ELSEVIER Bioelectrochemistry and Bioenergetics 39 (1996) 291-294

Short communication

A study on reduction potentials of porcine and bovine superoxide dismutase and dissociation of bridging imidazolate

Wen Qian, Qin-Hui Luo *, Meng-Chang Shen Coordination Chemistry Institute, Coordination Chemistry State Key Laboratory, Nanjing University, Nanjing 210093, People's Republic of China

Received 1 June 1995; in revised form 31 August 1995

Keywords: Superoxide dismutase; Imidazolase bridging; Platinum working electrode

1. Introduction

Superoxide dismutase (SOD) is a scavenger of the superoxide anion [1] and plays an important role in protect- ing cells from the toxic effects of the superoxide anion and various species derived from it [2-5]. Cu2Zn2SOD is composed of two subunits, each of which contains one copper(II) and one zinc(II) ion linked by a histidine imida- zolate bridge (his-63) [6,7]. Generally chemists believe that the catalytic dismutation of 02 by Cu2Zn2SOD involves alternate reduction and reoxidation of the Cu(II) centre [8,9]. ECu(II) + 02 --) ECu(I) + 02

ECu(I) + O~ + 2H +-'-) ECu(II) + n202

In this mechanism, reduction of copper is accompanied by protonation of the bridging imidazolate group. Reduction of copper results in the uptake of one proton per subunit [101.

Among the many factors that affect the activity of SOD, the oxidation-reduction potential of the copper(II) centre in Cu2Zn2SOD plays a major role. Although some data on oxidation-reduction potentials of Cu2Zn2SOD from bovine erythrocytes have been reported [11-14], there are differences among them. The reported potential values were in the range 0.28-0.42 V(NHE) (NHE, normal hy- drogen electrode) at pH 7. Some authors [13,14] pointed out that the variability in potential values depended on the protein preparation from different sources and redox medi- ators. However, other authors [12,15] indicated that the presence of mediators did not affect the reduction potential

* Corresponding author.

0302-4598/96/$15.00 © 1996 Elsevier Science S.A. All rights reserved SSDI 0302-4598(95)01 883 -2

of SOD. Therefore we studied the reduction potentials (midpoint potentials) E m of porcine and bovine superoxide dismutase (PE-SOD and BE-SOD) by coulometric titration with methyl vioiogen as mediator. The results show that PE-SOD and BE-SOD have the same reduction potential values within certain error limits, namely the same poten- tial values were obtained for SOD prepared from different sources. We studied systematically the relationship be- tween E m and the pH or standing time of freshly prepared solutions and obtained the protonation constants of the bridging imidazolate group of SOD in oxidized and re- duced forms (PKox and pK~d). The results are useful for interpreting the behaviour of the bridging imidazolate of SOD.

2. Experimental details

2.1. Materials

BE-SOD and PE-SOD were prepared from bovine and porcine blood respectively by methods of previous authors [1]. The raw extracts of SOD were purified by DEAE-cel- lulose 32 and Sephadex G 50 successively. Each of the purified BE-SOD and PE-SOD had only one homogeneous and clear band in polyacrylamide gel electrophoresis. The relative molecular mass of PE-SOD was measured as 32 000 by SDS-polyacrylamide gel electrophoresis; its Cu and Zn contents were measured by inductively coupled plasma (ICP) spectrometry as 0.39% and 0.40% respec- tively. The relative molecular mass of BE-SOD was mea- sured as 32 800 by gel electrophoresis; its Cu and Zn contents were measured by ICP spectrometry as 0.35% and 0.37% respectively. The specific activities were measured

292 W. Qian et aL / Bioelectrochemistry and Bioenergetics 39 (1996) 291-294

as 3500 unit mg- 1 for PE-SOD and 6000 unit mg- ~ for BE-SOD by the xanthine oxidase-cytochrome C assay method [1]. The parameters of electron paramagnetic reso- nance (EPR) and peak values of UV spectra were in agreement with those in previous reports [16,17]. Methyl viologen was purchased from Fluka Co. Buffer solutions with pH 6.0-8.0 consisted of phosphate and trihydrox- ymethylaminomethane ('Iris). Buffer solutions with pH < 6 consisted of phosphate and citrate. The ionic strength was kept constant by using KC1 (AR). All reagents were recrys- tallized in doubling distilled water.

