. I . Phys. Chem. 1994,98, 12621- 12629
12621
Three Rate Constants from a Single Kinetic Experiment: Formation, Decomposition, and Reactivity of the Chromium(V1)-Glutathione Thioester Intermediate Joaquin F. Perez-Benito,"Driss Lamrhari, and Conchita Arias Departamento de Quimica Fisica, Facultad de Quimica, Universidad de Barcelona, Marti i Franques, 1, 08028 Barcelona, Spain Received: July I S , 1994; In Final Form: September 19, I994@
The use of an exact integrated rate law has allowed the determination of the three rate constants associated with the formation (kl), decomposition (k-I), and reactivity (k2) of the thioester involved as a relativelystable intermediate in the reduction of chromium(V1) by glutathione in neutral or slightly acidic aqueous solution (pH = 4.0-7.2). The rate constants kl and k2 increase linearly as the concentration of glutathione increases, whereas k-1 does not change. Both kl and k-1 increase with rising concentration of buffer. The effect of three different buffering agents has been studied, the catalytic power of each buffer increasing in the order acetate < citrate < phosphate. At constant buffer concentration, the kl vs pH plots are bell-shaped, with a maximum at pH = 5-6. The three rate constants increase with rising ionic strength, and in the presence of acetate buffer, Zn2+ exerts a catalytic effect on both kl and k-1. The apparent activation energies associated with the rate constants kl, k-1, and k2 are 42.3 f 0.8, 61.6 f 1.4, and 35.7 f 3.3 kT mol-', respectively. A mechanism explaining the catalytic effects of both the buffer and Zn2+ and involving Cr(I1) as an unstable intermediate is proposed for the reaction. It is also suggested that either Cr(I1) or CxG2+ (formed from the reaction of the latter intermediate with oxygen) might be one of the mutagenic agents implicated in the carcinogenicity of chromium(V1) activated by glutathione.
state hypothesis to the thioester in the last stretch of the kinetic runs (once its maximum concentration has been r e a ~ h e d ) . ~ Chromium(V1) is one of the most active inorganic carcinoA more rigorous approach, however, can be attempted by gens,' and in recent years the elucidation of the mechanism of application of the matrix formulation of chemical reaction its carcinogenic action has received considerable a t t e n t i ~ n . ~ , ~ rates. 19s20 Besides the biological implications of the reaction However, it is usually accepted that the direct mutagenic agent studied, the solution to this kinetic problem may be taken as a is not chromate(V1) itself but some chromium species with a test for the conditions under which an integrated rate law lower oxidation state (probably, an intermediate) formed in its implicating the determination of three rate constants for each metabolic reduction inside the cell! The peptide glutathione kinetic experiment (an unusual situation in chemical kinetics) (y-glutamylcysteinylglycine) is a likely candidate as reductant, can be successfully used. since it is the most concentrated among the small-size cellular constituents capable of reducing chromium(V1) at physiological Experimental Section pH: according to the stoichiometry Glutathione was purchased from Sigma. All other chemicals were purchased from Merck (K2Cr207, NaCH3C02, K2HP04, 2Cr0:6GSH 10H+ = 2Cr3+ 3GSSG 8 H 2 0 (1) HCl, KOH, KCl, ZnSO4, MnS04, and gum arabic) or Aldrich (sodium citrate). The solvent water was purified by deionization GSH and GSSG standing for glutathione and glutathionyl followed by double distillation (the first time from a potassium disulfide, respectively.6 Moreover, an increase in the intrapermanganate solution). cellular level of glutathione has been shown to cause a dramatic The reaction was followed by monitoring the chromium(V1) increase in the number of chromium(V1)-induced DNA strand decay at 370 nm during a half-life. The A, value was measured break^.^ 2 days later after allowing the reaction to complete. The Consequently, the Cr(V1)-GSH reaction has been studied formation and decay of the thioester intermediate were detected in some depth, focusing on the search for reactive interby following the reaction at 430 nm. All the absorbances were m e d i a t e ~ ~ - as ' ~ well as on the determination of kinetic measured with a Varian Cary 219 UV-vis spectrophotometer parameter^.^.^^,^^-^* Due to the relative stability of one of the provided with a water-circulated cell holder, allowing us to carry intermediates (the thioester, GSCr03-), the kinetics of this out notably accurate determinations of the absorbance values reaction corresponding to the decay of Cr(V1) under a large (AA = kO.0001). The pH of the solutions was measured with excess of GSH presents the particularity of being adjustable to a Metrohm 605 pH meter provided with a glass-calomel a single-exponential function only under special conditions combination electrode. (HEPES buffer, pH = 7.0),17 and under most conditions it The solutions of glutathione were renewed daily. All the deviates from a first-order b e h a ~ i o r . Good ~ results have been experiments were done under a large excess of reductant with reported by the use of certain approximations: application of respect to oxidant (typically a 30-fold excess). In the series of the integrated rate law deduced by taking the formation of the experiments where the effect of pH was studied, the total thioester as an irreversible step13,16 or application of the steadyconcentration of buffer was kept constant by using a fixed concentration of a proper base: sodium acetate (NaAc), sodium citrate (Na3Cit), or K2HP04, whereas the pH was changed by Abstract published in Advance ACS Abstracts, November 1, 1994.
