Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4245-4249, September 1979
Biochemistry
pH-dependent migration of copper(II) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase (metalloprotein/electron spin resonance/histidyl imidazolate)
JOAN S. VALENTINE*, MICHAEL W. PANTOLIANO*, PETER J. MCDONNELL*, ALLAN R. BURGERt, AND STEPHEN J. LIPPARDt *Department of Chemistry, Rutgers, The State University, New Brunswick, New Jersey 08903; and tDepartment of Chemistry, Columbia University, New York, New York 10027
Communicated by Ronald Breslow, May 22, 1979 ABSTRACT Bovine erythrocyte superoxide dismutase (Cu2Zn2SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of two identical subunits each containing Cu2+ and Zn2+ in close proximity. We describe here electron spin resonance (ESR) and visible absorption spectroscopic studies of the zinc-free derivative of this protein, Cu2E2SODase (E = empty) over the pH range 6-10. The ESR spectrum of the zincfree protein at 77 K is markedly pH dependent. At pH < 8.0 the ESR spectrum is axial in appearance. At pH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum is very broad and resembles that of the four-copper derivative Cu2Cu2SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30°C are also consistent with formation of Cu(II)Im-Cu(II). A plot of changes in the signal amplitude of g1 for Cu2E2SODase as a function of pH gives an apparent pKa of 8.2 for the transition. The long-wavelength absorption with Xmax = 700 nm characteristic of Cu2E2SODase shifts with increasing pH to 800 nm and the resulting visible spectrum is identical to that of Cu2Cu2SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH are reversed when the pH is decreased with HNO3, although the approach to equilibrium is slow in the latter case. The results of these experiments are consistent with a reversible, pH-dependent migration of Cu2+ from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit. By contrast, native protein, Cu2Zn2SODase, and the four-copper protein, Cu2Cu2SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu2E2SODase and its thermal stability are reexamined in light of these new findings.
Apart from studies to determine the function, mechanism, and reactivity of this protein (4), there has also been considerable interest in various structural aspects of this and the other closely related Cu-Zn proteins isolated from various eukaryotic sources (4). The interaction between metal binding sites within each protein subunit is of particular interest (6). Previously we described conformational changes induced at the empty copper binding site when zinc is bound to the apoprotein (7). Here we present evidence for the reversible, pH-dependent migration of copper(II) ion from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit and discuss its possible significance. MATERIALS AND METHODS Cu2Zn2SODase was isolated from bovine erythrocytes and purified according to the procedure of McCord and Fridovich (8). The purified enzyme was dialyzed against doubly distilled water and stored as a lyophilized powder at -20'C. The specific activity, determined by using the epinephrine assay of Misra and Fridovich (9), was 85-90% of that reported by these authors. Most preparations were found to give one or two bands on polyacrylamide electrophoresis by a modification of the method of Rigo et al. (10) in which 0.25% rather than 2.5% (wt/vol) N,N'-methylenebisacrylamide was used. Copper and zinc contents were determined by atomic absorption photometry using a Techtron AA4 instrument. The native enzyme contained 0.85-0.92 mol of copper and 0.82-0.89 mol of zinc per mole of subunits; the protein concentration was based on weighed amounts of lyophilized powder. The apoprotein was prepared by dialysis against disodium EDTA at pH 3.8 as described by McCord and Fridovich (8) followed by exhaustive dialysis against 0.1 M NaCl to remove enzyme-bound EDTA (11). The metal-free enzyme typically contained 1-2% of the amounts of copper and zinc present in the native enzyme. The zinc-free derivative, Cu2E2SODase, was prepared by first diluting the apoprotein with 10 mM sodium acetate buffer at pH 3.8 to give a final concentration of 1 mg/ml. Then 3.15 mM CuSO4 was infused at the rate of 1 ml/hr (12) until 1.0 equivalent per subunit had been added. The resulting protein solution was exhaustively dialyzed against doubly distilled water and stored as a lyophilized powder at -200C. Weighed samples
Copper-zinc superoxide dismutase (Cu2Zn2SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1), as isolated from bovine erythrocytes, contains one copper(II) atom and one zinc(II) atom (1) in each of two identical subunits (2-4). The metal ions in each subunit are bound in close proximity to one another (5, 6) and are believed to share a common ligand, an imidazolate (Im) anion derived from the side chain of histidine-61 (6).
Cu2+ -N
Z92+
JC \
Abbreviations: Cu2Zn2SODase, native bovine erythrocyte superoxide dismutase; Cu2Cu2SODase, derivative of the native enzyme in which Cu(II) has been substituted for Zn(II); Cu2E2SODase, derivative of the native enzyme in which the zinc site is vacant (E = empty); Zn-free Cu2E2SODase, designates under conditions such that the Cu ions are not necessarily in their original locations; Im, imidazolate; ESR, electron spin resonance.
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Biochemistry: Valentine et al.
of this powder dissolved in water contained 0.95, 1.03, and 1.07 mol of copper per mole of subunits and 0.03, 0.01, and 0.05 mol of zinc per mole of subunits for three different preparations. The four-copper derivative of superoxide dismutase, Cu2Cu2SODase, in which Cu(II) is substituted for Zn(II), was prepared by two methods. The first procedure was that of Fee and Briggs (13), with CUSO4 substituted for CuC12. The other method involved the CUSO4 titration of Cu2E2SODase in 0.25 M sodium acetate buffer at pH 5.5. Both procedures gave the same results as judged by the characteristic electron spin resonance (ESR) spectrum at 77 K (see below). Adventitiously bound Cu2+ was removed by dialysis against doubly distilled water for 4 hr with no change of the dialyzate. Dialysis against acetate buffer, or further changes in the dialysate when water alone was used, caused changes in the ESR spectrum consistent with partial removal of Cu from Cu2Cu2SODase. The protein concentration of Cu2Cu2SODase was determined with the Bio-Rad protein assay kit and Cu2Zn2SODase as the standard. Metal analyses of the Cu2Cu2SODase sample used for studies described in this report indicated that it contained 1.81 equivalents of Cu and 0.04 equivalent of Zn per subunit of protein. ESR spectra
obtained by using a Varian E-12 spectrometer together with a Hewlett-Packard model 5255 A frequency meter. The magnetic field was calibrated by using the signal of Mn(II) naturally present as an impurity in strontium oxide (14). Room temperature ESR spectra were obtained by using an aqueous ESR cell from Wilmad. All ESR parameters are defined according to Malmstrom and Vannga'rd (15). Visible and near-infrared spectra were obtained by using a Cary 17D spectrophotometer. Samples were centrifuged before spectral observations in order to decrease the amount of light scattering due to small amounts of undissolved protein. The pH was adjusted with small amounts of 1 M NaOH and 1 M HNO3 and pH measurements were made with a Corning model 12 pH meter in conjunction with a thin (6 mm) Sensorex combination electrode. Doubly distilled water was used in the preparation of all solutions, and reagent grade chemicals were used throughout. were
Proc. Natl. Acad. Sci. USA 76 (1979)
Magnetic field,
gauss
FIG. 2. ESR spectrum of Zn-free SODase at pH 9.3 and 77 K. This spectrum is the same as curve c of Fig. 1 except that the gain was increased by a factor of 5.
