2236
J . Am. Chem. SOC.1984, 106, 2236-2239
Comparative Behavior in the Kinetics of Reduction by Superoxide and Dithionite Ions Z. BradiC and R. G. Wilkins* Contribution from the Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003. Received October 27, 1983
Abstract: Rate constants for reaction of superoxide and dithionite ions with a number of oxidants a r e reported. Potassium superoxide dissolved a t a pH 11 1.5 represents a source of 02-in aqueous solution which can be used for kinetic studies of the radical. Disproportionation rate data for superoxide ion a t p H 1-13 a r e in good agreement with values obtained by pulse ions a r e generally approximately constant radiolysis. It is found that the relative rate constants for reduction by SO2- and 02l o 3 has predictive value. and the ratio of
-
Ever increasing attention is being paid to the chemistry of the ion (02-) and to the related enzyme superoxide dismutase.’V2 M e t h o d s of g e n e r a t i ~ nof ~ ,02~ in aprotic solvents, in which is it stable, include electrochemicalreduction of dioxygen: as well as by direct use of salts including soluble Me4N+02-5and KO2 ‘solubilized” by crown ethers.6 The very rapid disproportionation of superoxide ion in aqueous solution, except at high pH, presents problems however in t h e study of its kinetic reactivity in t h a t medium. One has therefore to resort to specialized methods, of which t h e most used for kinetic studies’ involves pulse radiolysis.* Superoxide ion is produced within microsecs by e,, reduction of 02.The reaction of 02with substrate is examined in situg or after mixing in a stopped-flow apparatus.1° Reaction of O2with reduced flavins,ll dyes, and biochemical reductants,I2J3 differential mixing of 02-in Me2S0 with a large volume of water,14 and photochemical methods15 have also been employed t o generate superoxide ion in aqueous solution.15d A largely overlooked16 source of 02-ions in aqueous solution is by dissolution of KOz at pH 1 1 1.5, where disproportionation is slow. We have found that such a solution can be mixed with buffer, with or without substrate, and the rate of disproportionation of 02-or superoxide
(1) (a) Michelson, A. M.; McCord, J. M.; Fridovich, I. “Superoxide and Superoxide Dismutases”; Academic Press: New York, 1977. (b) Oberley, L. W., Ed. ‘Superoxide Dismutase”; CRC Press: Boca Raton, 1982; Vol. I and 11. (2) Sawyer, D. T.; Valentine, J. S. Acc. Chem. Res. 1981, 14, 393. (3) Fee, J. A,; Valentine, J. S. In ref la, pp 10-60. (4) (a) Sawyer, D. T.; Roberts, J. L., Jr. J . Electroanal. Chem. 1966, 12, 90. (b) Fee, J. A,; Hidenbrand, P. G. FEES Lett. 1974, 39, 79. (5) (a) Peters, J. W.; Foote, C. S. J . Am. Chem. SOC.1976, 98, 873. (b) McElroy, A. D.; Hashman, J. S. Inorg. Chem. 1964, 3, 1798. (c) Sawyer, D. T.; Calderwood, T. S.; Yamaguchi, K.; Angelis, C. T. Inorg. Chem. 1983, 22, 2577. (6) (a) Valentine, J. S.; Curtin, A. B. J . Am. Chem. SOC.1975, 97, 224. (b) Johnson, R. A.; Nidy, E. G. J . Org. Chem. 1975.40, 1680. (7) Farhataziz, R.; Ross, A. B. Natl. Stand. Ref. Data Ser. (US., Nut. Bur. Stand.) 1977, 59, 113. (8) (a) Shafferman, A,; Stein, G. Eiochem. Eiophys. Acta 1975, 416, 287. (b) Wilkins, R. G. Adu. Inorg. Eioinorg. Mechs. 