Reconstitution of Cu,Zn-Superoxide Dismutase by the Cu(I)aGlutathione Complex*

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I)*GSH com- plex was monitored by: (a) EPR and optical spectros- copy upon reoxidation of the enzyme-bound copper; (6) NMR spectroscopy following the broadening of the resonances of the Cu(I)*GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spec- troscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I),Co enzyme. than other low molecular ticular, chiometric

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I)*GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (6) NMR spectroscopy following the broadening of the resonances of the Cu(I)*GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I),Co enzyme. Cu(I)*GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(I1) complexes.
In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I)*GSH-protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper*GSH complexes in copper-overloaded hepatoma cells (Freedman, J. H., Ciriolo, M. R., and Peisach, J. (1989) J. Biol. Chem. 264, 559% 5605), Cu(1) l GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in viva.
Superoxide dismutases are metalloenzymes that play an essential role in the defense of the cell against potentially toxic derivatives of the biological activation of oxygen. They are ubiquitous enzymes; and in particular, the isoenzyme that carries one copper and one adjacent zinc ion in the catalytically active center on each of its two identical subunits is typical of, although not restricted to, eukaryotic cytosol (Bannister et al., 1987). A still unresolved problem in the comprehension of the physiological regulation of its activity is the biological mechanism by which the catalytically active copper is taken up by the protein moiety in the active-site pocket. Previous evidence (Cass et al., 1979) points to a role of zinc in giving the adjacent copper-binding site the geometry that is typical of the native holoenzyme and consists of a tetrahedrally distorted tetragonal array of histidines (Tainer et al., 1982). In fact, the inactive apoenzyme is more readily reactivated in vitro by Cu(I1) salts after prior addition of two zinc ions/protein molecule . However, the transfer of copper to the enzyme in uiuo poses a number of problems since at variance with redox-inert Zn(II), Cu(I1) may act as an oxidizing agent on -OH, -SH, and -CHO moieties of biomolecules, and Cu(1) may give rise to oxidizing oxygen radicals according to Fenton and Haber-Weiss-type reactions (Samuni et al., 1981;Halliwell and Gutteridge, 1984). Among the few biomolecules that are able to screen copper ions from reactions with oxygen and other ligands, metallothioneins have been suggested to participate in biological copper-transfer processes in addition to acting as copperstorage devices. However, copper-thionein, while found to reactivate several copper apoenzymes in vitro (Beltramini and Lerch, 1982;Morpurgo et al., 1983;Hartmann et al., 1983;Brutsch et al., 1984;Schechinger et al., 1986;Markossian et al., 1988), was apparently ineffective in reconstituting the holoenzyme from copper-free superoxide dismutase. Since glutathione has recently been shown to be involved in the resistance of cultured cells to copper , we have tried to reconstitute copper-free superoxide dismutase by using a copper complex with glutathione.
The process was followed by EPR spectroscopy monitoring the formation of the native Cu(I1) *enzyme complex and by NMR spectroscopy monitoring Cu(I)-protein binding. No transfer of copper to the copper-free enzyme was observed when the copperglutathione complex was in the oxidized form Cu(I1). GSSG, whereas the reduced complex, Cu(1). GSH, fully reconstituted the enzyme in a very efficient process apparently involving a Cu(1). GSH . protein intermediate.
This result is a strong indication that GSH may be able to donate Cu(1) to the copperfree enzyme in vivo. to McCord and Fridovich (1969), and its concentration was calculated either by the absorbance of the copper chromophore (McCord and Fridovich, 1969) or by the method of Lowry et al. (1951) using bovine serum albumin as a standard. The copper-cobalt derivative was prepared by addition of CoCl, to the Zn-free enzyme which was obtained as previously described (Valentine et al., 1979). The final concentration of the cobalt bound to the enzyme was controlled spectrophotometrically . The copper-free derivatives of either the holo-or copper-cobalt enzymes were prepared by reducing the copper with excess potassium ferrocyanide and dialyzing for 12 h at 4 "C against 0.1 M phosphate buffer containing 0.05 M KCN at pH 6.0 (Rigo et al., 1977). The samples were further dialyzed for 24 h at 4 'C against water. Final copper content was less than 2%. The reduced Cu(1) form of the enzyme was prepared by anaerobic treatment with NaBH, as previously described (Viglino et al., 1981). The enzyme activity was assayed with the polarographic method of Rigo et al. (1975 . GSSG complex (Postal et al., 1985), and at 680 nm, typical of Cu(II),Zn-superoxide dismutase (McCord and Fridovich, 1969). The reoxidation experiments were always carried out in the presence of 9.1 PM catalase to eliminate H202, a possible reaction product, which may interfere by both reducing and denaturing the enzyme (Bray et aZ., 1974;Hodgson and Fridovich, 1975). Optical spectra were recorded on a Perkin-Elmer Lambda 9 spectrophotometer.