RESULTS ESR spectra of Zn-free SODase at various pH values and 77 K in Fig. 1. At pH 7.8, the spectrum was characteristic of Cu(II) in an axially symmetric ligand environment with gll = 2.268, gm = 2.067, and All = 146 G. These values are in good are shown
agreement with
those previously reported for the zinc-free
protein (12, 13, 16). An extensive analysis of this spectrum by Beem et al. (12) has led to the conclusion that the Cu2+ ions are in the same sites as in the native Cu2Zn2SODase protein. This conclusion is also supported by the observation of distinctly
different ESR parameters for a silver-copper derivative of superoxide dismutase in which Ag+ resides in the native copper site and Cu(II) resides in the zinc-binding site (16). However, adjustment of the pH to 9.3 by slow addition of 1 M NaOH resulted in a broadening of the ESR spectrum with development of a broad trough at approximately 3600 G and the appearance of a weak signal at approximately 1540 G (Figs. 1 and 2). The final spectrum (Fig. 2) was similar to that of Cu2Cu2SODase (Fig. 3A) previously prepared by Fee and Briggs 1400 1800 2200 2600 3000 3400 3800 4200 4600
AA
B
2200 2600 3000 3400 3800 Magnetic field, gauss
FIG. 1. pH dependence of the ESR spectrum of Zn-free SODase at 77 K. The concentration of protein subunits was 2.0 mM in 2.0 ml
0.1 M potassium phosphate initially buffered at pH 7.8. Curves: ESR spectrum of the initial solution; b (pH 8.3) and c (pH 9.3), after addition of 10 and 20 ,ul of 1 M NaOH; d, after pH was readjusted downward from 9.3 to 6.5 by addition of 34 ,ul of 1 M HNO3. The spectra were recorded with 100-kHz field modulation at a modulation amplitude of 4.0 G. The microwave power was 50 mW at a frequency ot 9.13 GHz; the gain was 250 for each spectrum. The spectral variab)les for curves a, b, and d are: All = 146 G; g~l = 2.268; gm = 2.067. of
a,
2200 2400 2600 2800 3000 3200 3400 3600 3800 Magnetic field, gauss FIG. 3. ESR spectrum of Cu2Cu2SODase at 77 K (A) and at 30 ± 2°C (B). The concentration of protein subunits was 0.42 mM at pH 5.2 (unbuffered). The spectral conditions in A were similar to those in Fig. 1 except that the modulation amplitude was 10.0 G and the gain was increased by a factor of 8. In B the spectrum was similarly recorded except that the microwave power was 30 mW at a frequency of 9.41 GHz and a gain of 6300.
Biochemistry:
Valentine et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
(5, 13). This type of spectrum is characteristic of binuclear copper(II) complexes in which there is magnetic interaction between the two metals. Similar spectra have been observed for Im-bridged and other dicopper(II) complexes and have been interpreted in terms of dipolar coupling of Cu(II) ions (17-19). The weak signal at low field arises from the Am = 2 transition characteristic of a magnetic triplet (19). Increasing the pH of a solution of Zn-free SODase thus causes the formation of Imbridged dicopper(II) centers identical to those found in Cu2Cu2SODase and several model compounds. The structure that appears between 2600 and 3200 G in the spectrum of Zn-free SODase at pH 9.3 (Fig. 2) is much more pronounced than that observed in the spectrum of Cu2Cu2SODase (Fig. 3). It is attributed to subunits that contain only one copper, which suggests that conversion of Cu2E2SODase to dicopper(II) subunits at high pH is not complete. The changes in the ESR spectrum caused by an increase in pH were complete within the time necessary to record the ESR spectrum. Readjustment of the pH down to 6.5 by stepwise addition of 1 M HNO3 eventually resulted in the reversion of the spectrum to that characteristic of the low pH form (Cu2E2SODase) as shown by curve d of Fig. 1. The approach to equilibrium was considerably slower upon decreasing the pH, however. This time hysteresis was also seen in the ESR experiment at 30°C discussed below. The ESR spectrum at 30 I 2°C was also used to follow the pH titration of Cu2E2SODase. At this temperature, copper proteins often have ESR spectra similar to those obtained in the frozen state because the slow tumbling rate of the macromolecule does not cause total averaging of the g and A tensors (20). This situation obtained for Cu2E2SODase. The spectrum at pH 7.4 (Fig. 4) had gli = 2.267, gm = 2.073, and All = 140 G. The ESR spectrum of Cu2Cu2SODase, by contrast, showed no signal at 30°C (Fig. 3B), a feature commonly observed in the spectra of low molecular weight Cu(II) dimers (18, 19). Fig. 4 shows the effect of increasing the pH on the ESR spectrum of Zn-free SODase at 30°C. A marked decrease in signal intensity occurred but without the broadening observed with increasing pH in the frozen solution ESR spectra (compare Fig. 1). The signal intensity decreased with increasing pH until pH 9.4. Increasing the pH to 9.8 caused no further change in the spectrum. The gII (2.254) and A1I (145 G) of the species at pH 9.4 and 9.8 are slightly different from those of the initial
4247
a
b C
d e
2600 2800 3000 3200 3400 3600 3800 Magnetic field, gauss
FIG. 5. Reversibility of the pH-dependent transition of Zn-free SODase. To 0.5 ml of the pH 9.8 sample described in Fig. 4 was added 16 Ail of 1 M HNO3 to adjust the pH to 7.1. The spectrum before addition of acid is reproduced as curve a. ESR spectra of the sample at various times after the addition of acid are shown in curves b-e (b, 1.5 hr; c, 8.5; d, 10.5; e, 12.5). The pH of the solution after 14.5 hr was 7.2. Spectral conditions are exactly as described for Fig. 3B.