1983, 2, 139. (9) Beilski, B. H. J. J . Photochem. Photobiol. 1978, 28, 645 and previous citations. (10) Bielski, B. H. J.; Richter, H. W. J. Am. Chem. SOC.1977, 99, 3019. (11) (a) McCord, J. M.; Fridovich, I. J. Eiol. Chem. 1969, 244,6049. (b) Ballou, D.; Palmer, G.; Massey, V. Eiochem. Eiophys. Res. Commun. 1969, 36, 898. (12) (a) Bray, R. C. Eiochem. J . 1961, 81, 189. (b) Knowles, P. F.; Gibson, J. F.; Pick, F. M.; Bray, R. C. Eiochem. J . 1969, 111, 53. (13) Lee-Ruff, E. Chem. SOC.Reu. 1977, 6, 195. (14) McClune, G. J.; Fee, J. A. FEES Lett. 1976, 67, 294. (15) (a) Holroyd, R. A,; Bielski, B. H. J. J . Am. Chem. SOC.1978, 100, 5796. (b) Gebicki, J. M.; Bielski, B. H. J. Ibid. 1982, 104, 796. (c) McDowell, M. S.; Bakac, A.; Espenson, J. H. Inorg. Chem. 1983, 22, 847. (d) Bielski, B. H. J.; Arudi, R. L. Anal. Eiochem. 1983, 133, 170. Bielski et al. have developed methods for generating (by ionizing radiation or vacuumUV photolysis) and stabilizing alkaline aqueous and ethanolic superoxide solutions. They can be rendered anaerobic. (16) Marklund, S. J . Eiol. Chem. 1976,251,7504. Marklund used KO2 as a source of 02-for the study of the disproportionation of 02-from pH 8.9 to 12.7.
0002-7863/84/ 1506-2236$01SO10
of its reduction reactions measured. In this way, the rate constants for t h e reaction of 02-with a number of oxidants have been determined (or remeasured) and compared with those of SO2(or S2042-) ion. In reductions by dithionite, S20d2-or SO; or sometimes both are kinetically important ~ p e c i e s . l ~ Both - ~ ~ 02and SO; are small, singly charged ions, and they should generally have a constant comparative reactivity on t h e basis of t h e Marcus relationship for outer-sphere reactions.26 Experimental Section Chemicals used were the purest commercial products. Horse heart cytochrome c was Type I11 (Sigma) and potassium superoxide was 96.5% pure (Alfa). The complexes [Co(terpy),]Br3 and K[Mn(CyDTA)].2H20 were prepared by literature methods.27 Doubly distilled water was further purified by extraction with dithizone dissolved in CCI, and redistilled in the presence of 0.1 mM Na2EDTA. Kinetics. In a typical procedure, freshly powdered potassium superoxide ( - 5 mg) was dissolved in swirling water (100 mL) containing 0.1 mM EDTA at pH 11.5. The concentration of 0,- in the fresh solutions was -0.2 mM by using absorbance at 250 nm9 (e 2188 M-I cm-’) and represented -30% of the theoretical amount. The solution was transferred quickly to one of the syringes of a stopped-flow apparatus (solution A). For a study of disproportionation, solution A was mixed with buffer (10 mM citrate, acetate, phosphate, or borate) at the appropiate pH in the other syringe. The spectral change was monitored at 250 nm, and the second-order rate constant for loss of 02-was computed by using molar absorbance coefficients at various pH listed by B i e l ~ k i .For ~ the study of other 0,- reactions, solution A was mixed with substrate in the second syringe (usually 20.5 mM reactant at pH 9.2 by using 15-30 mM Na2B407solution containing 0.1 mM EDTA). The reaction was monitored at a