Introduction
+
+
+
+
@
0022-365419412098-12621$04.50/0
0 1994 American Chemical Society
Perez-Benito et al.
12622 J. Phys. Chem., Vol. 98, No. 48, 1994 adequate additions of either HC1 or KOH. When neccessary, the ionic strength was kept constant by addition of a large excess of potassium chloride. Given that the simultaneous detennination of three rate constants from each kinetic experiment makes them more sensitive than usual to both accidental and systematic errors (for instance, those related to a deficient thermostatization at the beginning of the kinetic runs, when the cell containing the sample was introduced into the spectrophotometer), the temperature of the laboratory was kept as close as possible to that corresponding to the experiments.
Determination of the Rate Constants General Kinetic Equations. The opening steps of the Cr(V1)-GSH reaction are a particular case of a more general situation that may be outlined as follows:
TABLE 1: Values of the Rate Constants at Various Initial Concentrations of Chromium(VIY lO"k2(s-') 103kl(s-l) 103k-1(s-l) lO"[Cr(VI)]o(M) 1.90 f 0.01 1.63 f 0.02 2.99 f 0.27 0.56 1.69 f 0.02 3.35 f 0.24 1.93 f 0.02 0.80 1.69 f 0.01 3.47 f 0.13 1.93 f 0.03 1.04 1.68 f 0.01 3.42 f 0.13 1.92 f 0.02 1.36 3.55 f 0.27 1.68 f 0.05 1.60 1.93 f 0.02 1.77 f 0.01 4.10 f 0.08 1.86 1.96 f 0.01 1.77 f 0.02 3.94 f 0.09 1.95 f 0.01 2.13 1.75 f 0.01 3.98 f 0.01 2.40 1.96 f 0.01 1.76 f 0.04 3.89 f 0.01 1.94 f 0.01 2.66 a
M, [Na@] = 6.80 x lo-* M, [HCl] = [GSH] = 4.84 x M, pH 6.59, ionic strength 0.41 M, 25.0 O C .
9.47 x
12.5
-
0
10.0 -
m
v
the integration of the differential kinetic equations corresponding to the three species involved being facilitated by the use of matrix algebra.lg If we assume that at the beginning of the reaction only species A1 is present (initial concentration, CO), the integrated equations are
88
7.5
-
5.0 -
2 2.5 -
"."
0.00
0.25
0.50
0.75
1.00
1.25
1O2[GSH] ( M )
.and [A31 = co - [AI] - [A2]. The parameters w1 and w2 are defined as
We can see that, although it has been shown that many different kinetic situations can be described by a. general integrated rate law involving a single-exponential function,21 the study of the evolution of the concentrations of species AI, A2, and A3 with time requires the use of a more complex, biexponential function. Application to the Chromium(V1)-Glutathione Reaction. In this case, A1 and A2 stand for the reactant Cr(V1) (whose initial concentration is CO) and the thioester GSCr03-, respectively, whereas k l , k-1, and k2 are the first-order rate constants (under a large excess of GSH). The nature of A3 depends on whether a second GSH molecule participates or not in the redox destruction of the thioester,18bimolecular decompositionleading to Cr(IV) and the unimolecular one to Cr(V), but the concentration of Cr(1V) [or Cr(V)] can not be calculated as co - [Cr(VI)I - [GSCr03-], since those species are later reduced to the ultimate inorganic product, Cr(II1). However, given that the reduction of Cr(1V) [or Cr(V)] to Cr(II1) takes place in fast, nondetermining steps, eqs 3 and 4 remain unaffected. The rate constants determined in this work have been obtained from nonlinear least squares fits (according to eq 3) of the absorbances at 370 nm vs time for a large number of experiments (total number, 1300). The nonlinear correlation coefficients were excellent (usually, 0.999 99 or higher). All the
Figure 1. Dependence of the rate constants on the concentration of glutathione for its reaction with chromium(V1) (1.64 x M) in the presence of NaAc (0.300M) at pH 6.55, ionic strength 2.10 M (KCl), and 25.0 "C. Rate constants: (squares) 10%; (triangles) lO3k-1; (circles) lO"k2. The experimental pH decreased from 6.55 to 6.31 as the concentration of glutathione increased; the values plotted for the rate constants have been corrected to discount the pH dependence. experiments were duplicated, and the reproducibility of the rate constants always was in the order kl > k-1 >> k2. In fact, it was possible to obtain good values of the rate constant kl under most conditions, whereas the reproducibility of k-1 was somehow worse and the accidental errors associated to k2 were too big in all experiments except those performed at pH > 6. It should be noticed that, according to eqs 5 and 6, the absolute value of the first exponential parameter (loll) is higher than that of the second (Iw;ll), and from our results, it seems that, with an integrated rate equation such as eq 3, involving the determination of three rate constants from each kinetic experiment, it is possible to obtain accurate values of the three of them only when the values of the exponential parameters are different enough (lull >> Iwzl), which in the case of the Cr(V1)GSH reaction happened actually at pH > 6, the accidental errors associated to k2 increasing dramatically as the difference 1011 - lw2l decreases.
