A kinetic investigation on Fe and Cu,Zn superoxide dismutases by polarography

ELSEVIER

Bioelectrochemistry and Bioenergetics 36 (1995) 165-170

Short communication

A kinetic investigation on Fe and Cu,Zn superoxide dismutases by polarography Emanuele Argese *, Ligia M. Moretto, Catia Granito, Emilio F. Orsega Faculty of Sciences, University of Venice, Dorsoduro 2137, 30123 Venice, Italy Received 3 August 1994; revised 22 September 1994

Keywords: Polarographic method; Enzyme activity; Iron superoxide dismutase; Copper; Zinc superoxide dismutase; Saturation kinetics

1. Introduction

0 2 is unstable in protic solvents owing to its fast dismutation process:

Superoxide dismutases (SODs) form a class of metalloenzymes which efficiently catalyse the dismutation of the superoxide ion 02. They contain Cu,Zn or Fe or Mn ions at the active site [1]. It has been demonstrated that for all types of SODs the dismutation reaction proceeds via a common cyclic oxidation-reduction mechanism [2-5]: Mn++ O~-

kl '

M(n-t)++ 02

M("-~)++Of+2H +

k2 , M n + + H 2 0 2

(1) (2)

where M denotes Cu, Fe or Mn and n is 2, 3 and 3 for Cu, Fe and Mn respectively. However, each type of SOD shows differences in the details of the catalytic process. In the case of Cu,Zn-SOD, several studies have shown that the reaction is diffusion limited [3,6,7]; in fact no saturation has been observed, even at very high 0 2 concentrations. Hence the reaction rate is given by

d[O;] -

dt

= ks[SOD][O2]

(3)

where k s is the experimental rate constant. In contrast, Fe-SOD and Mn-SOD show saturation kinetics [5], which can be interpreted according to the MichaelisMenten formalism. The measurement of SOD activity is not trivial since

* Corresponding author. Fax 0039 41 5298594. 0302-4598/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 0 3 0 2 - 4 5 9 8 ( 9 4 ) 0 1 7 6 4 - 6

0 2 + HO2 H+ ' ,02 + H202

(4)

As a consequence, such a measurement requires the use of methods which are able to both generate and detecte 0 2 in aqueous solution. Photochemical, enzymatic and electrochemical techniques have been employed for this purpose [8-11]. Among these techniques, the generation of 0 2 by the electrochemical reduction of molecular oxygen appears to be a convenient method since no by-products are generated and the equipment required is simple and cheap. The short drop time mercury electrode (SDTME) [12] has been used in the presence of a surfactant such as triphenylphosphine oxide (TPO) both to generate considerable flows of 0 2 and to measure its SOD-catalysed dismutation rate. This method permits rapid and accurate measurements of enzymatic activity in the pH range 7.5-12, despite the high instability of the 0 2 ion in aqueous solution. Until now this technique has been restricted to Cu,Zn-SOD kinetic studies [7,12,13]. In the work reported here the STDME method [12] was utilized to study the kinetic behaviour of Fe-SOD enzyme. Although the value of the experimental kinetic rate constant k s for Fe-SOD can be calculated using the theory of catalytic currents [14], this cannot be done by a trivial analogy with Cu,Zn-SOD. In fact, the dependence of the dismutation rate of 0 2 on its concentration is linear for Cu,Zn-SOD, while it follows a Michaelis-Menten relationship for Fe-SOD. In this paper we demonstrate that, despite this, the mathematical model employed to calculate k s for Cu,Zn-SOD is

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E. Argese et al. / Bioelectrochemistry and Bioenergetics 36 (1995) 165-170

still valid for Fe-SOD provided that the above-mentioned dependence on the substrate concentration and the related kinetic parameters are taken into account.

. [. . . . . . . hX'xX~xJ\i - ~ x~x~ I g.,, f.,:!

2. Experimental

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21d

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d---

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c. . . .

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2.1. Materials All the solutions were prepared from analytical grade chemicals and water purified using a Millipore MilliRO Milli-Q system (Milan, Italy). Cu,Zn-SOD and Fe-SOD were purchased from Sigma (St. Louis, MO, USA). Triphenylphosphine oxide (TPO), hydroxymethyl aminomethane (Tris) and borate buffers were purchased from Fluka (Buchs, Switzerland).