wavelength usually determined by the spectral characteristics of the substrate and of the reduced product. Good first-order kinetics were obtained, and the first-order rate constant was linearly dependent on substrate concentration. The latter was determined by weight or by using known absorbance coefficients. That used for azurin was 5.7 X lo3 M-I cm-’ at 625 nm.28 The final reaction pH was quite close to that of the contents of the second syringe. Reactions of dithionite were studied anerobically by using reductant in excessI9 and with the same conditions as used in the superoxide experiments. A Gibson-Dionex stopped-flow apparatus linked to an OLIS data collecting system was used to measure
-
(17) Lambeth, D. 0.;Palmer, G. J. Eiol. Chem. 1973, 248, 6095. (18) Creutz, C.; Sutin, N. Inorg. Chem. 1974, 13, 2041. (19) Olivas, E.; de Waal, D. J. A,; Wilkins, R. G. J . Eiol. Chem. 1977, 252, 4038. (20) Cox, R. P.; Hollaway, M. R. Eur. J . Eiochem. 1977, 74, 575. (21) Scaife, C. W. J.; Wilkins, R. G. Inorg. Chem. 1980, 19, 3244. (22) Mehrotra, R. N.; Wilkins, R. G. Inorg. Chem. 1980, 19, 2177. (23) Kazmi, S. A.; Shorter, A. L.; McArdle, J. V. J . Inorg. Eiochem. 1982, 17, 269. (24) Jones, G. D.; Jones, M. G.; Wilson, M. T.; Brunori, M.; Colosimo, A.; Sarti, P. Eiochem. J . 1983, 209, 175. (25) Balahura, R. J.; Wilkins, R. G. Eiochim. Eiophys. Acta 1983, 724, 465. (26) Marcus, R. A. J . Phys. Chem. 1963, 67, 853. (27) (a) Baker, B. R.; Basolo, F.; Neumann, H. M. J . Phys. Chem. 1959, 63, 371. (b) Hamm, R. E.; Suwyn, M. A. Inorg. Chem. 1967, 6, 139. (28) Goldberg, M.; Pecht, I. Biochemistry 1976, 15, 4197.
0 1984 American Chemical Society
J . Am. Chem. SOC.,Vol. 106, No. 8, 1984 2237
Reduction by Superoxide and Dithionite Ions
w
u z
4
\
.42
.35
0 .28
a a
.21
g
.14
I-I
.07
0
-.07 220
2.28
236
W~AVVEENG?~ 2?6
244
284
292
300
Figure 1. Rapid scan of 02after rapid mixing of 02-at pH 11.5 with
buffer at pH 9.2 (16 spectra in 5 s, 7.0-ms sweep time, 220-300 nm). Final reference spectrum after 35 s. Data at 25 ' C .
1
9
5
13
PH the rates of all reactions studied. The rapid scan spectral stopped-flow apparatus used a Harrick Rapid Scan Monochromator and was designed by Dr. DeSa (OLIS Jefferson, GA).
Results Disproportionation of 02-at pH 11.5 and above is quite slow, requiring many minutes for substantial completion of the reaction. It was found that increasing the ionic strength with NaCl had a small effect on the disproportionation rate constant at pH 11.5 (20 mM NaC1, k = 64 M-l s-l; 400 m M NaCl, k = 85 M-' s-l; 1.0 M NaCl, k = 91 M-' s-l). However, N a 2 S 0 4 markedly accelerated the rate constant (10mM Na2S04, k = 101 M-I s-l; 330 mM Na2S04,k = 652 M-' SI). In any ionic strength adjustments, NaCl was therefore used. A rapid scan of the spectrum of 0, when a solution at pH 11.5 was rapidly mixed with a borate buffer at pH 9.1 (final pH 9.2) is shown in Figure 1. A maximum at 248 nm was observed. Use of the appropriate absorbance coefficient (2.25 X lo3 M-' cm-' ) leads to k = 4.6 X lo3 M-' s-l. Kinetic data were always obtained by continual observation at 250 nm. The variation with pH of the second-order rate constant for disproportionation of superoxide ion is shown in Figure 2 and is interpreted in terms of reactions 1-4.9 The computed constant