Results Influence of the Reactants. Although (in the absence of systematic errors) the three rate constants are expected to be independent of the Cr(VI) initial concentration (in the presence of a large excess of GSH), the method was checked by following the reaction for different values of the initial concentration of the rate-monitoring species; the values obtained for the rate constant kl remained essentially the same for a fivefold increase of [Cr(VI)]o, whereas those of k-1 and k2 showed a slight increase (Table 1). On the other hand, as seen in Figure 1, both kl and k2 increased linearly with the reductant concentration, whereas k-1
Chromium(V1)-Glutathione Thioester Intermediate
J. Phys. Chem., Vol. 98, No. 48, 1994 12623
a, t
n " 4.5
I
5.4
PH
6.3
7.2
Figure 2. Dependence of the rate constants kl (top) and k-1 (bottom) M) by on pH for the reduction of chromium(V1) (1.64 x glutathione (4.82 x low3M) in the presence of NaAc at ionic strength 2.10 M (KC1) and 25.0 "C. Total acetate concentrations: (open triangles) 6.88 x M; (solid triangles) 0.200 M; (open circles) 0.250 M; (solid circles) 0.300 M; (rhombuses) 0.350 M.
was essentially independent of that magnitude (except for a very slight increase coming probably from a minor systematic error). Influence of pH and Euffer. It has been reported that the nature of the buffering agent affects the possibility of detection of Cr(V) as an intermediate of the Cr(V1)-GSH reaction.16 Especially interesting is the finding that it also has a strong influence on the extent of DNA strand breaks produced by the combination of Cr(V1) and GSH.22 Consequently, we have dedicated most of our work to study the mechanism through which the buffer participates in the reaction. It can be seen in Figures 2-4 that both rate constants kl and k-1 increase with increasing concentration of the buffer (acetate, citrate, or phosphate); an important feature of the kl vs pH plots is that they have a bell-shaped profile [in agreement with the results reported for the reduction of Cr(V1) by other thiols23] that becomes more definite as the buffer concentration increases. With respect to the other rate constant, it has been possible to obtain reproducible values of k2 only at pH > 6, where k2 decreases with increasing pH (Figure 5 ) and it is rather independent of the buffer concentration (Table 11s in the supplementary material). At a pH fixed, both kl and k-1 increase linearly with the buffer concentration (Figures 1S-3s in the supplementary material), according to the equations
where [BIT is the total concentration of buffer, whereas al, bl, a-1, and b-1 are fitting parameters. It can be seen in Figure 6 that the parameter a1 is independent of the nature of the buffer used and increases with increasing pH, and also that it is parameter bl that is the one that is responsible for the bell-
4
5
PH
6
7
Figure 3. Dependence of the rate constants kl (top) and k-1 (bottom) on pH for the reduction of chromium(V1) (1.64 x M) by glutathione (4.82 x M) in the presence of NasCit at ionic strength 2.10 M (KC1) and 25.0 "C. Total citrate concentrations: (open triangles) 1.36 x M; (solid triangles) 2.72 x M; (open circles) 4.08 x M; (solid circles) 5.44 x M; (rhombuses) 6.80 x M.
shaped profile of the kl vs pH plots, the catalytic power of the buffer increasing through the sequence acetate < citrate < phosphate
(9)
which is exactly the same as that found for the reduction of chromium(V1) by DL-peni~illamine.~~ Figure 7 shows that the parameter a-1 is similar to a1 in the sense that it increases with increasing pH, but although both parameters should be independent of the buffer used (because they are extrapolations of the corresponding rate constants at [BIT= 0), there exists some systematicerror in the measurement of a-1, since only the data for acetate and phosphate buffers are coherent. On the other hand, the parameter b-1 follows the same sequence for the catalytic power of the buffer as parameter bl (eq 9). Influence of Electrolytes. The rate constants kl and k-1 increased with increasing ionic strength when KCl was used as bottom electrolyte, the increase being more pronounced for citrate buffer than for acetate buffer (Figure 8). The rate constant k2 also increased with increasing ionic strength (Figure 9). When acetate was used as buffer, addition of ZnS04 to the solution resulted in an increase of both kl and k-1. This must be regarded as a catalytic effect specific to zinc ion, since for the same concentrations of MnS04 no effect on the rate constants was observed (Figure lo), thus evidencing that the increase of the rate constants was not due to a change of the ionic strength (too small at the concentrations of ZnS04 used) nor to an effect of sulfate ion. Influence of Temperature. The three rate constants fulfilled the Arrhenius law (Figure 4 s in the supplementary material). The corresponding apparent activation parameters are given in Table 2.
Perez-Benito et al.
12624 J. Phys. Chem., Vol. 98, No. 48, 1994
E
t
1.8
2%
IU
1.4
n
7
In
6 -
I .o
W
AT-
% &
4 -
0.6
2 -
0 0
I
.
I
.
I
.
1
.
4 -
c
In W d
4
m
2
2 -
" 3.5
4.5
5.5
6.5
7.5
4.0
PH Figure 4. Dependence of the rate constants kl (top) and k-1 (bottom) on pH for the reduction of chromium(V1) (1.64 x M) by glutathione (4.82 x M) in the presence of K 2 m 4 at ionic strength 2.10 M (KC1) and 25.0 "C. Total phosphate concentrations: (open M; (solid triangles) 2.45 x M; (open circles) triangles) 1.64 x M; (solid circles) 4.04 x M; (rhombuses) 4.98 x 3.28 x M.
