A pulse radiolysis study of the catalytic dismutation of superoxide ion by a superoxide dismutase model compound [CU(aptn)](ClO4)2

Radiat. Phys. Chem. Vol. 49, No. 5, pp. 527-530, 1997

~

Pergamon

PII:S0969-806X(96)00183-1

© 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0969-806X/97 $17.00- 0.00

A P U L S E R A D I O L Y S I S S T U D Y OF THE C A T A L Y T I C D I S M U T A T I O N O F S U P E R O X I D E ION BY A S U P E R O X I D E D I S M U T A S E M O D E L C O M P O U N D [CU(APTN)](CLO4)2 JUN WANG, 1 QIN-HUI LUO,t I JIAN-JUN ZHANG, I M E N G - C H A N G SHEN, 1 A N - D O N G LIU, 2 HONG-CHUN GU, 2 FENG-MEI LF and SHAO-JIE DF ~Coordination Chemistry State Key Laboratory, Coordination Chemistry Institute, Nanjing University, Nanjing 210093, P. R. China and 2Institute of Low Energy Nuclear Physics, Beijing Normal University, Beijing 100875. P. R. China (Accepted 11 November 1996)

Abstract--The kinetics of catalytic dismutation of superoxide ion by N,N'-butylene bis-(2-acetyl-pyridine iminato) diperchlorate copper [Cu(aptn)]CIO4)2 was studied by pulse radiolysis techniques. The experimental results show that the catalytic mechanism of the coordination compound is different from Cu-Zn superoxide dismutase (SOD) which is a first order reaction with regard to "Oz-. A reverse reaction is involved here in the catalytic mechanism, namely, re-oxidation of the reduced product [Cu~(aptn)](CIO4) can not be ignored. The rate constants k=t, kl, k2 and k_ are reported. © 1997 Elsevier Science Ltd

INTRODUCTION

Cu(I) + 'O2--~H~CU(II) + H202.

Superoxide ion is one of the metabolic products of oxygen. The accumulation of " 0 2 in vivo can cause inflammation, cancer, and some other diseases (Digiuseppi and Fridovich, 1984). Superoxide dismutase (SOD) was an effective scavenger of superoxide anion (McCord and Fridovich, 1969) and has the function of protecting living bodies from damage of -O£, but the clinical application of SOD enzyme is limited due to its high prices and difficulty in permeating through cell membrane. Studies show that many copper(II) complexes with low molecular weights also have SOD-like function for dismutating •O2 (Tian et al., 1992; Vrgtle and Weber, 1986), therefore people pay attention to these complexes for as SOD mimics. We have synthesized a series of Schiff base copper(II) complexes (Luo et al., 1993; Lu et al., 1993) by condensation of 2-acetylpyridine with polyamines in the presence of Cu(C104)2, and studied the relationships between the structures of complexes and their activities of .O£ dismutation. In this work, the pulse radiolysis method was used to investigate the catalytic mechanism of "O2 dismutation by the representative complex of the Schiff base ligand N,N'-butylene bis-(2-acetylpyridine iminato) diperchlorate copper [Cu(aptn)](C104)2 (Fig. 1). The catalytic mechanism of Cu,Zn superoxide dismutase (SOD) has been shown to involve alternate reduction and reoxidation of the copper ion (II): Cu(II) + .O£ ~ C u ( I ) + O2 tTo whom all correspondence should be addressed.

A lot of studies (Jouini et al., 1986; Younes et al., 1978) showed that the catalytic mechanisms of ' 0 2 dismutation by low molecular weight copper (II) complexes were similar to that of Cu,Zn-SOD enzyme. But our copper(II) complex exhibited a different catalytic mechanism. The catalytic process involved a reverse reaction, i.e. re-oxidation of the reduced product [Cu~(aptn)](C104) by oxygen molecule can not be ignored. In this paper the novel kinetic mechanism is discussed and it might provide helpful information for design of new model complexes.