solution at pH 7.4. These small changes began to occur above pH 8.4. The loss of signal intensity as the pH is increased is the consequence of converting most of the mononuclear copper subunits of Cu2E2SODase to ESR silent forms. This behavior is exactly that expected if binuclear copper subunits form because subunits of this kind give no ESR signal at 30°C in solutions of Cu2Cu2SODase (Fig. 3B). The small changes that occur in the lineshape of the ESR spectrum at high pH may reflect deprotonation of histidyl ligands or of water bound to copper, or they may be due to small pH-dependent, reversible, conformational changes. The reversibility of the pH titration at 30'C is illustrated in Fig. 5. The rate of reverse reaction was slow. Reestablishment of equilibrium after adjustment of the pH from 9.8 to 7.2 required 12.5 hr at room temperature. Increasing the pH to 10.3 resulted in the irreversible formation of a purple solution. The signal amplitude (peak-to-peak height) in the gI region of the spectra shown in Fig. 4 is plotted as a function of pH in
a
b
A
d
f 2600 2800 3000 3200 3400 3600 3800
Magnetic field,
gauss
FIG. 4. pH dependence of the ESR spectrum of Zn-free SODase at 30 2°C. The protein subunit concentration was 1.3 mM in 0.1 M potassium phosphate initially buffered at pH 7.4 (curve a). The pH then was adjusted downward with 1 M NaOH to give spectra b-f (b, j)H 8.0; c, 8.4; d, 9.0: e, 9.4; f, 9.8). A total of 24 ,l of base was added to 1.0 ml of protein solution. The spectra were recorded as in Fig. 3B. For a-c: All = 140 G, g~l = 2.267, gm = 2.073. For f: All = 145 G, gII = 2.254.
pH FIG. 6. pH dependence of signal amplitude in the gI region of the ESR spectrum of Zn-free SODase at 30 20C. The data were obtained from three separate experiments similar to the experiment described in Fig. 4. In two experiments (0 and ,), the protein subunit concentration was 1.9 mM. In the third (-), the concentration was 1.3 mM. The line represents a theoretical acid-base titration curve for a single ionizing group of pKa = 8.2. Error bar represents largest measured range. +
4248
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Valentine et al.
Fig. 6. If it is assumed Zn-free SODase is partially converted to an ESR silent form as the pH is increased, the signal amplitude may be used to estimate the concentration of the remaining mononuclear copper subunits. A strict quantitative comparison of relative intensities at various pH values is not possible because of lineshape changes in the high pH spectrum (curve f in Fig. 4). Because the change in peak-to-peak width is small, however, the peak-to-peak amplitudes of the derivative spectra should remain proportional to intensities (21). Any error introduced is unlikely to be larger than the inherent irreproducibility of the measured signal amplitude, denoted by error bar in Fig. 6. As the pH increased, the signal amplitude decreased until it leveled off at pH > 9.4 at 25% of its original value. This result indicates that 25% of the subunits contain mononuclear copper at high pH. The presence of mononuclear copper is also evident in the high pH ESR spectrum at 77 K as discussed above. The shape of the plot of signal amplitude versus pH (Fig. 6) resembles that of a titration curve with an apparent pKa of 8.2. Changes in the visible absorption spectrum of Zn-free SODase were also studied as a function of pH. The longwavelength absorption at XmaX = 700 nm characteristic of Cu2E2SODase (12, 13) shifted, with increasing pH, to t800 nm. The resulting spectrum was identical to that of Cu2Cu2SODase (13) (Fig. 7). In addition to the long-wavelength band, both spectra showed a shoulder at 450 nm, also exhibited by native Cu2Zn2SODase (22) but not by Cu2E2SODase (13). This shoulder is thought to be characteristic of protein derivatives in which Im bridges two metal ions (22). The visible spectral results are consistent with the formation of binuclear copper subunits in Zn-free SODase at high pH.
DISCUSSION Increasing the pH of solutions of Cu2E2SODase brings about changes in the ESR spectra of solutions at 30'C and of frozen solutions (77 K) as well as in the visible absorption spectrum. Spectra characteristic of the four-copper derivative Cu2Cu2SODase are observed. The changes are fully reversed when the pH is decreased, although the rate of approach to equilibrium is considerably slower when the pH is decreased than when it is increased. These observations demonstrate that a reversible pH-dependent migration of copper(II) ion from one subunit to the zinc site of another subunit occurs.
2
Cu2+ _Nk, N H
N
§VN--H
The pH titration of the ESR signal amplitude at 30°C (Fig. 6) indicates that the apparent pKa for this transition is 8.2. The leveling off of the signal amplitude at 25% of its original value above pH 9.4 suggests that at high pH 25% of the subunits remaining contain mononuclear copper. The absence of similar pH-dependent spectral changes for Cu2Cu2SODase supports this interpretation. It is instructive to compare the pH-dependent behavior of native Cu2Zn2SODase and Cu2Cu2SODase with that described above for Cu2E2SODase. In the first two cases, both metal binding sites in each subunit are occupied. In those cases, no pH-dependent spectral changes are observed until the pH is
.M
I
00.25
400
500
600 700 800 Wavelength, nm
900
1 000
FIG. 7. Visible absorption spectra: - -, Cu2Zn2SODase, 0.96 mM (subunits), pH = 6.3; -, Zn-free SODase, 0.96 mM, pH = 6.2; - - -, Zn-free SODase, 0.96 mM pH 10.1 by addition of 95 ,l of 1 M NaOH to 1.5 ml of protein solution; - - -, Cu2Cu2SODase, 0.42 mM (subunits), pH 5.2 (in dilute sodium acetate buffer; all others in 0.1 M potassium phosphate).