4.5
5.0
5.5
fl
6.0
6.5
7.0
7.5
Figure 6. Dependence of the parameters a1 (top) and bl (bottom) on pH for the reduction of chromium(V1) (1.64 x M) by glutathione (4.82 x M) in the presence of several buffers at ionic strength 2.10 M (KCI) and 25.0 "C. Buffers: (triangles) NaAc; (solid circles) Na3Cit; (open circles) K2HP04. 5
4 -
4
6.0
I
6.5
0
7.0
PH
Figure 5. Dependence of the rate constant k2 on pH for the reduction M) in of chromium(V1) (1.64 x M) by glutathione (4.82 x the presence of Na3Cit (6.80 x M) at ionic strength 2.10 M (KC1) and 25.0 "C.
Influence of Gum Arabic. Some redox reactions (such as the oxidations of formate ion by iodinez5 and of triethylamine by permanganatez6) are known to be catalyzed by soluble colloids. Given that the intracellular medium is more similar to a colloidal solution than to an ordinary one, we have tried to check if the reaction between chromium(V1) and glutathione takes place on the surface of colloidal particles better than in the bulk solution. Our results with gum arabic as water-soluble colloid indicate that this polysaccharide has no catalytic effect on the Cr(V1)-GSH reaction; in fact, a slight inhibition effect (decrease in rate constant kl and increase in k-1) was found when gum arabic was added to the solution (Figure 5 s in the supplementary material), which might be explained by a change in the viscosity of the medium.
241 d
2
0
PH Figure 7. Dependence of the parameters a-1 (top) and b-1 (bottom) on pH for the reduction of chromium(V1) (1.64 x M) by M) in the presence of several buffers at ionic glutathione (4.82 x strength 2.10 M (KCI) and 25.0 "C. Buffers: (triangles) NaAc; (solid circles) Na3Cit; (open circles) K2HP04.
Cr(V1)-Buffer Interaction. Given that the integrated rate law for the decay of the chromium(V1) concentration (eq 3) contains four fitting parameters (two preexponential factors and
J. Phys. Chem., Vol. 98, No. 48, 1994 12625
Chromium(V1)-Glutathione Thioester Intermediate 0.3
I
t 0.0
0.2
log& :.k
0.1
0.0
-0.1
7.0
3.5
104[M2+] ( M ) Figure 10. Dependence of the rate constants kl (circles) and k-1 (triangles) on the concentrations of zinc (solid points, pH 6.21-6.15) and manganese(I1) (open points, pH 6.21-6.19) ions for the reduction M) by glutathione (4.82 x low3M) in of chromium(V1) (1.64 x the presence of NaAc (0.200 M) at ionic strength 0.21 M and 25.0 "C.
1
0.38
I
0.41
I
1
0.44
0.47
0.50
( MI'* )
l+Yi Figure 8. Dependence of the rate constants kl (top) and k-1 (bottom) on ionic strength for the reduction of chromium(V1) (1.64 x M) by glutathione (4.82 x M) in the presence of NaAc (0.350 M, pH 5.23 f 0.01, triangles) and NasCit (6.80 x M, pH 5.785.53, circles) buffers at 25.0 "C. The ionic strength was changed by additions of KC1; kIO and k-I0 are the values of the respective rate constants in the absence of that electrolyte.
TABLE 2: Apparent Activation ParameterV rate constant E, (Mmol-') AH*"(kJ mol-') -A&" (J K-' 39.8 f 0.8 119 f 3c ki 42.3 f 0.8 99 f 5 k- 1 61.6 f 1.4 59.1 f 1.4 33.2 f 3.3 1 5 4 f 11' k2 35.7 f 3.3 M, [GSH] = 4.85 x M, [Na3Cit] = [Cr(VI)]o = 1.60 x M, pH 6.59, ionic strength 0.41 M, [HCl] = 9.47 x 6.80 x M, temperature 14.8-35.2 "C. The activation entropies associated with kl and kz have been obtained after dividing the experimental first-order rate constants by the concentration of GSH, whereas that associated with k-1 has been obtained using the experimental rate constant as such. Referred to the 1 M standard state. 0.8
0.6
A $0 0.4
4.5
-"..,
A 7
0.39
I
0.44
l+fi
0.49
(MIn)
Figure 9. Dependence of the rate constant kZ on the ionic strength for M) by glutathione (4.82 the reduction of chromium(V1) (1.64 x M) at pH 5.78x M) in the presence of Na3Cit (6.80 x
5.53 and 25.0 "C. The ionic strength was changed by additions of KC1; kzo is the value of the rate constant in the absence of that electrolyte. two exponential parameters), besides the values of the three rate constants it has also been possible to obtain the value of the initial absorbance of Cr(V1) at 370 nm (AO370) for the conditions corresponding to each kinetic experiment (Figures 11 and 6S10s in the supplementary material). As shown in Figure 11, the '4'370 vs pH plots corresponding to acetate buffer revealed the existence of an interaction between the buffer and chromium(VI), since at each pH the A0370 value increased with increasing acetate concentration. This effect was also observed for citrate buffer (Figure 8s in the supplementary material), but it was not so clear in the case of phosphate buffer (Figure 10s in the supplementary material).