I

1

-1.0

-0.5

E / V vs SCE

2.Z Apparatus Polarographic waves were recorded using an Amel model 472 multipolarograph equipped with a Linseis L-800 x-y recorder. The concentration of each enzyme was calculated on the basis of the amount of metal ion found in dialysed enzyme aliquots using a Varian model SpectrAA-10 atomic absorption spectrometer equipped with a GTA-96 graphite furnace. Enzyme concentrations were also checked spectrophotometrically with a Beckman DU-7 spectrophotometer using the following extinction coefficients: e350 1850 [metal] -~ cm -1 for Fe-SOD [15] and e6s0 = 310 [metal] -1 cm -1 for Cu,ZnSOD [16].

Fig. 1. Polarographic waves of 0 2 reduction to O 2 in air-saturated solution containing 5 x 1 0 -4 M TPO: t g = 0 . 1 s; T = 25+0.1°C; i d limiting current due to the monoelectronic reduction of 0 2 to O~, calculated as half of 2id, which corresponds to the bielectronic reduction of 0 2 to H 2 0 2 ; i¢, increment of i d due to spontaneous a n d / o r SOD-catalysed O~" dismutation; i l, diffusive current i d plus the current i c due to spontaneous a n d / o r SOD-catalysed dismutation. Curves: (b) reduction wave of 0 2 in 0.01 M sodium borate (pH 9.3); (c), (d), (e), (f), (g), (h), (i), (j): as (b) plus 12, 25, 37, 49, 62, 78, 99 and 120 nM of Fe-SOD respectively; (a) as (k) without 02.

=

Z 3. Methods Tris and borate buffers were used to regulate pH in the range 7.7-10.1. Enzyme activity was measured at 25.0 + 0.1°C in aqueous thermostated buffered solutions containing 300 mg 1-1 of TPO. In order to obtain known constant concentrations of 02 the measurement solutions were equilibrated in the polarographic cell in certified gaseous 0 2 + N 2 mixtures with 0 2 contents ranging from 5% to 100%.

3. Results and discussion

3.1. Fe-SOD activity measurement The essential theoretical background to the polarographic method of the catalytic currents and the related applications to SOD enzymes are extensively described in previous papers [11,12,14], to which the reader should refer for basic information on SOD activity measurements.

Briefly, the molecular oxygen dissolved in aqueous solution is reduced to 0 2 at a drop mercury electrode in the presence of a surfactant such as TPO. The resulting polarographic limiting current (Fig. 1) is the sum of two terms: the first one (diffusive current id) is due to the oxygen diffusing toward the electrode from the bulk solution and is calculated as half the bielectronic current of the reduction of 02 to H202; the second (catalytic current ic) is ascribed to the oxygen generated by spontaneous a n d / o r SOD-catalysed dismutation of 0 2 diffusing from the electrode towards the bulk solution. A rearrangement of Koutecky's treatment of the catalytic currents [14], taking into account both the spontaneous and SOD-catalysed dismutation processes, leads to a straightforward approximate relationship which correlates the catalytic current with the SOD activity: 7.42R

f ( R ) -- 1 - 1.25R = ks[SOD]tg + k°tg

(5)

where R = (il/i d - 1 ) , tg is the drop time, k 0 is the kinetic rate constant of the spontaneous dismutation of 0 2 and k~ is the experimental rate constant of the SOD-catalysed dismutation. The value of k s can be calculated from the slope of the linear plot of f ( R ) vs. [SOD] (Fig. 2) and the contribution of the spontaneous

E. Argese et aL / Bioelectrochemistry and Bioenergetics 36 (1995) 165-170

dismutation of 0 2 represented by the intercept can be discriminated from that due to the catalysed reaction. Eq. (5) is valid, within 1%, in the range 0 < R < 0.5, i.e. f(R) < 9.9. As a consequence no SOD activity measurement is possible when the contribution of the spontaneous 0 2 dismutation makes R > 0.5, i.e. i~ > 1.5id (Fig. 1). Such a contribution increases with the DH of the test solution and decreases with t~. In a previous paper [12] it was demonstrated that with SDTME (tg = 0.1 s) the contribution of the spontaneous dismutation of 0 2 is such that the i I value is lowered below 1.5, even at pH values near 7, so that Eq. (5) is still applicable. The above-mentioned conditions allowed the Cu,Zn-SOD activity to be measured in the pH range 7.4-12.5, at different ionic strength, with an error below 3%. The kinetic characteristics of Cu,Zn-SOD have been explained in terms of the presence of positively charged groups around the active site that drive the 0 2 ion toward the reaction centre. This accounts for the equality of kinetic constants k~ and k 2 appearing in Eqs. (1) and (2) and for the value of k s that is higher than the maximum value that can be calculated for the pure diffusive process [7]. Such a mechanism cannot be represented by the classic Michaelis-Menten formalism, at least in our measurement conditions, i.e. for 02 bulk concentration in the range 5 x 10-5-1 × 10 - 3 M and an SOD concentration of ca. 10 -8 M. As a consequence, the 0 2 dismutation rate is expressed by Eq. (3). In contrary Fe-SOD exhibits saturation kinetics, so that the SOD-catalysed dismutation rate of O 2 is expressed by [5] d[O2]