relatively low. In the case of Cu2Zn2SODase, the bridging histidine-61 apparently is protonated at pH < 4, breaking the bond between the imidazole nitrogen and the zinc atom (22-24). In the case of Cu2Cu2SODase, at pH < 4.5, Cu2+ apparently dissociates from the native zinc site to give Cu2E2SODase (unpublished results). The situation is different for Cu2E2SODase because each subunit has one occupied and one unoccupied metal binding site. Thus, in this zinc-free derivative, there is competition between the two metal binding sites for the Cu2 . At low pH, the native copper binding site apparently has a higher affinity for Cu2+ than does the zinc-binding site. At higher pH, however, there is apparently a preference for binding Cu2+ as Im-bridged binuclear pairs. This preferred configuration for the rearrangement of the metals at high pH requires deprotonation of histidine-61. The reaction is remarkably similar to that observed in the pH titration of equimolar imidazole and tetramethyldiethylenetriamine Cu2+ (17) or CH3Hg+ (25). Here also as the pH is increased the Imbridged bis-metal derivative is favored. Although deprotonation of histidine-61 and formation of Im-bridged binuclear copper pairs is the most likely explanation for the pH-dependent spectral changes, a pH-dependent conformational change cannot be absolutely ruled out. The shape of Fig. 6 strongly implies that only one deprotonation is involved, however, and the spectrum of Fig. 2 is uniquely characteristic of the Cu(II)-Im-Cu(II) moiety (17). Other examples of pH-dependent migration of metal ions in metalloproteins occur in the literature and are expected of, proteins that contain several binding sites with different proton and metal ion affinities (reviewed in ref. 26). The present case is unique, however, because Cu(II) migrates only to subunits that already contain a Cu(II) ion. Some previous results concerning the activity of the zinc-free protein as well as the effect of metal ions on its thermal stability must be reexamined in light of the results described above. Other investigators (12, 13) have measured the activity of this protein derivative at pH 7.8 and obtained widely varying results. This variation probably was due at least in part to the migration of copper at high pH as described above. Cu2+ in the zinc-binding site gives little or no superoxide dismutase activity. This conclusion arises from the observation that Ag2Cu2SODase, in which copper is in the native zinc site, has almost no activity
Biochemistry: Valentine et al. (16) and from the fact that Cu2Cu2SODase has the same activity as Cu2Zn2SODase (13) in spite of the fact that Cu has been substituted for Zn, thereby doubling the total Cu concentration. Thus, the migration that we observe takes Cu from a site on the protein where it is catalytically active to another site where is is not, resulting in a lowering of the overall superoxide dismutase activity of the protein. We have measured the activity of our preparation of zinc-free enzyme at pH 6.0, where most of the Cu is in the native Cu site, by using a modification of the 6-hydroxydopamine assay of Heikkila and Cabbat (27). This assay is based on the inhibitory effect of superoxide dismutase on the initial rate of autoxidation of 6-hydroxydopamine and allows measurements in the pH range in which the copper of Cu2E2SODase is predominantly in the native binding site. At pH 6.0 it was found that Cu2E2SODase is at least 80 i 5% as active as the native enzyme. It would appear, therefore, that the presence of Zn2+ in Cu2Zn2SODase does not greatly enhance the activity of the neighboring Cu as previously reported (13). The observed lower activity (12, 13) is presumably due to the fact that most superoxide dismutase activity measurements are carried out at relatively high pH, at which a rearrangement of Cu ions has occurred. The other point to be considered is contained in a report (28) that Cu2Zn2SODase is more stable to thermal inactivation at pH 7.8 than is Zn-free SODase. From Fig. 6 it can be estimated that at pH 7.8 at least 10% of the subunits are in the apo form. The apoprotein is much less stable to thermal inactivation than are derivatives that contain metals (28, 29). It is possible, therefore, that the source of the apparent thermal instability of the Zn-free SODase at pH 7.8 relative to the native protein is the apo subunits that are present at that pH. The metal ion migration in Cu2E2SODase may have little physiological significance if the protein is fully saturated with metals in vivo and its primary function is to catalyze superoxide dismutation. Doubts have been expressed, however, concerning the true function of this protein owing to the absence of a chemical explanation for the source of the proposed toxicity of siperoxide (30-33). If dismutation of superoxide is not the primary function of this protein, one is led to consider alternative functions. The kinetic lability and thermodynamic affinities of the metal binding sites of this protein may in fact be }ighly relevant to the physiological function of this protein if, after all, it turns out that its primary function is to transport or store copper and zinc ions rather than to catalyze superoxide dismutation. We thank J. Burstyn and G. Fox for technical assistance and M. Nappa for his programming skills in computer graphics. J.S.V. also thanks Professor J. A. Fee for helpful discussions. Financial support from the Charles and Johanna Busch Memorial Fund (to J.S.V.), U.S. Public Health Service Grants GM 24759 (to J.S.V.) and GM 16449 (to S.J.L.), National Science Foundation Grant CHE 75-14463 (to J.S.V.), a Johnson and Johnson Fellowship in the Biological Sciences for 1978-79 (to M.W.P.), and National Institutes of Health Training Grant GMO 7216 (to A.R.B.) is gratefully acknowledged. J.S.V. also acknowledges receipt of a U.S. Public Health Service Research Career Development Award. 1. Carrico, R. J. & Deutsch, H. F. (1970) J. Biol. Chem. 245, 723-727. 2. Richardson, J. S., Thomas, K. A. & Richardson, D. C. (1975) Biochem. Biophys. Res. Commun. 63,986-992. 3. Richardson, J. S., Thomas, K. A., Rubin, B. H. & Richardson, D. C. (1975) Proc. Natl. Acad. Sci. USA 72, 1349-1353.
Proc. Natl. Acad. Sci. USA 76 (1979)
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4. Fridovich, I. (1974) Adv. Enzymol. 41,35-97. 5. Fee, J. A. (1973) Biochim. Biophys. Acta 295,107-116. 6. Fee, J. A. (1977) in Superoxide and Superoxide Dismutases, eds. Michelson, A. M., McCord, J. M. & Fridovich, I. (Academic, London), pp. 173-192. 7. Lippard, S. J., Burger, A. R., Ugurbil, K., Pantoliano, M. W. & Valentine, J. S. (1977) Biochemistry 16, 1136-1141. 8. McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055. 9. Misra, H. P. & Fridovich, I. (1972) J. Biol. Chem. 247, 31703175. 10. Rigo, A., Viglino, P., Calabrese, L., Cocco, D. & Rotilio, G. (1977)
Biochem. J. 161,27-30. 11. Forman, H. J., Evans, H. J., Hill, R. L. & Fridovich, I. (1973) Biochemistry 12, 823-827. 12. Beem, K. M., Rich, W. E. & Rajagopalan, K. V. (1974) J. Biol. Chem. 249, 7298-7305. 13. Fee, J. A. & Briggs, R. G. (1975) Biochim. Biophys. Acta 400,
439-450. 14. Bolton, J. R., Borg, D. C. & Swartz, H. M. (1972) in Biological Applications of Electron Spin Resonance, eds. Swartz, H. M., Bolton, J. R. & Borg, D. C. (Wiley-Interscience, New York), pp. 63-118. 15. Malmstrom, B. G. & Vannga'rd, T. (1960) J. Mol. Biol. 2,118124. 16. Beem, K. M., Richardson, D. C. & Rajagopalan, K. V. (1977) Biochemistry 16, 1930-1936. 17. O'Young, C.-L., Dewan, J. C., Lilienthal, H. R. & Lippard, S. J. (1978) J. Am. Chem. Soc. 100, 7291-7300. 18. Boas, J. F., Dunhill, R. H., Pilbrow, J. R., Srivastava, R. C. & Smith, T. D. (1969) J. Chem. Soc. A, 94-108. 19. Smith, T. D. & Pilbrow, J. R. (1974) Coord. Chem. Rev. 13, 173-278. 20. Vannga'rd, T. (1972) in Biological Applications of Electron Spin Resonance, eds. Swartz, H. M., Bolton, J. R. & Borg, D. C. (Wiley-Interscience, New York), pp. 411-447. 21. Wertz, J. E. & Bolton, J. R. (1972) Electron Spin Resonance:
Elementary Theory and Practical Applications (McGraw-Hill, New York), pp. 32-36. 22. Fee, J. A. & Phillips, W. D. (1975) Biochim. Biophys. Acta 412, 26-38. 23. Rotilio, G., Calabrese, L., Bossa, F., Barra, D., Finazzi-Agr6, A., & Mondovi, B. (1972) Biochemistry 11, 2182-2187. 24. Calabrese, L., Cocco, D., Morpurgo, L., Mondovi, B. & Rotilio, G. (1975) FEBS Lett. 59, 29-31. 25. Evans, C. A., Rabenstein, D. L., Geier, G. & Erni, I. W. (1977) J. Am. Chem. Soc. 99,8106-8108. 26. Breslow, E. (1973) in Inorganic Biochemistry, ed. Eichhorn, G. L. (Elsevier, Amsterdam), pp. 227-249. 27. Heikkila, R. E. & Cabbat, F. (1976) Anal. Biochem. 75, 356362. 28. Forman, H. J. & Fridovich, I. (1973) J. Biol. Chem. 248, 2645-2649. 29. Ulmer, D. D. & Vallee, B. L. (1971) in Advances in Chemistry Series 100, ed. Gould, R. F. (Am. Chem. Soc., Washington, DC),
pp.187-218. 30. Fee, J. A. & Valentine, J. S. (1977) in Superoxide and Superoxide Dismutases, eds. Michelson, A. M., McCord, J. M. & Fridovich, I. (Academic, London), pp. 19-60. 31. Fee, J. A. & McClune, G. J. (1978) in Mechanisms of Oxidizing Enzymes, eds. Singer, T. P. & Ondarza, R. N. (Elsevier, New York), pp. 273-284. 32. Fee, J. A., Lees, A. C., Bloch, P. L. & Neidhardt, F. C. (1979) in Oxygen: Clinical and Biochemical Aspects, ed. Caughey, W. S. (Academic, New York), in press. 33. Valentine, J. S. (1979) in Oxygen: Clinical and Biochemical Aspects, ed. Caughey, W. S. (Academic, New York), in press.