2.2. Methods

The electrochemical cell consisted of a glass cell and four electrodes, namely a platinum foil working electrode with an approximately 2 cm 2 surface area, a Pt coil wire auxiliary electrode separated from the bulk solution by a salt bridge, a Pt foil measuring electrode and a saturated calomel reference electrode (SCE). The working and auxil- iary electrodes were connected to a galvanostat and the measuring and reference electrodes were connected to a Coming Model 130 pH meter for measuring the potential values. The measured potential values (vs. SCE) were converted into values vs. NHE. pH values were calibrated by using standard phosphate buffer solution (pH 6.88, 25°C). The efficiency of the coulometric titration was checked several times to be about 99.5% by using 12 - Na2S203 solutions. Before each experiment the Pt work- ing electrode was polished carefully with chromatographic grade A1203 and then electrolysed, acting as anode and cathode alternately, in 0.1 mol dm -3 HNO 3 for 1-2 min. The Pt working electrode was treated repeatedly until a stable potential value was obtained [18]. The solutions approximately contained 2 × 10 -5 mol dm -3 SOD, 2.6 × 10 -3 mol dm -3 methyl viologen, 0.1 mol dm -3 KC1 and 5 × 10 -2 mol dm -3 Tris-phosphate buffer. Superpure ni- trogen gas was bubbled through freshly prepared solutions in the electrochemical cell for about 5 min and coulometric titration curves were recorded up to at least 1.5 h after the solutions had been prepared. However, for investigating the variation in E m with the standing time of solutions, the curves were recorded after the freshly prepared solutions had stood for predetermined times at 25°C. It was con- finned that the activities of the SOD solutions did not vary during standing.

3. Results and discussion

E (V.vs. NHE)

0.30t I

0 . 4 0

0 . 5 0

o'.5 Iio 1'.5 215 31o a ( equ / rnol )

Fig. 1. Coulometric titration curves of PE-SOD. 1, pH 6.0; 2, pH 7.0; 3, pH 7.5. All solutions contained 2.0X 10 -5 moldm -3 PE-SOD, 5.0X l0 -2 mol dm -3 Tris-phosphate, 0.1 mol dm -3 KCI and 2.6× l0 -3 tool dm -3 MV +. The titration current was 250 #A.

the normal behaviour for SOD. The two copper centres in SOD were not distinguishable from one another by coulo- metric titration. The reduction of Cu(II) was completed in the range a = 2.0-2.3. The net reduction stoichiometry is slightly greater than 2 equ. /mol of SOD and is probably due to the reduction of H + by the MV + radical [19].

Table 1 lists the midpoint potentials E m of PE-SOD and BE-SOD obtained by the method in Ref. [20] The number of electrons, n, of the reduction reaction was calculated from the slope of the straight line obtained by plotting E vs. log[Cu(I)/eu(II)] (Table 2).

From Table 1 it may be seen that BE-SOD and PE-SOD have the same redox potentials, i.e., SOD samples pre- pared from different sources have the same values of redox potential. Our results are very close to the values reported by Fee and DiCorleto [11] and St. Clair et al. [12]. This implies that values of E m have no obvious variance with different experimental methods. Because our experimental conditions (such as pH and ionic strength of solutions) were different from those of the above authors, our results are slightly different from theirs. Lawrence and Sawyer [13] used impure SOD which showed three bands in the gel electrophoretic assay. Their titration curves showed anamalous behaviour for SOD and a break appeared in the region a < 1. The existence of impurities in SOD caused an erroneous response of the electrodes. In our experi- ments the SOD samples were purified strictly and the electrodes in the coulometric titration were treated rigor- ously.

3.1. Reduction potentials 3.2. Binding of imidazolate bridge with protons

Fig. 1 shows the coulometric titration curves of PE-SOD at pH 6.0, 7.0 and 7.5 which were obtained by plotting the reduction potential values E vs. a, the equivalents of Cu(I) per mole of SOD, i.e. a = Cu(I)/Cso o. They display

It was found that the E m values changed with the standing time t of the SOD solutions (Fig. 2). In Fig. 2 for t < 20 min and t > 75 rain the E m values were constant, but in the range t = 20-75 min they increased rapidly with

