio, G. & Calabrese, L. (1974) Biochem. J . 139, 43-48. 12. Rotilio, G., Morpurgo, L., Giovdgnoh, C., Calabrese, L. & Mondovi, B. (1972) Biochemistry, 11, 2187-2192. 13. Fee, J. A. & Gaber, B. P. (1972) J . Biol. Chern. 247,60- 65.

14. Terenzi, M., Rigo, A,, Franconi, C., Mondovi, B., Calabrese, L. & Rotilio, G. (1974) Biochim. Biophys. Acta, 351,230-236. 15. Haffner, P. H. & Coleman, J. E. (1975) J . Bid. Chem. 250, 996-1005. 16. Kennedy, S. F., Hill, H. A. O., Kaden, T. A. & Vallee, B. L. (1972) Biochem. Biophys. Res. Commun. 48, 1533- 3539.

L. Calabrese, D. Cocco, and G. Rotilio, Centro di Biologia Molecolare del C.N.R. e Istituto di Chimica Biologica dell’Universita, Citta Universitaria, 1-00185 Roma, Italy L. Morpurgo, Centro di Biologia Molecolare del C.N.R., Universita degli Studi, Citti Universitaria, 1-00185 Roma, Italy

B. Mondovi, Istituto di Biochimica Applicata, Universita degli Studi, Citta Universitaria, 1-00185 Roma, Italy