Biochemistry
pH-dependent migration of copper(II) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase (metalloprotein/electron spin resonance/histidyl imidazolate)
JOAN S. VALENTINE*, MICHAEL W. PANTOLIANO*, PETER J. MCDONNELL*, ALLAN R. BURGERt, AND STEPHEN J. LIPPARDt *Department of Chemistry, Rutgers, The State University, New Brunswick, New Jersey 08903; and tDepartment of Chemistry, Columbia University, New York, New York 10027
Communicated by Ronald Breslow, May 22, 1979 ABSTRACT Bovine erythrocyte superoxide dismutase (Cu2Zn2SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of two identical subunits each containing Cu2+ and Zn2+ in close proximity. We describe here electron spin resonance (ESR) and visible absorption spectroscopic studies of the zinc-free derivative of this protein, Cu2E2SODase (E = empty) over the pH range 6-10. The ESR spectrum of the zincfree protein at 77 K is markedly pH dependent. At pH < 8.0 the ESR spectrum is axial in appearance. At pH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum is very broad and resembles that of the four-copper derivative Cu2Cu2SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30°C are also consistent with formation of Cu(II)Im-Cu(II). A plot of changes in the signal amplitude of g1 for Cu2E2SODase as a function of pH gives an apparent pKa of 8.2 for the transition. The long-wavelength absorption with Xmax = 700 nm characteristic of Cu2E2SODase shifts with increasing pH to 800 nm and the resulting visible spectrum is identical to that of Cu2Cu2SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH are reversed when the pH is decreased with HNO3, although the approach to equilibrium is slow in the latter case. The results of these experiments are consistent with a reversible, pH-dependent migration of Cu2+ from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit. By contrast, native protein, Cu2Zn2SODase, and the four-copper protein, Cu2Cu2SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu2E2SODase and its thermal stability are reexamined in light of these new findings.
Apart from studies to determine the function, mechanism, and reactivity of this protein (4), there has also been considerable interest in various structural aspects of this and the other closely related Cu-Zn proteins isolated from various eukaryotic sources (4). The interaction between metal binding sites within each protein subunit is of particular interest (6). Previously we described conformational changes induced at the empty copper binding site when zinc is bound to the apoprotein (7). Here we present evidence for the reversible, pH-dependent migration of copper(II) ion from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit and discuss its possible significance. MATERIALS AND METHODS Cu2Zn2SODase was isolated from bovine erythrocytes and purified according to the procedure of McCord and Fridovich (8). The purified enzyme was dialyzed against doubly distilled water and stored as a lyophilized powder at -20'C. The specific activity, determined by using the epinephrine assay of Misra and Fridovich (9), was 85-90% of that reported by these authors. Most preparations were found to give one or two bands on polyacrylamide electrophoresis by a modification of the method of Rigo et al. (10) in which 0.25% rather than 2.5% (wt/vol) N,N'-methylenebisacrylamide was used. Copper and zinc contents were determined by atomic absorption photometry using a Techtron AA4 instrument. The native enzyme contained 0.85-0.92 mol of copper and 0.82-0.89 mol of zinc per mole of subunits; the protein concentration was based on weighed amounts of lyophilized powder. The apoprotein was prepared by dialysis against disodium EDTA at pH 3.8 as described by McCord and Fridovich (8) followed by exhaustive dialysis against 0.1 M NaCl to remove enzyme-bound EDTA (11). The metal-free enzyme typically contained 1-2% of the amounts of copper and zinc present in the native enzyme. The zinc-free derivative, Cu2E2SODase, was prepared by first diluting the apoprotein with 10 mM sodium acetate buffer at pH 3.8 to give a final concentration of 1 mg/ml. Then 3.15 mM CuSO4 was infused at the rate of 1 ml/hr (12) until 1.0 equivalent per subunit had been added. The resulting protein solution was exhaustively dialyzed against doubly distilled water and stored as a lyophilized powder at -200C. Weighed samples
Copper-zinc superoxide dismutase (Cu2Zn2SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1), as isolated from bovine erythrocytes, contains one copper(II) atom and one zinc(II) atom (1) in each of two identical subunits (2-4). The metal ions in each subunit are bound in close proximity to one another (5, 6) and are believed to share a common ligand, an imidazolate (Im) anion derived from the side chain of histidine-61 (6).
Cu2+ -N
Z92+
JC \
Abbreviations: Cu2Zn2SODase, native bovine erythrocyte superoxide dismutase; Cu2Cu2SODase, derivative of the native enzyme in which Cu(II) has been substituted for Zn(II); Cu2E2SODase, derivative of the native enzyme in which the zinc site is vacant (E = empty); Zn-free Cu2E2SODase, designates under conditions such that the Cu ions are not necessarily in their original locations; Im, imidazolate; ESR, electron spin resonance.