W. Qian et a l . / Bioelectrochemistry and Bioenergetics 39 (1996) 291-294

Table 1 Midpoint potentials (V (NHE)) of PE-SOD and BE-SOD at 25°C

293

pH 6.00 pH 6.50 pH 7.00 pH 7.50 pH 8.00

PE-SOD (20 min) 0.436 + 0.005 0.414 + 0.003 0.406 + 0.005 0.390 + 0,002 0.375 + 0.003 BE-SOD (20 rain) 0.435 -1- 0.003 0.404 + 0.004 0.372 + 0.005 PE-SOD (75 rain) 0.488 + 0.005 0,466 + 0.005 0.435 + 0.005 0.405 + 0.005 0.370 + 0.005 BE-SOD (75 min) 0.484 + 0.008 0.436 + 0.005 0.370 -1- 0.006 BE-SOD [ 13] 0.35, 0.28 0.30, 0.21 BE-SOD [11] 0.422, (pH 6.56) 0.42 0.433 (pH 8.7) BE-SOD [ 12] 0.403 BE-SOD [14] 0.32 (pH 7.4)

Table 2 Number of electrons, n, of reduction reactions of PE-SOD and BE-SOD (t < 20 min)

PE-SOD BE-SOD

E m (V) pH Slope n Era (V) pH Slope n

0.406 7.00 0.069 0.86 0.435 6.00 0.063 0.94 0.390 7.50 0.065 0.91 0.404 7.00 0.065 0.91 0.376 8.00 0.063 0.94 0.372 8.00 0.065 0.91

increasing time. Therefore we measured the pH-dependent changes in E m under conditions where the freshly pre- pared solutions were allowed to stand for less than 20 min and for more than 75 min. By plotting E m vs. pH, curves 1 and 2 in Fig. 3 were obtained. Between about pH 5.0 and 8.2, E m was a linear function of pH, the slopes of the straight lines being 0.029 (curve 1) and 0.059 (curve 2). The average numbers of protons bound to the bridging imidazolate were obtained as p = 0.5 and 1.0 for curves 1 and 2 respectively. This implies that protonation of the bridging imidazolate accompanied the reduction of SOD, but that protonation was incomplete within 20 min, the

Em(V.v$. NHE)

0.43

0.42

0,41

20 60 ,oo ,8o

t (rain)

Fig. 2. Variation in midpoint potential of PE-SOD with standing time at 25°C, pH 7.0. All conditions were the same as in Fig. I.

bridging imidazolate of each subunit of SOD only taking up half a proton an average; after 75 min each subunit took up one proton. Thus the two bridging imidazolates of SOD took up two protons in two steps, one of them binding the proton rapidly and the other one slowly. Previous authors [21,22] investigated the time course of pH-dependent spec- tral changes in BE-SOD solution and found that equilib- rium was reached after at least 1 h. Their results are in agreement with ours. Furthermore, both curves 1 and 2 in Fig. 3 turned at about pH 5 and 8.2, and in the ranges pH < 5 and pH > 8 the change in E m with pH was less. This means that at pH ~ 5.0 protonation of the imidazolate nitrogen facing zinc caused the bridging ligand to break [22], while at pH >i 8.2 protonation of the bridging imida- zolate did not occur during the reduction of Cu(II) of SOD. Curves 1 and 2 crossed at about pH 7.8. At this pH the E m values did not depend on the standing time of the SOD solution and the bridging imidazolate absorbed protons very fast. Therefore it is obvious that in vivo the bridging imidazolate of SOD also takes up protons fast at physio- logical pH. Based on the above discussion and previous

E,~(V.v=. NHE)

0.48

0 .44

0 .40

0 .36

2

I

3'.o 4'.o 5'.o e'.o 7'.o 91o pH

Fig. 3. Variation in midpoint potential of PE-SOD with pH. 1, determined within 20 rain ( p = 0.5); 2, determined after 90 min ( p = 1.0). All conditions were the same as in Fig. 1.

294 W. Qian et al. / Bioelectrochemistry and Bioenergetics 39 (1996) 291-294

Table 3 pK a values of bridging imidazolate of oxidized and reduced PE-SOD

PKox pKred

p = 0.5 p = 1.0 p = 0.5 p = 1.0

PE-SOD 5.36 + 0.04 5.39 + 0.03 8.12 + 0.30 8.04 + 0.14 BE-SOD [14] 8.2 +0 .6

authors' suggestions [22,23], we present the reduction pro- cess of SOD as follows:

5 < p H < 8

p H > 8

(Cut ,__NF-~N--Znn) + e - . " (Cu, N / r ' ~ N - - Z n ~')

(l) p H < 5

z n , ) + e

.