6.3
5.4
7.2
PH Figure 11. Dependence of the initial absorbance at 370 nm on pH for M) by glutathione (4.82 the reduction of chromium(V1) (1.64 x x M) in the presence of NaAc at ionic strength 2.10 M (KCl) and 25.0 "C. Total acetate concentrations: (open circles) 6.88 x lo-* M; (solid circles) 0.350 M. Inset: Dependence of the second pK, of chromic acid on the total concentration of buffer (NaAc) at ionic strength 2.10 M (KC1) and 25.0 "C.
From those A0370 vs pH plots, it has been possible to obtain the value of the second pKa of chromic acid. It decreased slightly as the total concentration of buffer increased, this effect being more pronounced in the case of acetate buffer (Figure 11, inset). Extrapolation of the pK, values at [BIT = 0 led,to very consistent results for the three buffers, the average value for the second pKa of chromic acid in the absence of buffer, at ionic strength 2.10 M (KC1) and 25.0 "C, being 5.66 f 0.02. Intermediates. Chromium(V1) and glutathione form a relatively-stable thioester intermediate whose accumulation and decay can be conveniently followed at 430 nm.12J5 The maximum absorbance observed at that wavelength (A"430) for each set of experimental conditions may be taken as a measurement of the stability of the thioester for those conditions. As
12626 J. Phys. Chem., Vol. 98, No. 48, 1994
Perez-Benito et al.
0.20
1
From the mechanism proposed, it follows that: U l=
0.101
4.5
'
'
5.0
'
'
5.5
'
'
6.0
'
'
6.5
'
1
7.0
PH Figure 12. Dependence of the maximum absorbance at 430 nm on pH for the reduction of chromium(V1) (1.64 x M) by glutathione M) in the presence of Na3Cit (5.44 x lo-* M) at ionic (4.82 x strength 2.10 M (KCl) and 25.0 "C.
shown in Figure 12, the A"430 vs pH plot at a constant concentration of buffer reproduced the bell-shaped profile of the kl vs pH plots (see Figures 2-4, top), with a maximum at around the same pH. On the other hand, it has been reported that, in contrast with the belief sustained by many authors until re~ently?~ the general mechanism for the reduction of Cr(V1) by most organic reductants in acidic aqueous solution involves the formation of Cr(I1) as an unstable intermediate. This has been demon~ t r a t e d ~ by * , ~UV-spectroscopy ~ detection of chromium(II1) superoxide, C102~+, formed from the combination of that intermediate with molecular oxygen Cr2+
+ 0, - Cr0,2f
(10)
as well as (in a more indirect manner) from a kinetic study of the inhibition effect caused by Mn2+ on many reactions of chromi~m(VI).~~~~~ In a recent we have reported that can also be detected in the reduction of Cr(V1) with biological thiols (DLpenicillamine, L-cysteine, and glutathione) at near-physiological pH in the presence of a low concentration of 0 2 , thus evidencing that Cr(I1) is also involved as an intermediate in those reactions.
Discussion Formation of the Thioester in the Absence of Buffer. For this part of the mechanism, that must explain the a1 portion of the rate law (eq 7), we propose the following steps: HCrO,-
2CrOt-
+ H+
(1 1)
HCr0,-
+ GSH slow GSCrO,- + H 2 0
(12)
Cr0;-
+ GSH slow GSCr03- + OH-
(13)
kU
kn[Hf]
+ K km[GSH] I
KI + [H+l
in agreement with Figure 6 (top) provided that km > kn. In fact, according to that figure and eq 14, the values that can be M-' s-l estimated for those rate constants are kn = 8.0 x and k n ~= 0.36 M-' s-l. This certainly contrasts with the situation found for the reaction between Cr(V1) and DLpenicillamine in the absence of buffer, where HCr04- reacts much better than Cr042-,24 but it is in good agreement with the results reported by Connett and Wetterhahn for the reduction of Cr(V1) by other thiols such as cysteine.23 Thus, it seems that the nature of the thiol plays an important role in determining which of the Cr(V1) species is more favorable to form the thioester. Formation of the Thioester in the Presence of Buffer. For the part of the mechanism that must explain the bl portion of the rate law (eq 7) we propose the following steps:
A ~ B + H +
HC~O,-
+ A 2 c + H,O
c + GSH 2 D D
+ OH- -GSCr03- + B + H 2 0 kvJl
slow
(15) (16) (17) (18)
were A and B stand, respectively, for the acidic and basic forms of the buffer, whereas C and D are two different complexes. We have proposed that the participation of the buffer in the mechanism takes place through the formation of a complex between the acidic forms of that buffer and the oxidant. The capacity of Cr(V1) to form condensation complexes similar to the one proposed in eq 16 (C) with a great variety of organic33 and inorganic34substances is well-known. Moreover, the A0370 vs pH plots confirm the existence of a Cr(V1)-buffer complex in the case of both acetate (Figure 11) and citrate (Figure 8s in the supplementary material) buffers. Although the plots corresponding to phosphate buffer (Figure 10s in the supplementary material) do not give any evidence for the existence of a complex with Cr(VI), there are several report^^^,^^ demonstrating that the condensation of HCr04- and H2P04- leads to the complex [H03P -0-Cr0312-. From the mechanism proposed it follows that:
km
We have taken into consideration that, given the value of the pKa of hydrogen chromate ion determined for the experimental conditions of this study (pK1 = 5.66 f.0.02), in the pH range covered in it there are two predominant forms of the oxidant, HCr04- and Cr0d2-, and, according to Figure 6 (top), both of them contribute to the total reaction. The reductant also contains several groups with acid-base properties, but the plot shown in that figure seems to follow very closely the deprotonation curve of hydrogen chromate ion (see Figure 1l),thus indicating that the only acid-base forms necessary to explain the experimental behavior are those corresponding to Cr(V1).