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of this powder dissolved in water contained 0.95, 1.03, and 1.07 mol of copper per mole of subunits and 0.03, 0.01, and 0.05 mol of zinc per mole of subunits for three different preparations. The four-copper derivative of superoxide dismutase, Cu2Cu2SODase, in which Cu(II) is substituted for Zn(II), was prepared by two methods. The first procedure was that of Fee and Briggs (13), with CUSO4 substituted for CuC12. The other method involved the CUSO4 titration of Cu2E2SODase in 0.25 M sodium acetate buffer at pH 5.5. Both procedures gave the same results as judged by the characteristic electron spin resonance (ESR) spectrum at 77 K (see below). Adventitiously bound Cu2+ was removed by dialysis against doubly distilled water for 4 hr with no change of the dialyzate. Dialysis against acetate buffer, or further changes in the dialysate when water alone was used, caused changes in the ESR spectrum consistent with partial removal of Cu from Cu2Cu2SODase. The protein concentration of Cu2Cu2SODase was determined with the Bio-Rad protein assay kit and Cu2Zn2SODase as the standard. Metal analyses of the Cu2Cu2SODase sample used for studies described in this report indicated that it contained 1.81 equivalents of Cu and 0.04 equivalent of Zn per subunit of protein. ESR spectra
obtained by using a Varian E-12 spectrometer together with a Hewlett-Packard model 5255 A frequency meter. The magnetic field was calibrated by using the signal of Mn(II) naturally present as an impurity in strontium oxide (14). Room temperature ESR spectra were obtained by using an aqueous ESR cell from Wilmad. All ESR parameters are defined according to Malmstrom and Vannga'rd (15). Visible and near-infrared spectra were obtained by using a Cary 17D spectrophotometer. Samples were centrifuged before spectral observations in order to decrease the amount of light scattering due to small amounts of undissolved protein. The pH was adjusted with small amounts of 1 M NaOH and 1 M HNO3 and pH measurements were made with a Corning model 12 pH meter in conjunction with a thin (6 mm) Sensorex combination electrode. Doubly distilled water was used in the preparation of all solutions, and reagent grade chemicals were used throughout. were
Proc. Natl. Acad. Sci. USA 76 (1979)
Magnetic field,
gauss
FIG. 2. ESR spectrum of Zn-free SODase at pH 9.3 and 77 K. This spectrum is the same as curve c of Fig. 1 except that the gain was increased by a factor of 5.
RESULTS ESR spectra of Zn-free SODase at various pH values and 77 K in Fig. 1. At pH 7.8, the spectrum was characteristic of Cu(II) in an axially symmetric ligand environment with gll = 2.268, gm = 2.067, and All = 146 G. These values are in good are shown
agreement with
those previously reported for the zinc-free
protein (12, 13, 16). An extensive analysis of this spectrum by Beem et al. (12) has led to the conclusion that the Cu2+ ions are in the same sites as in the native Cu2Zn2SODase protein. This conclusion is also supported by the observation of distinctly
different ESR parameters for a silver-copper derivative of superoxide dismutase in which Ag+ resides in the native copper site and Cu(II) resides in the zinc-binding site (16). However, adjustment of the pH to 9.3 by slow addition of 1 M NaOH resulted in a broadening of the ESR spectrum with development of a broad trough at approximately 3600 G and the appearance of a weak signal at approximately 1540 G (Figs. 1 and 2). The final spectrum (Fig. 2) was similar to that of Cu2Cu2SODase (Fig. 3A) previously prepared by Fee and Briggs 1400 1800 2200 2600 3000 3400 3800 4200 4600
AA
B
2200 2600 3000 3400 3800 Magnetic field, gauss
FIG. 1. pH dependence of the ESR spectrum of Zn-free SODase at 77 K. The concentration of protein subunits was 2.0 mM in 2.0 ml
0.1 M potassium phosphate initially buffered at pH 7.8. Curves: ESR spectrum of the initial solution; b (pH 8.3) and c (pH 9.3), after addition of 10 and 20 ,ul of 1 M NaOH; d, after pH was readjusted downward from 9.3 to 6.5 by addition of 34 ,ul of 1 M HNO3. The spectra were recorded with 100-kHz field modulation at a modulation amplitude of 4.0 G. The microwave power was 50 mW at a frequency ot 9.13 GHz; the gain was 250 for each spectrum. The spectral variab)les for curves a, b, and d are: All = 146 G; g~l = 2.268; gm = 2.067. of
a,
2200 2400 2600 2800 3000 3200 3400 3600 3800 Magnetic field, gauss FIG. 3. ESR spectrum of Cu2Cu2SODase at 77 K (A) and at 30 ± 2°C (B). The concentration of protein subunits was 0.42 mM at pH 5.2 (unbuffered). The spectral conditions in A were similar to those in Fig. 1 except that the modulation amplitude was 10.0 G and the gain was increased by a factor of 8. In B the spectrum was similarly recorded except that the microwave power was 30 mW at a frequency of 9.41 GHz and a gain of 6300.
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Valentine et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
(5, 13). This type of spectrum is characteristic of binuclear copper(II) complexes in which there is magnetic interaction between the two metals. Similar spectra have been observed for Im-bridged and other dicopper(II) complexes and have been interpreted in terms of dipolar coupling of Cu(II) ions (17-19). The weak signal at low field arises from the Am = 2 transition characteristic of a magnetic triplet (19). Increasing the pH of a solution of Zn-free SODase thus causes the formation of Imbridged dicopper(II) centers identical to those found in Cu2Cu2SODase and several model compounds. The structure that appears between 2600 and 3200 G in the spectrum of Zn-free SODase at pH 9.3 (Fig. 2) is much more pronounced than that observed in the spectrum of Cu2Cu2SODase (Fig. 3). It is attributed to subunits that contain only one copper, which suggests that conversion of Cu2E2SODase to dicopper(II) subunits at high pH is not complete. The changes in the ESR spectrum caused by an increase in pH were complete within the time necessary to record the ESR spectrum. Readjustment of the pH down to 6.5 by stepwise addition of 1 M HNO3 eventually resulted in the reversion of the spectrum to that characteristic of the low pH form (Cu2E2SODase) as shown by curve d of Fig. 1. The approach to equilibrium was considerably slower upon decreasing the pH, however. This time hysteresis was also seen in the ESR experiment at 30°C discussed below. The ESR spectrum at 30 I 2°C was also used to follow the pH titration of Cu2E2SODase. At this temperature, copper proteins often have ESR spectra similar to those obtained in the frozen state because the slow tumbling rate of the macromolecule does not cause total averaging of the g and A tensors (20). This situation obtained for Cu2E2SODase. The spectrum at pH 7.4 (Fig. 4) had gli = 2.267, gm = 2.073, and All = 140 G. The ESR spectrum of Cu2Cu2SODase, by contrast, showed no signal at 30°C (Fig. 3B), a feature commonly observed in the spectra of low molecular weight Cu(II) dimers (18, 19). Fig. 4 shows the effect of increasing the pH on the ESR spectrum of Zn-free SODase at 30°C. A marked decrease in signal intensity occurred but without the broadening observed with increasing pH in the frozen solution ESR spectra (compare Fig. 1). The signal intensity decreased with increasing pH until pH 9.4. Increasing the pH to 9.8 caused no further change in the spectrum. The gII (2.254) and A1I (145 G) of the species at pH 9.4 and 9.8 are slightly different from those of the initial
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a
b C
d e
2600 2800 3000 3200 3400 3600 3800 Magnetic field, gauss
FIG. 5. Reversibility of the pH-dependent transition of Zn-free SODase. To 0.5 ml of the pH 9.8 sample described in Fig. 4 was added 16 Ail of 1 M HNO3 to adjust the pH to 7.1. The spectrum before addition of acid is reproduced as curve a. ESR spectra of the sample at various times after the addition of acid are shown in curves b-e (b, 1.5 hr; c, 8.5; d, 10.5; e, 12.5). The pH of the solution after 14.5 hr was 7.2. Spectral conditions are exactly as described for Fig. 3B.