kv[SOD][O2]

dt

KM + [O2]

(6)

where k v is the composite constant of the kinetic decomposition constants of the e n z y m e - substrate complexes in the products. As a consequence, relationship (5) between the catalytic limiting current and the SOD concentration should not be valid in principle. In fact, from a comparison of Eq. (3) with (6), k s should depend on the mean 0 2 concentration [O~-]m experienced by SOD near the electrode surface and should obey the relation kv k s = KM + [O~_]r "

(7)

However, the trend in the i~ values of the polarographic waves obtained on addition of known amounts of Fe-SOD (Fig. 1) follows Eq. (5). The linearity of the experimental data plotted in Fig. 2 shows that such a relationship is also applicable to Fe-SOD, within the validity limits mentioned above, and the slope of the straight line should denote a value of k s defined by Eq. (7) which is constant at varying SOD concentrations.

15

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,

./ 12

9

6

3

0

0.0

i

I

I

,~

0.1

0.2

0.3

0.4

0.5

[FeSOO] / ,u,M Fig. 2. Linearization of current values, obtained from Fig. 1, as a function of S O D concentration according to Eq. (5). Same experimental conditions as in Fig. 1.

This leads us to infer that, in our experimental conditions and at a constant 02 bulk concentration [O2]b, the value of [O2] m in the reaction layer remains constant as the SOD concentration varies. This can be theoretically supported starting from the concept of reaction layer introduced by Brdicka and Wiesner [17] in their classical approximate treatment of polarographic currents. The reaction layer thickness is the effective distance/z through which the 0 2 ions move within their mean lifetime z before reacting with the enzyme. As the SOD concentration increases, both the reaction layer and 0 2 concentration decrease at any value of x + 0, while at the electrode surface (x = 0) the O f concentration is equal to the 02 bulk concentration, for any value of added SOD, as long as the diffusion coefficients Do2 and Do2_ are equal [18]. This entails that the gradient of O 2 concentration at the electrode, proportional to the limiting current, increases with increasing SOD. Brdicka and Wiesner [17] assumed that this gradient is linear, so that the 0 2 concentration profile is represented by a straight line with/z as the x axis intercept. The [O~-]m value in the reaction layer can be obtained independently from /~ (as is evident from calculating the integral mean value of [02] over the distance /~), and hence from the SOD concentration, and is equal to [O2]b/2. It is easy to demonstrate that [O2] m does not depend on SOD concentration even if a non-linear 0 2 concentration profile is assumed (e.g. a negative exponential or quasi-exponential function of x), taking into account that the reaction layer thickness is actually finite [19]. In any case [O2] m appears to be proportional to [O2] b and independent of /z and hence the SOD concentration.

E. Argese et aL / Bioelectrochemistry and Bioenergetics 36 (1995) 165-170

168

In conclusion, both the experimental results and the theoretical arguments lead us to state that the mean 0 2 concentration experienced by SOD depends only on [O2]b- Hence, during the activity measurement [O~-]m does not vary with progressive SOD additions, and the relevant k s value is a constant at fixed [02] b. As a consequence, the catalytic limiting current is related to the SOD concentration according to Eq. (5), provided that k s is expressed as kv

5.0 4.5 ,.-

4.0

.,.-

3.5

•

3.0 ~o

,5

2.5 2.0

ks = r , , + p [ o , ] , ,

(8) 1.5

where p is the proportionality factor between [O~]r~ and [O2]b. Therefore the plot of 1 / k s vs. [O2] b is expected to be linear, for Fe-SOD. It is worth noting that, on the basis of the above arguments, p is expected to have the same value for both Cu,Zn-SOD and Fe-SOD and to be independent of their concentrations. These results show that the short drop time polarographic method is suitable for Fe-SOD kinetic studies, provided that one accounts for the 0 2 concentration when the kinetic constants k v and K M are calculated from Eq. (5). 3.2. Comparison of Cu, Zn-SOD and Fe-SOD activities at variable 0 2 concentration