were KW is the water ionic product. It is easy to demonstrate that, according to eq 19, the bl vs pH plots must be bell-shaped (see Figure 6, down), with a maximum at
that can be estimated for each plot from the pKa values for hydrogen chromate ion and the acidic form of each buffer.37It can be seen in Table 3 that there is excellent agreement between the calculated and experimental values of pH,, for both acetate and citrate buffers (in the latter case when the value chosen for pKw is the second pKa of citric acid). In the case of phosphate
J. Phys. Chem., Vol. 98, No. 48, 1994 12627
Chromium(V1)-Glutathione Thioester Intermediate
TABLE 3: Estimation of the pHbuffer acetate citrate phosphate
PKN" 4.75 4.77 (6.39)d 7.21e
Values
PHmax(calc)b 5.2 5.2 (6.0) 6.4
pHmax(exp)C 5.2
5.3 5.7
L2 From ref 37. Values of pH,, calculated according to eq 20. Experimental values of pH,,, (Figure 6, bottom). The value out of parentheses corresponds to the dissociation of HzCit-, and the one in parentheses, to that of HCit2-. e Corresponding to the dissociation of HzPOd-.
buffer, although the agreement is not so good (probably due to a very pronounced effect of the ionic strength of the medium on the second pKa of phosphoric acid), eq 20 can at least explain a clear experimental finding: that the value of pH,, for phosphate buffer is higher than those for both acetate and citrate. Since species A is a molecule of acetic acid in the case of acetate buffer and either H2Cit- or HCit2- in the case of citrate buffer, complex C must be anionic (see eq 16). Moreover, since the predominant acid-base form of GSH in the pH range of our study is probably anionic (glutathione behaves as an acid in aqueous solution), complex D must also be anionic (see eq 17). Therefore, the rate-detemining step (eq 18) requires the encounter between two like-charged species (D and OH-), in agreement with the increase observed in rate constant kl as the ionic strength increases (Figure 8, top). The finding that the effect of the ionic strength is more pronounced for citrate buffer than for acetate is probably a reflection of the fact that in the former case the absolute value of the electrostatic charge of complex D is higher (because the charge of complex C is -2 or -3 for citrate buffer and -1 for acetate). Moreover, eqs 14 and 19 are in agreement with the experimental finding that the rate constant kl increases linearly with the concentration of glutathione (see Figure 1). Formation of the Thioester in the Presence of Zinc Ion. The catalytic effect caused by this ion can be explained by the following steps: Zn2+ + Ac-
%Zn2+-Ac-
(21)
+ GSH 3 Ac- -Zn2+-GSH (22) Ac- - Zn2+-GSH + CrV'= GSCr0,- + Zn2+-AcZn2'-Ac-
kX
(23) We have supposed that a complex is formed in eq 22 with zinc ion as metal center and acetate ion and glutathione as ligands. The capacity of Zn2+to form complexes with a carboxyl group of glutamic acid and with the sulfhydryl group of cysteine (two of the amino acids constituting the peptide glutathione) is wellknown, and it is one of the most important factors in determining the biological role of that ion in enzyme catalysis.38 On the other hand, it seems that acetate ion is another ligand in that complex (which might be tetradentate, with one or two H20 molecules as ligands, depending respectively on whether GSH participates with two groups or only one as ligands), since it has been reported that Zn2+ catalyzes the reaction between Cr(V1) and a similar thiol (DL-peniCillamine) only when acetate is present as the buffering agent.24 According to the mechanism proposed, the expression deduced for the rate constant kl is
k , = k,'
+ K,,,,K,,k,[GSH]
[Ac-I [Zn2+1
(24)
in agreement with Figure 10 (in eq 24, k1° is the value of the rate constant in the absence of zinc ion).