solution at pH 7.4. These small changes began to occur above pH 8.4. The loss of signal intensity as the pH is increased is the consequence of converting most of the mononuclear copper subunits of Cu2E2SODase to ESR silent forms. This behavior is exactly that expected if binuclear copper subunits form because subunits of this kind give no ESR signal at 30°C in solutions of Cu2Cu2SODase (Fig. 3B). The small changes that occur in the lineshape of the ESR spectrum at high pH may reflect deprotonation of histidyl ligands or of water bound to copper, or they may be due to small pH-dependent, reversible, conformational changes. The reversibility of the pH titration at 30'C is illustrated in Fig. 5. The rate of reverse reaction was slow. Reestablishment of equilibrium after adjustment of the pH from 9.8 to 7.2 required 12.5 hr at room temperature. Increasing the pH to 10.3 resulted in the irreversible formation of a purple solution. The signal amplitude (peak-to-peak height) in the gI region of the spectra shown in Fig. 4 is plotted as a function of pH in
a
b
A
d
f 2600 2800 3000 3200 3400 3600 3800
Magnetic field,
gauss
FIG. 4. pH dependence of the ESR spectrum of Zn-free SODase at 30 2°C. The protein subunit concentration was 1.3 mM in 0.1 M potassium phosphate initially buffered at pH 7.4 (curve a). The pH then was adjusted downward with 1 M NaOH to give spectra b-f (b, j)H 8.0; c, 8.4; d, 9.0: e, 9.4; f, 9.8). A total of 24 ,l of base was added to 1.0 ml of protein solution. The spectra were recorded as in Fig. 3B. For a-c: All = 140 G, g~l = 2.267, gm = 2.073. For f: All = 145 G, gII = 2.254.
pH FIG. 6. pH dependence of signal amplitude in the gI region of the ESR spectrum of Zn-free SODase at 30 20C. The data were obtained from three separate experiments similar to the experiment described in Fig. 4. In two experiments (0 and ,), the protein subunit concentration was 1.9 mM. In the third (-), the concentration was 1.3 mM. The line represents a theoretical acid-base titration curve for a single ionizing group of pKa = 8.2. Error bar represents largest measured range. +
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Biochemistry: Valentine et al.
Fig. 6. If it is assumed Zn-free SODase is partially converted to an ESR silent form as the pH is increased, the signal amplitude may be used to estimate the concentration of the remaining mononuclear copper subunits. A strict quantitative comparison of relative intensities at various pH values is not possible because of lineshape changes in the high pH spectrum (curve f in Fig. 4). Because the change in peak-to-peak width is small, however, the peak-to-peak amplitudes of the derivative spectra should remain proportional to intensities (21). Any error introduced is unlikely to be larger than the inherent irreproducibility of the measured signal amplitude, denoted by error bar in Fig. 6. As the pH increased, the signal amplitude decreased until it leveled off at pH > 9.4 at 25% of its original value. This result indicates that 25% of the subunits contain mononuclear copper at high pH. The presence of mononuclear copper is also evident in the high pH ESR spectrum at 77 K as discussed above. The shape of the plot of signal amplitude versus pH (Fig. 6) resembles that of a titration curve with an apparent pKa of 8.2. Changes in the visible absorption spectrum of Zn-free SODase were also studied as a function of pH. The longwavelength absorption at XmaX = 700 nm characteristic of Cu2E2SODase (12, 13) shifted, with increasing pH, to t800 nm. The resulting spectrum was identical to that of Cu2Cu2SODase (13) (Fig. 7). In addition to the long-wavelength band, both spectra showed a shoulder at 450 nm, also exhibited by native Cu2Zn2SODase (22) but not by Cu2E2SODase (13). This shoulder is thought to be characteristic of protein derivatives in which Im bridges two metal ions (22). The visible spectral results are consistent with the formation of binuclear copper subunits in Zn-free SODase at high pH.
DISCUSSION Increasing the pH of solutions of Cu2E2SODase brings about changes in the ESR spectra of solutions at 30'C and of frozen solutions (77 K) as well as in the visible absorption spectrum. Spectra characteristic of the four-copper derivative Cu2Cu2SODase are observed. The changes are fully reversed when the pH is decreased, although the rate of approach to equilibrium is considerably slower when the pH is decreased than when it is increased. These observations demonstrate that a reversible pH-dependent migration of copper(II) ion from one subunit to the zinc site of another subunit occurs.
2
Cu2+ _Nk, N H
N
§VN--H
The pH titration of the ESR signal amplitude at 30°C (Fig. 6) indicates that the apparent pKa for this transition is 8.2. The leveling off of the signal amplitude at 25% of its original value above pH 9.4 suggests that at high pH 25% of the subunits remaining contain mononuclear copper. The absence of similar pH-dependent spectral changes for Cu2Cu2SODase supports this interpretation. It is instructive to compare the pH-dependent behavior of native Cu2Zn2SODase and Cu2Cu2SODase with that described above for Cu2E2SODase. In the first two cases, both metal binding sites in each subunit are occupied. In those cases, no pH-dependent spectral changes are observed until the pH is
.M
I
00.25
400
500
600 700 800 Wavelength, nm
900
1 000
FIG. 7. Visible absorption spectra: - -, Cu2Zn2SODase, 0.96 mM (subunits), pH = 6.3; -, Zn-free SODase, 0.96 mM, pH = 6.2; - - -, Zn-free SODase, 0.96 mM pH 10.1 by addition of 95 ,l of 1 M NaOH to 1.5 ml of protein solution; - - -, Cu2Cu2SODase, 0.42 mM (subunits), pH 5.2 (in dilute sodium acetate buffer; all others in 0.1 M potassium phosphate).