3.3. Protonation constants

The protonation constants pKox and PKre d may be calculated via Eqs. (4) and (5) below using the values of pH and corresponding E m of curves 1 and 2 in Fig. 3. The protonation constants are defined by

[H + l[ox] Ko,~= [Ho~ ] (2)

[H + ][red] Kred = [Hred ] (3)

where [ox] and [red] denote the concentrations of oxidized and reduced Cu forms in SOD respectively and [Ho~ ] and [Hred] denote the concentrations of their respective proto- nated species. Then

p H > 5 Em = E°m

p H < 8 Em-- El°m + ~

00 9 / Ko,/ - - p log 1 + (4)

n ~ [ H + ] /

0.059 ([H+]) p log 1 + (5)

n Kre d

where p = 0.5 or 1 and El°m denotes the limit value of E m. In Table 3 we give the values of PKox and pKre a obtained by fitting curve 1 or 2 in Fig. 3 by Eq. (4) or (5).

The pK a (pKre d or PKo~) values in Table 3 were calculated from the E m and corresponding p values. From Table 3 it is seen that the pK a values calculated from the

two curves ( p = 0.5 or 1.0) in Fig. 3 are very approxi- mate. This means that although the bridging imidazolates of the two subunits bind protons in two steps, they have the same pK a values after the protonation has reached equilibrium. The value of PKox is reported for the first time. The values of PK~d are in agreement with those obtained by cyclic voltammetry [14].

Acknowledgements

This project was supported by the Natural Science Foundation of China and Fuzhou State Key Laboratory of Structural Chemistry.

References

[I] J.M. McCord and I. Fridovich, J. Biol. Chem., 224 (1969) 6049. [2] J.S. Valentine and M.W. Pantoliano, in T.G. Spiro (ed.), Copper

Proteins, Vol. 3, Wiley, New York, 1981, p. 291. [3] H.M. Hassan and I. Fridovich, Arch. Biochem. Biophys., 196 (1979)

385. [4] B. Halliwell and J.M.C. Gutteride, Arch. Biochem. Biophys., 246

(1986) 501. [5] B.P. Sharonov and I.V. Churilova, Biochem. Biophys. Res. Com-

mun., 189 0992) 1129. [6] J.A. Tainer, E.D. Getzoff, K.M. Beem, J.S. Richardson and D.C.

Richardson, J. Mol. Biol., 160 (1982) 181. [7] J.S. Valentine, J. Chem. Educ., 62 (1985) 990. [8] S. Goldstein and G. Czapski, Inorg. Chem., 89 (1985) 1087. [9] J. Rabani, D. Klug-Roth and J. Lille, J. Phys. Chem., 77 (1973)

1169. [I0] K.G. Strothkamp and S.J. Lippard, Acc. Chem. Res., 15 (1982) 318. [l I] J.A. Fee and P.E. DiCorleto, Biochemistry, 2 (1973) 4893. [12] C.S. St. Clair, H.B. Gray and J.S. Valentine, Inorg. Chem., 31

(I 992) 925. [13] G.D. Lawrence and D.T. Sawyer, Biochemistry, 18 (1973) 3045. [14] H.A. Azab, L. Banci, M. Borsari, C. Lachinat, M. Sola and M.S.

Viezzoli, Inorg. Chem., 31 (1992) 4649. [15] C.S. St. Clair, Ph.D. Thesis, California Institute of Technology,

1989. [16] M.W. Pantoliano, J.S. Valentine and L.A. Nafie, J. Am. Chem. Soc.,

I04 (1982) 6310. [17] I. Bertini, L. Banci and M. Piccili, Coord. Chem. Rev., I00 (1990)

67. [18] I.M. Kolthoff and N. Tanaka, Anal. Chem., 26 (1954) 632. [19] I.V. Shelepin and O.A. Ushakov, Russ. J. Phys. Chem., 49 (1975)

I021. [20] R. Frederic and P. Michel, J. Chem. Educ., 69 (1992) 245. [21] M.W. Pantoliano, J.S. Valentine, RJ. Mammone and D.M. Scholler,

J. Am. Chem. Soc., 104 (1982) 1717. [22] J.A. Fee and W.D. Phillips, Biochem. Biophys. Acta, 412 (1975) 26. [23] J.A. Tainer, E.D. Getzoff, J.S. Richardson and D.C. Richardson,

Nature, 306 (1983) 284.