Decomposition of the Thioester. Obviously, the Cr(V1)GSH thiolate complex decomposes via the elementary reactions opposite to the rate-determiningsteps proposed for its formation. Therefore, the general expression deduced for the rate constant k-1 is
where k-11, k-m, k-w, and k-x are the rate constants associated with the reactions opposite to eqs 12, 13, 18, and 23, respectively. Equation 25 can also be written as
k-, = k-,'
+ K,,nk-x[Ac-][Zn2+]
(26)
in agreement with the linear increase of the rate constant k-1 with the rising concentration of zinc ion found in Figure 10. From both the expression corresponding to k-1° (the value of the rate constant k-1 in the absence of zinc ion) and eq 8 it follows that
According to eqs 27 and 28, an increase of both parameters a-1 and b-1 with rising pH would be expected. This is indeed in agreement with the situation found in Figure 7 (top) but not with that found in Figure 7 (bottom), since it can be seen in the latter that parameter b-1 increases with increasing [OH-] only for some pH intervals. It seems that the b-1 vs pH plots require for their explanation one to take into account two or more of the different acid-base forms of the thioester (including protonation or deprotonation of the chromate group as well as of the different functional groups of the glutathione residue) and not only one as done to deduce eq 28; thus, the latter must be considered as an approximate equation. On the other hand, eqs 27 and 28 are in agreement with the finding that the rate constant k-1 is independent of the GSH concentration (see Figure l), as well as with the increase of k-1 found when the ionic strength increases (Figure 8, bottom), since, according to the mechanism proposed, most of the decomposition of the thioester in the absence of zinc ion takes place by the encounter of two like-charged species, one of which is the thioester (GSCr03-) and the other is either OH- (for the reaction associated to the rate constant k-111) or the basic form of the buffer (B, for the reaction associated to k-vn). Moreover, since the absolute value of the electrostatic charge of the latter species is higher for citrate buffer (B = HCit2- or Cit3-) than for acetate (B = Ac-), k-1 is expected to be more affected by the ionic strength in the case of citrate buffer (see Figure 8, bottom). Reactivity of the Thioester. All the experimental evidence found by other authors and ourselves points to a rather complex sequence of events for the redox steps of the mechanism. The ones proposed by us are the following:
+ GS'
GSCr"0,-
+ GSH slow HCr"0,- -tGSSG CrVO,- + GSH - HCr"0,- + GS'
GSCrvlO,-
km
(30) (31)
Perez-Benito et al.
12628 J. Pkys. Chem., Vol. 98, No. 48, 1994 HCr"0,GSCr"0,-
+ GSH + GSH
GSCP0,-
-
HCr"0,-
+H20
(32)
+ GSSG
(33)
+ 3H+ Cr2+ + 2 H 2 0 Cr2+ + Cr"' - c r 3 + + Crv
HC?'02-
-
(34) (35)
2GS' GSSG (36) In order to clarify the redox transformation from Cr(V1) to Cr(III), we have indicated with an appropriate superscript the oxidation state of each chromium atom. The intermediates Cr(V) and GS' have been detected by other A glutathionyl chromate(IV) authors by EPR intermediate similar to that formed in eq 32 has also been included in the mechanism proposed by Bose et al. for the Cr(V1)-GSH reaction13and is required to explain the formation of Cr(I1) (see eq 33). To the best of our knowledge, this is the first time that a mechanism involving the formation of Cr(II) as an intermediate of the Cr(V1)-GSH reaction has been proposed. This is supported by the detection of Cr0z2+when that reaction takes place in the presence of oxygen, as reported by us in a previous work.32 It should be noticed that eq 34 does not represent an elementary step but the overall result of several steps; however, those steps are situated after the ratedetermining ones (eqs 29 and 30), so that the rate of reaction is not affected by them. According to the mechanism proposed, the expression corresponding to the rate constant k2 is
(37) and agrees well with the finding that k2 increases linearly with Concentration of glutathione (see Figure 1). The big accidental errors committed in the determination of that rate constant (especially at the higher GSH concentrations) make difficult an accurate estimation of k x ~and kx11, but it seems that at the values of [GSH] used in our work the contribution of the latter rate constant is very predominant, so that most of the redox transformation suffered by the thioester takes place via a bimolecular reaction with GSH (eq 30) rather than by intemal electron transfer (eq 29), in agreement with the results reported by O'Brien and Wang.'