The conclusions reported above are corroborated by the experimental results. Moreover, Fe-SOD activity measurements in the presence of catalase enzyme demonstrated that H202 produced by 0 2 dismutation did not influence the enzymatic activity values at the 0 2 concentrations used in this work. The data plotted in Fig. 3 show that the experimental value of k, for Fe-SOD decreases with increasing [O2]b, suggesting a Michaelis-Menten effect of the substrate according to Eq. (8). By rearranging the Eq. (8), the following linear relationship between 1 / k s and [O2]b is obtained: 1

KM - -

k,

kv

1.0 0.0 0

-

-

t

0.6

I

0.9

12

[02]b/raM

Fig. 3. Activity of Fe-SOD as a function of bulk concentration of 0 2. The activity measurenaents were carried out in a solution containing TPO and buffered with 0.04 M Tris (hydroxymethyl)aminomethane at p H 8.1 and T = 25±0.1°C.

constant k, for bovine Cu,Zn-SOD at different values of [O2]b, are reported in Table 1 for pH 7.9 and 8.9 and ionic strength 0.04 M. These data show that k s is almost constant at increasing [O2] b in the range 5 x 10-5-1 × 10 -a M. This means that, unlike FeSOD, the k s value for Cu,Zn-SOD is independent of the 0 2 concentration.

I

;

P[O2] b +

I

03

I

I

../

I

04 I-

S

-I

(9)

kv

The linear plot in Fig. 4 demonstrates the agreement of this equation with the experimental data; the ratios K M / k v and p / k v were calculated from the intercept and the slope respectively. Using the literature value of 5.2 × 10 4 S -1 [5], for k v calculated from the protein concentration, it was possible to calculate the values p = 0.27 5:0.02 and K M = 8.0(5:1.3) × 10 -5 M. The latter is in excellent agreement with the result obtained by Bull and Fee [5]. The value found for p indicates that the O~- concentration profile is quasi-exponential rather than linear, as expected from Koutecky's exact solution [14]. The values of the rate

-0.3

o.o

0.3

0.6

0.9

1.2

[02]b/mM Fig. 4. Linearization of the dependence of k s on bulk 0 2 concentration according to Eq. (9). The :dotted lines represent 95% confidence interval calculated using the Student distribution. Same experimental conditions as in Fig. 3.

E. Argese et al. / Bioelectrochemistry and Bioenergetics 36 (1995) 165-170 Table 1 Kinetic constants of Cu,Zn-SOD measured at different mean O 2 concentrations [O~ ]m' 0 2 (%)

[O~" ]m (mM)

kv[SOD][O2] Vo -----

5 10 21 40 70 100

0.01 0.03 0.06 0.11 0.20 0.28

2.98 2.90 3.17 3.25 2.87 3.27

0.03 0.06 0.11 0.20 0.28

3.30 3.25 3.17 3.14 3.19

pH S.9 10 21 40 70 100

The activity measurements were carded out in solutions buffered with Tris (pH 7.9) and sodium borate (pH 8.9) at an ionic strength of 0.04 M and T = 25 +0.1°C.

3.3. Steady-state analysis of Cu,Zn-SOD and Fe-SOD Both Cu,Zn-SOD and Fe-SOD catalyse O~ dismutation according to the following reaction mechanism [5]: Eox + O 2 ~

kl

k_l

~Eox" 0 2

k3 ERe d + 0 2 <

k_ 3

k2 ~ ERe d + O 2

k4 > ERe d " 0 2

> Eox + H 2 0 2

In summary a common cyclic oxidation-reduction mechanism is valid for both Cu,Zn-SOD and Fe-SOD: it probably consists of an initial step which involves the binding of 0 2 to the metal, followed by various intermediate steps involving electron redistribution and proton exchange, and a final step involving the release of products. The different kinetic behaviour of the two enzymes is probably due to different rate-limiting steps. For Cu,Zn-SOD the rate-limiting step is the encounter of 0 2 with the copper ion, in agreement with the fact that no saturation is observed even at very high [ 0 2 ] and with the hypothesis of the presence of an electrostatic force driving the 0 2 towards the enzy/natic reaction centre [6,7,20]. In the case of Fe-SOD the observed saturation kinetics can be interpreted in terms of a slow intermediate step limiting the reaction rate; this has been suggested to be proton donation to an enzyme-substrate intermediate complex [5].