'H NMR spectra were recorded at 400 MHz in 20% deuteriated solutions of 0.05 M phosphate buffer, pH 7.4, with a Bruker AM-400 spectrometer.
The spectra of GSH and the Cu(1). GSH complex were recorded with solvent suppression by selective presaturation of I.7 s of the water proton resonance. 128 decays were accumulated on a spectral width of 6 kHz using a time domain of 8,000 data points. Cu (I1) and Cu-free,Zn-superoxide dismutase were added in minute amounts directly to the NMR tube. The spectra of the isotropically shifted resonances of the Cu,Co-superoxide dismutase (Banci et al., 1987) were obtained by using a modified pulse sequence (Hochmann and Kellerhals, 1980) I). The broadening depends on the decrease of the Tz spin-lattice proton relaxation time, which is primarily due to the reduced mobility of GSH in the complex-bound form. From the NMR spectra, it is immediately obvious that the two complexes are identical. In spectrum 6, the typical resonances of GSSG are also detectable after complex formation.
Addition of Cu(I1) causes oxidation of stoichiometric amounts of GSH to GSSG and complexation of the resulting Cu(1) with the remaining GSH. At all GSH:copper ratios tested (2:1,3:1,&l, 10:1,5&l, and 500:1), GSH was able to form very stable complexes with Cu(1) even in the presence of oxygen. The 2:l complex (0.7 mM copper) started to oxidize in air only after 5 h of incubation in a shaking water bath at 37 "C; the same behavior was observed with the 3:l complex starting with GSH and Cu(II) regardless of whether it was formed in air or under nitrogen I~~",~~~~,""I,~"I'~"I""i"~'I atmosphere. The reoxidation time was longer for complexes with higher GSH:Cu(I) ratios. Fig. 2 shows the EPR spectrum of a 0.25 mM solution of Cu-free,&-superoxide dismutase in 0.05 M phosphate buffer, pH 7.4, and that obtained immediately after the addition of the 1:3 Cu(1). GSH complex in air (spectra A and B, respectively). No Cu(II) signal was detectable in either case at the instrumental settings used. After 1 h of incubation at 37 "C in air, an EPR signal accounting for 10% of the total copper appeared (spectrum C), with a heterogeneous line shape consisting of that of the native enzyme (spectrum D) plus another form of the Cu(I1) complex. Only after exposure of the sample to air for at least 24 h did an EPR signal accounting for 100% reconstitution of the enzyme appear (spectrum D), and the sample was 100% enzymatically active as compared to the native protein. The same extent of reconstitution was obtained when the incubation mixture as in spectrum B was immediately subjected to chromatography on a Sephadex G-25 column, demonstrating that copper was already bound to the enzyme at the beginning of the process. Fig. 2 also shows the EPR spectrum (spectrum E) of the Cu(1) .GSH complex (25 mM copper) at any time between mixing and 5 h of incubation in air. EPR experiments (not shown) also demonstrated that 200-fold GSH excesses were not able to remove the copper from either the oxidized or reduced enzyme. Reoxidation of Cu(1). GSH (0.70 mM copper) in the presence of 0.35 mM Cu-free,Zn-superoxide dismutase was followed continuously by optical spectroscopy: 50% Cu(I1) enzyme was detected after 5 h of incubation in air. Since reoxidation of Cu(I),Zn-superoxide dismutase by oxygen has been shown to be faster under comparable conditions (50% oxidized enzyme after 30 min of incubation in air of the enzyme fully reduced by borohydride; Viglino et al., 1986) and, under our conditions, a similar behavior was found for the enzyme (0.35 mM) reduced anaerobically by GSH, the much slower reoxidation rate of the Cu(I) a GSH . Cu-free,Znsuperoxide dismutase incubation mixture may suggest the occurrence of a Cu(1) -GSH -protein intermediate in the reconstitution process. To investigate this possibility, the reconstitution of the Cu-free enzyme with Cu(I).GSH was also followed by NMR spectroscopy. Fig. 3 shows that each addition of Cu-free,Zn-superoxide Cu(I).GSH comnlex (1.8 mM Conner) in 0.05 M nhosphate buffer, pH 7.4'(20% DzO); >pect& b-d, same as