relatively low. In the case of Cu2Zn2SODase, the bridging histidine-61 apparently is protonated at pH < 4, breaking the bond between the imidazole nitrogen and the zinc atom (22-24). In the case of Cu2Cu2SODase, at pH < 4.5, Cu2+ apparently dissociates from the native zinc site to give Cu2E2SODase (unpublished results). The situation is different for Cu2E2SODase because each subunit has one occupied and one unoccupied metal binding site. Thus, in this zinc-free derivative, there is competition between the two metal binding sites for the Cu2 . At low pH, the native copper binding site apparently has a higher affinity for Cu2+ than does the zinc-binding site. At higher pH, however, there is apparently a preference for binding Cu2+ as Im-bridged binuclear pairs. This preferred configuration for the rearrangement of the metals at high pH requires deprotonation of histidine-61. The reaction is remarkably similar to that observed in the pH titration of equimolar imidazole and tetramethyldiethylenetriamine Cu2+ (17) or CH3Hg+ (25). Here also as the pH is increased the Imbridged bis-metal derivative is favored. Although deprotonation of histidine-61 and formation of Im-bridged binuclear copper pairs is the most likely explanation for the pH-dependent spectral changes, a pH-dependent conformational change cannot be absolutely ruled out. The shape of Fig. 6 strongly implies that only one deprotonation is involved, however, and the spectrum of Fig. 2 is uniquely characteristic of the Cu(II)-Im-Cu(II) moiety (17). Other examples of pH-dependent migration of metal ions in metalloproteins occur in the literature and are expected of, proteins that contain several binding sites with different proton and metal ion affinities (reviewed in ref. 26). The present case is unique, however, because Cu(II) migrates only to subunits that already contain a Cu(II) ion. Some previous results concerning the activity of the zinc-free protein as well as the effect of metal ions on its thermal stability must be reexamined in light of the results described above. Other investigators (12, 13) have measured the activity of this protein derivative at pH 7.8 and obtained widely varying results. This variation probably was due at least in part to the migration of copper at high pH as described above. Cu2+ in the zinc-binding site gives little or no superoxide dismutase activity. This conclusion arises from the observation that Ag2Cu2SODase, in which copper is in the native zinc site, has almost no activity
Biochemistry: Valentine et al. (16) and from the fact that Cu2Cu2SODase has the same activity as Cu2Zn2SODase (13) in spite of the fact that Cu has been substituted for Zn, thereby doubling the total Cu concentration. Thus, the migration that we observe takes Cu from a site on the protein where it is catalytically active to another site where is is not, resulting in a lowering of the overall superoxide dismutase activity of the protein. We have measured the activity of our preparation of zinc-free enzyme at pH 6.0, where most of the Cu is in the native Cu site, by using a modification of the 6-hydroxydopamine assay of Heikkila and Cabbat (27). This assay is based on the inhibitory effect of superoxide dismutase on the initial rate of autoxidation of 6-hydroxydopamine and allows measurements in the pH range in which the copper of Cu2E2SODase is predominantly in the native binding site. At pH 6.0 it was found that Cu2E2SODase is at least 80 i 5% as active as the native enzyme. It would appear, therefore, that the presence of Zn2+ in Cu2Zn2SODase does not greatly enhance the activity of the neighboring Cu as previously reported (13). The observed lower activity (12, 13) is presumably due to the fact that most superoxide dismutase activity measurements are carried out at relatively high pH, at which a rearrangement of Cu ions has occurred. The other point to be considered is contained in a report (28) that Cu2Zn2SODase is more stable to thermal inactivation at pH 7.8 than is Zn-free SODase. From Fig. 6 it can be estimated that at pH 7.8 at least 10% of the subunits are in the apo form. The apoprotein is much less stable to thermal inactivation than are derivatives that contain metals (28, 29). It is possible, therefore, that the source of the apparent thermal instability of the Zn-free SODase at pH 7.8 relative to the native protein is the apo subunits that are present at that pH. The metal ion migration in Cu2E2SODase may have little physiological significance if the protein is fully saturated with metals in vivo and its primary function is to catalyze superoxide dismutation. Doubts have been expressed, however, concerning the true function of this protein owing to the absence of a chemical explanation for the source of the proposed toxicity of siperoxide (30-33). If dismutation of superoxide is not the primary function of this protein, one is led to consider alternative functions. The kinetic lability and thermodynamic affinities of the metal binding sites of this protein may in fact be }ighly relevant to the physiological function of this protein if, after all, it turns out that its primary function is to transport or store copper and zinc ions rather than to catalyze superoxide dismutation. We thank J. Burstyn and G. Fox for technical assistance and M. Nappa for his programming skills in computer graphics. J.S.V. also thanks Professor J. A. Fee for helpful discussions. Financial support from the Charles and Johanna Busch Memorial Fund (to J.S.V.), U.S. Public Health Service Grants GM 24759 (to J.S.V.) and GM 16449 (to S.J.L.), National Science Foundation Grant CHE 75-14463 (to J.S.V.), a Johnson and Johnson Fellowship in the Biological Sciences for 1978-79 (to M.W.P.), and National Institutes of Health Training Grant GMO 7216 (to A.R.B.) is gratefully acknowledged. J.S.V. also acknowledges receipt of a U.S. Public Health Service Research Career Development Award. 1. Carrico, R. J. & Deutsch, H. F. (1970) J. Biol. Chem. 245, 723-727. 2. Richardson, J. S., Thomas, K. A. & Richardson, D. C. (1975) Biochem. Biophys. Res. Commun. 63,986-992. 3. Richardson, J. S., Thomas, K. A., Rubin, B. H. & Richardson, D. C. (1975) Proc. Natl. Acad. Sci. USA 72, 1349-1353.
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pp.187-218. 30. Fee, J. A. & Valentine, J. S. (1977) in Superoxide and Superoxide Dismutases, eds. Michelson, A. M., McCord, J. M. & Fridovich, I. (Academic, London), pp. 19-60. 31. Fee, J. A. & McClune, G. J. (1978) in Mechanisms of Oxidizing Enzymes, eds. Singer, T. P. & Ondarza, R. N. (Elsevier, New York), pp. 273-284. 32. Fee, J. A., Lees, A. C., Bloch, P. L. & Neidhardt, F. C. (1979) in Oxygen: Clinical and Biochemical Aspects, ed. Caughey, W. S. (Academic, New York), in press. 33. Valentine, J. S. (1979) in Oxygen: Clinical and Biochemical Aspects, ed. Caughey, W. S. (Academic, New York), in press.