EXPERIMENTAL Materials

[Cu(aptn)]ClO4)2 was synthesized by condensation of 2-acetylpyridine with 1,4-butanediamine in the presence of Cu(C10#)2 (molar ratio 2:1:1) in methanol solution according to the previously reported methods (Luo et al., 1993). Elemental analysis, conductivity and IR spectrum of the complex are consistent with its chemical formula. HCOONa, KH2PO4 and K2HPO4 were of A.R. and recrystallized twice in twice distilled water. The twice distilled water was used in all reaction solutions. The concentration of the complex were about 2.4 x 10 -5 and 1.2 × 1 0 - 4 m o l d m -3. The solutions of complex contained 10% excess of ligand over the complex, 0.1 tool dm -3 HCOONa and 0.05 mol dm -3 phosphate buffer for controlling pH values. The slight

527

H3c

Jun Wang et al.

528

(CH2)4"~ N v

the electron beam injected into solutions. "02 concentration produced by one single electron pulse was calculated to be 7.2 x 10 -5 mol dm -3.

CH

\cu /

(CIO4) 2 RESULTS AND DISCUSSION

After the electron beam radiolyzed the oxygenated aqueous solutions, the following species are primarily formed: Fig. 1. Structure formula of [Cu(aptn)]ClO4)> H20--*'OH, "H, H2, "eaq, H202, excess of ligand over complex was used to scavenge the possible trace metal ions in solutions.

At sufficiently high concentrations, the formate anion immediately converts the free radicals into .O£ according to the following reaction:

Apparatus and experimental methods

"OH + H C O O

The apparatus consists of a BF-5 linear electron accelerator and optical monitor system. Experimental methods have been reported (Luo et al., 1995) and the dose of electron pulse was determined t o be 136 Gy. Before the electron beam was injected into the reaction cell, which was made of Suprasil quartz (diameter 1.0 cm, length 1.5 cm), superpure oxygen gas was bubbled into the solutions for 15 min, therefore the oxygen concentration in solutions was about 1.5 × 10 - 3 mol dm 3 at 15°C and 1.7 × 10 -3 mol dm -3 at 10°C. The yield of .O£ in the oxygenated formate solutions was assumed to be six molecules/100 eV (Bielski et al., 1977). The molar extinction coefficient of .O£ at 250 nm is 2000 mol-1 dm 3 c m - i. The concentrations of complex in experiments were 2.36 × 10 -5 mol dm -3 at 15°C and 1.18 × 10 -4 mol dm -3 at 10°C. The optical density O D of "02 were recorded immediately after

7~ 0.8 I-

"HO2~-~H + + " 0 2 , p k = 4.8 (Getoff and Prucha, 1983) " C O O - + O 2 ~ ' O £ + CO2 "eaq + O 2 - - - ~ ' O 2 - .

The trace metal ions in the solutions would enhance the rate of -O£ dismutation and lead to incorrect results, therefore a blank solution (the solution without copper complex) at p H 7.4 was measured and the second-order reaction rate constant of spontaneous dismutation of .O£ of k ~ = 3 . 9 × 105mol ~dm 3s-~ was obtained. The result is in agreement with that reported by Bielski et al. (1977) (ks = 4.0 × 105 mol ~ dm 3 s - ~, p H 7.0).

.~.~1~

d

'

~H20 + "COO-

•H + O2---~'HO2

~ 2.0 ~- (a)

10

H30 +

-

~ 2.0 ~ (b) 7"~ 0.8 F 04

0

S

O

t

I

I

I

I

I

1

2

3

4

5

6

ms Fig. 2. Plot of concentration of .O£ vs time t. [Cu(aptn)]CIO4)_,: 2.36 x 10 -5 mol dm-3; aptn: 2.4 x 10 -6 mol dm-3; HCOONa: 0.1 mol dm-3; pH = 7.0. Inset (a): 1n[-O2-]0-- ln[.O£], vs t. Inset (b): l / [ ' O ~ ] t - 1/['O2-]0 vs t.