spectrum* a immediately after successive additions of 0.11 mM Cu-free,Zn-superoxide dismutase. The peaks of the Cu(1) . GSH complex are marked with arrows. For the NMR conditions, see "Experimental Procedures." dismutase (up to 0.33 mM) to the 1:2 Cu(1) . GSH complex (1.8 mM copper) under nitrogen atmosphere further broadened the resonances marking the Cu(1) +GSH complex by reducing its internal mobility. Furthermore, the NMR spectrum of the Cu(1). GSH complex was not altered by the addition of native Cu(I),Zn-superoxide dismutase, which is expected not to bind copper. In fact, as shown in Fig. 4, the height of the resonance at 3.83 ppm (typical of the Cu(1) .GSH complex) was decreased to 30% of its original value after the addition of Cufree,Zn-superoxide dismutase (spectrum c), whereas it was not altered (92%) by the addition of twice as concentrated Cu(I),Zn-superoxide dismutase (spectrum d). These results actually support the formation of a ternary complex between the copper-free enzyme and the Cu(I). GSH complex.
On the other hand, NMR spectroscopy proved to be very useful in monitoring the changes occurring at the enzyme active site during the reconstitution process. To this purpose, the copper-free enzyme with Co(I1) substituting for the zinc ion was examined. The use of this approach is advantageous in two respects. First, it is possible to monitor the reconstitution process by observing the changes of the active-site geometry through the modification of the protons of the histidines bound to the metal cluster (Banci et al., 1987) which are subjected to isotropic shifts instead of being broadened to undetectability because of the short Co(I1) relaxation time (paramagnetic NMR). These shifts are an extremely sensitive probe for even small changes of the coordination geometry around the copper ion. Second, this approach, at variance with EPR spectroscopy, does not require the active site to be in the Cu(I1) state and therefore gives direct information on the binding of Cu(1) to the enzyme, a process which has not yet been studied in detail.  with increasing Cu(1) .GSH (1:3) concentrations up to 1:l (copper-protein monomer) molar ratio (spectra b-d), the spectrum changed until the final spectrum was similar to that of the Cu(I),Co enzyme as obtained by anaerobic reduction of the Cu(II),Co protein with borohydride in the presence of 20% D,O (Fig. 6, (Bertini et al., 1985).
Under the experimental conditions of Fig, 5 (spectrum d), these groups could be in a faster exchange with the solvent, resulting in the disappearance of their proton resonances. If this explanation is correct, the effect is likely to be related to a greater solvent accessibility and may be considered as a further, although indirect, indication for the presence of bulky ligand, such as Cu(1). GSH, in the enzyme active site.
Reoxidation of the Cu(1) . GSH . protein complex restored the geometry of the coordination sphere of the native active site as monitored by paramagnetic NMR spectroscopy (Fig.  6). Also in this case, the process was accelerated by chromatography of the sample on Sephadex G-25.
Since full reconstitution of the protein was achieved in the experiments described above by adding copper as Cu(1). GSH at a 1:l ratio with respect to the available copper-binding sites of the protein, the relative reconstitution efficiency of Cu (I1) complexes under comparable conditions was tested. In fact, Cu,Zn-superoxide dismutase is usually reconstituted from its apoprotein by adding inorganic cupric salts. The results obtained with CuSO,, Cu(I1). GSSG, and Cu(I1). methionine are shown in Table I ently of the GSH:copper ratio used between 2 and 500. In conclusion, our results show that a very stable complex between copper and GSH is obtained even in the presence of oxygen and that this complex is able to donate Cu(1) to Cufree,Zn-superoxide dismutase, most likely through the formation of a ternary complex, giving 100% reconstitution of the holoenzyme under conditions where Cu(1) *cysteine is unstable and Cu(I1) complexes are not able to fully reconsti-