H 0 2 z H+ + 02-
+ HO2 H02 + 0 2 0 2 - + 0,
HO2
-
-
+ H20, 0 2 + H020 2 + 0220 2
Figure 2. Experimental values of second-order disproportionationconstant ( k o M )vs. pH at 25 ' C . Total superoxide = 0.4-1.5 mM. Buffers in 0.1 mM EDTA were 10 mM sodium citrate, acetate, phosphate, and borate. A 250 nm (also 240 and 270 nm). The full line fits the equation kobd = ( k 2 + k3Kl[H+]-I) (1 + K1[H+]-')-' + k4 by using values in the text. I
I
I
I
(1)
K1 kz
(2)
k3
(3)
k4
(4)
m M Substrate
1
.u
Figure 3. Variation of kobd (s-') with substrate concentration for reacfor reactions 1-4 compared with literature values at 23 OC9 (in tions of 02-at pH 9.2 using 15 mM sodium borate, 0.1 mM EDTA, and 25 ' C . Substrates: azurin (0); ferricytochromec Fe(CN),3- ( 0 ) ; (2.1 X parentheses) are as follows: K, = 2.23 X M; Mn(CyDTA)- (A);Co(terpy)p (B); nitro blue tetrazolium (A);DCIP k2 = 1.1 X lo6 (8.6 X lo5)M-I s-l; k, = 1.8 X lo8 (1.0 X lo*) (0). For Mn(CyDTA)- there is an additional point at 0.3 mM substrate, M-' s-I; k4 = 5.0 (C0.35) M-' s-l. The larger values of k4 probably k = 202 arises from catalysis by impurities in the potassium s u p e r ~ x i d e . ~ ~ ~ ~ ~ s-'. For ferricytochromec, the concentration ordinate is 0.15 mM not 1.5 mM. For azurin, concentration ordinate is 0.15 mM and The studies of reduction of substrate by 0, were mostly carried rate constant ordinate is 10 s-I not 100 SC'. out at pH 9.2. Here disproportionation of 02-was sufficiently slow, especially at the low concentrations examined (C50 kM), Co(terpy)?+ and nitro blue tetrazolium were also studied at pH not to interfere in the study of the pseudo-first-order rates of 6.0 and 7.0, respectively, and no difference from the rate constants reaction of 02-with substrate in excess (>lo0 kM). The plots at pH 9.2 was observed. Reduction by dithionite ion of the of kow vs. the concentration of substrate were linear (Figure 3), substrates examined (azurin, nitro blue tetrazolium, and 2,6-diand from these the second-order rate constants were computed chlorophenol indophenol) all conformed to the two-term rate law (Table I). As disproportionation of 0, occurred in the solution (5). The linear plots of koM[S20~-]-'/2 vs. [S2042-]1/2 (see Figure standing at pH 11.5, the absorbance change of the examined rate = kl[substrate] [S2042-] k2[substrate][S2042-]1/2 = reaction decreased, but the second-order (for disproportionation) kobd[substrate] (5) or first-order rate constants remained invariant. This allowed for easy recognition of the 02-reaction. The reactions of 02-with 4) yield values from the intercept of k2 and from the slope, k l . (0);
+
2238 J . Am. Chem. Soc., Vol. 106, No. 8, 1984 Table I. Rate Constants (M-' s - ' ) for Reactions of 02-, SO;,
Bradit and Wilkins and S 2 0 4 2 -Ions at 25 "C
1% ( k s o,-I oxidant
ko2-
ks0,-
9.3 x 103a*b
Pseudomonos aerirginosa azurin horseheart ferricytochrome c horseradish ferriperoxidase h or seheart metmyoglobin Fe(CN),3-
1.8 x 105,a3d 1.0 x 1 o 5 f > 1o 7 ; , b -1 0 8 h
5.0
6.9 x 103,a,d 2.7 X IO2" 1.3 X l o 6 "