Catalytic dismutation of superoxide ion

529

20

16

12

o

I

0

1

! 2

I 3

I 4

I 5

I 6

ms

Fig. 3. Plot of OD ('02-) at 250 nm vs t under same condition as Fig. 2. The broken line denotes the experimental data, smooth curve denotes the fitted line. Therefore, the impurities existing in the tested solution can be ignored. Dismutation o f superoxide ion by [Cu(aptn)]Cl04)2 Figure 2 represents the decay o f - O ~ - against time t in the presence of complex recorded at 250 nm. The insets (a) and (b) display the plots of ln['Of]0 - ln['OT], and 1/[-O2-],- 1/['O2-]0 vs t, respectively. They showed that the reaction is neither first-order nor second-order in observed time, but it can be seen that Aln['O2-] vs t has the linear relationship at initial time of reaction when "02concentration is high. The dismutation of "02- is a first order reaction initially, but, with decrease of ' 0 2 concentration, '02- decay changed into second order reaction. Therefore, we suggest a reaction mechanism involving in the reversible equilibrium between superoxide ion and the complex: [CuI'aptn]2+ + OZ k~..~-'[Cu'aptn] + + 02 k k-I

F r o m equation (2), it can be seen that when ['OT]<>KM, i.e. (k~ + k2)['Oz-]>>k_ 1, the O2- decay is first order. Taking into account the spontaneous dismutation of "02,

- d[-O{l/dt = ks[.O2] z + k~Co[.0212/(['0~1 + KM) (3) upon rearrangement and integration, we have, t = c + KM/(koatCo + ksKM)/['Or] + k , tCo/ (kcatC0 q- k~KM)2" ln(1/['O2-] + kd(kca, Co + ksKM) (4) where c is the integration constant and t is time

(ms). In equation (4), t =f(['O2-], kcat, KM). A computer program of nonlinear least square method was used to minimize the sum of error square U,

[Cu'aptn] + + OF ~-H]~[Cu"aptn] 2+ + H202 using a steady-state approximation, we obtain the dismutation rate equation of superoxide: - d['O2- ]/dt = 2k~kz[-Of ]2[Cu"aptn]0/

((k, + k~)[.O~-] + k_,[O~])

(1)

In the pulse radiolysis experiments, the concentration of 02 in solution is approximately constant at 1.5 x 10 -3 tool dm-3(15°C) and 1.7 × 10 -3 tool dm -3 (10°C) (William, 1965). Taking k¢,t = 2k,kd (k, + k2), KM = k_ ,[O2]/(k, + k2), Co = [Cu"aptn]0, equation (1) becomes equation (2): - d[.O(]/dt = kc,tCo['O{]'-/(['Oi-] + KM).

(2)

U = ~[f(['O2-],, kc~t, KM) -- ti]2 i ---- 1,2 ..... n Where i denotes the n u m b e r of experiment points, ti is the time of point i. The correlation coefficient R is given by: R = 1 -- U(min)/Zt~.

(5)

For convenience, the OD curves were fitted directly. It was unnecessary to convert OD into ['02-]. Figure 3 presents an example of computer fitting of experimental OD curve. The values of kc,,, KM and related data were obtained and are given in Table 1.

Jun Wang et al.

530

Concentration (mol dm -3) 2.36 x 10 s 2.36 × 10-5 1.18 × 10-4 2.36 x 10-5 2.36 × 10-5

pH 7.0 7.0 7.0 7.8 7.8

Table 1. Rate constants of catalytic dismutation of OS by [Cu"(aptn)]-'+ KM kc,~ k~ k2 k (mol dm -3) (mol -R dm3 s -j) (tool-~ dm3 s -I) (moi J dm3 s -~) (mol-I dm3 s I) 5.95 x 10 6 4.20 x 107 1.57 × 108 2.42 × 107 7.23 x 105 5.51 × 10-~ 4.04 × 107 1.04 x 108 2.51 × 107 4.76 x 105 6.70 × l0 -6 0.48 × 107 0.13 × 108 0.28× 107 0.97 × I05 6.45 x 10-6 3.62 × 107 2.86 × 108 1.92 × 107 1.31 x 106 6.52 × 10 6 3.65 × l07 3.68 x 108 1.92 × 10 7 1.68 × 106