* Since in all probability the predominant form of GSH in the pH range of our study is anionic (see above), km is the rate constant associated with a reaction between two like-charged species (eq 30), which explains the increase of k2 with rising ionic strength (see Figure 9). On the other hand, the decrease found in that rate constant as the pH increases (Figure 5 ) indicates that at least two different acid-base forms of either the thioester or the reductant (or both) are involved in eqs 29 and 30. Given that protonation results in a decrease in the electron density of an oxidizing agent (and, so, in an enhancement of its oxidizing properties), the most probable explanation for the results found in Figure 5 is that protonation of the chromate group of the thioester favors its ability to suffer either an intemal electron transfer (eq 29) or a redox attack by a GSH molecule (eq 30). Biological Implications. Some evidence has been reported on the involvement of a Cr(V) species [formed as an intermediate in the intracellular reduction of Cr(V1) by glutathione] in the appearance of DNA single-strand breaksz2 We wish to suggest the possibility that either Cr2+ (a strong reducing agent formed as an intermediate in the same reaction) or Cr0z2+ (an
oxidizing agent formed from the reaction of the latter intermediate with oxygen, eq 10) might also collaborate in the genesis of DNA mutations. In particular, the hypothesis of chromium(III) superoxide being one of the active mutagenic agents might be supported by the well-known toxicity of the superoxide free radical, 02-.39 Moreover, the combination of that free radical with Cr(III), as in Cr0z2+, might be a way of avoiding the action of the enzyme superoxide dismutase.40 Conclusion. Equation 3 can be used as an integrated rate law for the determination of the rate constants associated with the formation (kl), decomposition (k-l), and reactivity (k2) of the thioester involved as a relatively-stable intermediate in the reduction of chromium(V1) by glutathione. However, the precision with which those rate constants are determined (especially as far as k2 is concerned) depends notably on whether the difference between the absolute values of the exponential parameters w1 and wz is large enough. Acknowledgment. This work was supported by a grant from the Spanish Ministry of Education and Science (DGICYT PB910243). Supplementary Material Available: Deduction of the kinetic equations; values of rate constant k2 at constant pH and various concentrations of citrate buffer; dependence of the rate constants kl and k-1 on the total concentrationof buffer (acetate, citrate, and phosphate); Arrhenius plots; influence of gum arabic; and A0370 vs pH plots for the three buffers (14 pages). Ordering information is given on any current masthead page. References and Notes (1) Oesterberg, R.; Persson, D. J . Trace Elem. Exp. Med. 1989,2,211. (2) Bianchi, V.; Levis, A. G . Toxicol. Environ. Chem. 1984, 9, 1. (3) Gao, M.; Binks, S. P.; Chipman, J. K.; Levy,L. S. Toxicology 1993, 77, 171. (4) Tsapakos, M. J.; Wetterhahn, K. E. Chem.-Biol. Inferact. 1983, 46, 265. (5) Connett, P. H.;Wetterhahn, K. E. J. Am. Chem. SOC.1985, 107, 4282. (6) Wiegand, H. J.; Ottenwaelder, H.;Bolt, H. M. Toxicology 1984, 33, 341. (7) C u p , D. Y.; Wetterhahn,K. E. Proc. Natl. Acad. Sci. U S A . 1985, 82, 6755. (8) O'Brien, P.; Barrett, J.; Swanson, F. Znorg. Chim. Acta 1985,108, L19. (9) Kitagawa, S.; Seki, H.;Kametani, F.; Sakurai, H. Znorg. Chim. Acta 1988, 152, 251. (10) Shi, X.; Dalal, N. S. Biochem. Biophys. Res. Commun. 1988,156, 137. (11) Aiyar, J.; Borges, K. M.; Floyd, R. A,; Wetterhahn, K. E. Toxicol. Environ. Chem. 1989, 22, 135. (12) Brauer, S. L.; Wetterhahn, K. E. J. Am. Chem. Sot. 1991, 113, 3001. (13) Bose, R. N.; Moghaddas, S.; Gelerinter, E. Znorg. Chem. 1992,31, 1987. (14) O'Brien, P.; Wang, G. Polyhedron 1993, 12, 1409. (15) McAuley, A.; Olatunji, M. A. Can. J. Chem. 1977,55, 3328; 1977, 55, 3335. (16) O'Brien, P.; Ozolins, 2.Znorg. Chim. Acta 1989, 161, 261. (17) O'Brien, P.; Wang, G.; Wyatt, P. B. Polyhedron 1992, 11, 3211. (18) O'Brien, P.; Wang, G . J . Chem. Soc., Chem. Commun. 1992,690. (19) Pogliani, L.; Terenzi, M. J. Chem. Educ. 1992, 69, 278. (20) Perez-Benito, J. F.; Arias, C.; Lamrhari, D.; Anhari,A. Znt. J . Chem. Kiner. 1994, 26, 587. (21) Lavabre, D.; Pimienta, V.; Levy, G.; Micheau, J. C. J . Phys. Chem. 1993, 97, 5321. (22) Kortenkamp, A.; Ozolins, Z.; Beyersmann, D.;O'Brien, P. Murat. Res. 1989, 216, 19. (23) Connett, P. H.;Wetterhahn, K. E. J . Am. Chem. Sot. 1986, 108, 1842. (24) Perez-Benito, J. F.; Lamrhari, D.; Arias, C. Can. J . Chem. 1994, 72, 1637. (25) Perez-Benito, J. F.; Arias, C.; Brillas, E. J. Chem. Res., Synop. 1990, 380; J . Chem. Res., Miniprint 1990, 2901. (26) Perez-Benito, J. F.; Arias, C. Znt. J . Chem. Kinet. 1991, 23, 717.
Chromium(V1)-Glutathione Thioester Intermediate (27) Beattie, J. K.; Haight, G. P. In Inorganic Reaction Mechanisms; Edwards, J. O., Ed.; Wiley: New York, 1972; Part 11, p 96. (28) Scott, S. L.; Bakac, A.; Espenson, J. H. J . Am. Chem. SOC.1991, 113, 7787; 1992, 114, 4205. (29) Bakac, A,; Espenson, J. H. Acc. Chem. Res. 1993, 26, 519. (30) Perez-Benito, J. F.; Arias, C.; Lamrhari, D. J . Chem. Soc., Chem. Commun. 1992, 472. (31) Perez-Benito, J. F.; Arias, C. Can. J . Chem. 1993, 71, 649. (32) Perez-Benito, J. F.; Arias, C.; Lamrhari, D. New J . Chem. 1994, 18, 663. (33) Haight, G. P.; Huang, T. J.; Shakhashiri, B. Z. J . Inorg. Nucl. Chem. 1971, 33, 2169.
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