Acknowledgements

(11)

The authors are grateful to Mr. R.V. Zonta and Mr. G. Giurin for helpful technical assistance. L.M.M. is grateful for the provision of a grant from CAPES and IPEN-CNEN, Brazil.

[2k2k4/(k4 + kz)][ET]' [ 0 2 l VO [(k l + k 2 ) k 3 k 4 + ( k _ 3 + k 4 ) k t k 2 ] / ( k 4 + k 2 ) k l k 3 + [ 0 2 ] (12)

which can be rewritten in classical Michaelis-Menten form as

KM+ [ 0 2 ]

(14)

kM

(10)

where Eox and ERea represent SOD in the oxidized and reduced states respectively. By applying the steady-state approximation to the intermediate enzyme-substrate complex and taking into account the enzymatic mass balance, the following expression for v 0 is obtained:

v°=

conditions. Therefore [0 2 ] is negligible with respect to the apparent K M value and Eq. (13) reduces to

10-gks (M -1 s -1)

pH 7.9

169

(13)

where k v and K M are composite constants dependent on the kinetic constants for the single steps of the overall reaction. The proposed mechanism accounts for the experimental dependence of activity on 0 2 concentration reported above for both Cu,Zn-SOD and Fe-SOD. In fact the non-saturation kinetics found for Cu,Zn-SOD can be justified with an apparent K M value much higher than 0.3 mM, which corresponds to the maximum 0 2 concentration achievable in our experimental

References [1] I. Fridovich, Adv. Enzymol. 41 (1974) 35. [2] D. Klug-Roth, I. Fridovich and G. Rabani, J. Am. Chem. Soc., 95 (1973) 2786. [3] E.M. Fielden, P.B. Roberts, R.C. Bray, D.J. Lowe, G.N. Mautner, G. Rotilio and L. Calabrese, Biochem. J., 139 (1974) 49. [4] F. Lavelle, M.E. McAdam, E.M. Fielden, P.D. Roberts, K. Pudget and A.M. Michelson, Biochem. J., 165 (1977) 71. [5] C. Bull and J.A. Fee, J. Am. Chem. Soc., 107 (1985) 3295. [6] A. Cudd and I. Fridovich, J. Biol. Chem., 257 (1982) 11443. [7] E. Argese, P. Viglino, G. Rotilio, M. Scarpa and A. Rigo, Biochemistry, 26 (1987) 3224. [8] D.P. Ballou, G. Palmer and V. Massey, Biochem. Biophys. Res. Commun., 38 (1986) 898. [9] J.M. McCord and I. Fridovich J. Biol. Chem., 244 (1969) 6049. [10] G. Rotilio, R.C. Bray and E.M. Fielden, Biochim. Biophys. Acta, 268 (1972) 604. [11] A. Rigo, P. Viglino and G. Rotilio, Anal. Biochem., 68 (1975) 1. [12] E. Argese, E.F. Orsega, B. De Carli, M. Scarpa and A. Rigo, Bioelectrochem. Bioenerg., 13 (1984) 385. [13] E. Argese, R. Girotto and E.F. Orsega, Biochem. J., 292 (1993) 451. [14] J. Koutecky, R. Brdicka and V. Hanus, Collect. Czech., Chem. Commun., 18 (1953) 611.

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E. Argese et al. /Bioelectrochemistry and Bioenergetics 36 (1995) 165-170

[15] T.O. Slykhouse and J.A. Fee, J. Biol. Chem., 251 (1976) 5472. [16] M.W. Pantoliano, J.S. Valentine and L.A. Nefic, J. Am. Chem. Soc., 104 (1982) 6310. [17] R. Brdicka and K. Wiesner, Collect. Czech. Chem. Commun., 12 (1947) 138. [18] P. Delahay, New Instrumental Methods in Electrochemistry, Krieger, Huntington, NY 1980, p. 102.

[19] E.F. Odemann and D.M.H, Kern, J. Am. Chem. Soc., 75 (1953) 3058. [20] W.H. Koppenol in M.A. Rodgers and Powers (eds.) Oxygen and Oxy-radicals in Chemistry and Biology, Academic Press, New York, 1981, p. 671.