Table 1 shows t h a t the fitting results o f KM a n d kc,t are satisfactory. All the kinetic c o n s t a n t s u n d e r conditions of different p H values were close with each other. Because the dissociation c o n s t a n t o f [-HO2-] is Ka = 104.8 (Getoff a n d Prucha, 1983), [ . H O f ] m i g h t be ignored a n d .O2 conc e n t r a t i o n o f solutions would n o t change with v a r i a t i o n of p H values. T h e complex [Cu(aptn)]C104)2 is stable in neutral a n d weakly alkaline solutions, this m e a n s t h a t the state o f the complex was u n c h a n g e d at different p H values. Therefore, the kinetic c o n s t a n t s at p H = 7.0 a n d 7.8 are very close. Furthermore, when concentrations of complex a n d oxygen were changed, the m e c h a n i s m still obeyed the m e c h a n i s m o f re-oxidation o f [Cu'(aptn)] +. E q u a t i o n (3) d e m o n s t r a t e s t h a t the decay rate o f .02- is n o t only related to kca t b u t is also affected by [-02-]/([.O2-] + KM). Therefore, w h e n ['02-] < > KM, i.e. ['02-] > > 6 × 10 -6 m o l d m -3, it obeys first order reaction. W e also o b t a i n e d kl, k2 a n d k _ ~ f r o m k0at, K~ a n d redox potential o f couple 02/'02- at p H = 7.0 (E°(O2/.O2-) = - 0.16 v (SHE)) a n d redox potential o f couple Cull(aptn) 2 + / C C ( a p t n ) ÷ at neutral solution (E°(Cun(aptn) 2 + / C C ( a p t n ) +) = - 0 . 0 2 2 v (SHE)) (Lu et al., 1993). The results showed t h a t k _ ~value c a n n o t be ignored a n d re-oxidation reaction o f reduced p r o d u c t [Cu~(aptn)](C104) by oxygen molecules led to decrease in activity o f the complex. Therefore, good S O D model c o m p o u n d s should have the p r o p e r t y t h a t the re-oxidation o f their reduced p r o d u c t s by oxygen molecules is difficult.

R 0.9973 0.997l 0.9983 0.9973 0.9973

REFERENCES

Bielski, B. H. J. and Allen, A. O. (1977) Mechanism of the disproportionation of superoxide radicals, J. Phys. Chem. 81, 1048. Digiuseppi, J. and Fridovich, I. (1984) The toxicology of molecular oxygen. Crit. Rev. Toxicol. 12, 315. Getoff, N. and Prucha, M. (1983) Spectroscopic and kinetic characteristics of HO and "02 species studied by pulse radiolysis. Z. Naturforsch 38, 589. Jouini, M., Lapluye, G., Heut, J., Julien, R. and Ferradini, C. (1986) Catalytic activity of a copper(II)-oxidized glutathione complex on aqueous superoxide ion dismutation. J. Inorg. Biochem. 26, 269. Lu, Qin, Shen, Chen-Yu and Luo, Qin-Hui. (1993) A study on the schiff base Cu2Zn2SOD model complexes--the relationship between structure and activity. Polyhedron 12, 2005. Luo, Qin-Hui, Lu, Qin, Dai, An-Bang and Huang, Lian-Ren. (1993) A study on the structure and properties of a new model compound of Cu(II)-Zn(II) superoxide dismutase. J. Inorg. Biochem. 51, 655. Luo, Qin-Hui, Zhu, Shou-Rong, Sheng, Meng-Chang and Wang, Jun. (1995) A pulse radiolysis study of catalytic dismutation of superoxide anion by copper(II) complex of biscyclam dioxotetraamine. Radiat. Phys. Chem. 45, 247. McCord, M. and Fridovich, I. (1969) Superoxide dismutase: an enzyme function for erythrocuperin. J. Biol. Chem. 244, 6049. Tian, Ya-Ping, Fang, Yun-Zhong, Luo, Qin-Hui, Shen, Meng-Chang and Shen, Wen-Mei. (t992) The inhibitory effects of 21 mimics of superoxide dismutase on luminol-mediated chemiluminescence emitted from PMA-stimulated polymorph0nuclear leukocyte. Free Rad. Biol. Med. 13, 533. V6gtle, FI and Weber, E. (1986) Molecular and Functional Aspect of Superoxide Dismutase in Biomimetic and Bioorganic Chemistry H, p. 40. Springer, Berlin, William, F. L. (1965) Solubilities, Inorganic and Metal-Organic Compounds, Vol. II, 4th edn, p. 1228. American Chemical Society, Washington, D. C. Younes, M., Lengfelder, E., Zienau, S. and Weser, U. (1978) Pulse radiolytically generated superoxide and Cu(II)-salicylates. Biochem. Biophys. Res. Commun. 81, 576.