J . Phys. Chem. 1991, 95, 684-687
684
Klnetlcs and Energetlcs of One-Electron-Transfer Reactions Involving Tryptophan Neutral and Cation Radicals Slobodan V. Jovanovic,*il Steen Steenken? and Michael G. Simic3 Laboratory 030, The Boris Kidric Institute of Nuclear Sciences, P. 0. Box 522, 11001 Beograd, Yugoslavia, Max- Planck-Institut fur Strahlenchemie, Miilheim a. d . Ruhr, West Germany, and Center for Radiation Research, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 (Received: December 18, 1989; In Final Form: August 15, 1990)
Reversible one-electron-transfer reactions involving the tryptophan cation and neutral radicals were investigated by pulse radiolysis. The one-electron-reduction potential of the neutral tryptophan radical at pH 7 was determined to be E7 = 1.01 f 0.03 V by using the bis( 1,4,7-triazacyclononane)nickel(III/II) redox couple with E = 0.95 V as a standard. The value obtained with promethazine ( E = 0.98 V) as a standard at pH 6 was E6 = 1.1 1 V. From these measurements, a mean value E7 = 1.03 V results, in agreement with some earlier determinations (1.05 and 1.08 V) obtained in experiments with inorganic redox standards. The kinetics and energetics of the reactions of the tryptophan neutral and cation radicals with selected electron donors were also investigated. The reactivity of the tryptophan radical cation was found to be 1-2 orders of magnitude higher than that of the neutral radical. The lower reactivity of the neutral tryptophan radical is explained by the entropy loss due to protonation of the tryptophan anion, resulting from the changes in the solvation shell.
Introduction Tryptophan, an essential amino acid, is often crucial for biological functioning of proteins and enzymes. It is also a metabolic precursor of vitamin B5 (niacin) and of the hormone and neurotransmitter 5-hydroxytryptamine (serotonin). The hydroxylated tryptophan metabolites 5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindole-3-acetic acid have recently been postulated as endogenous antio~idants.49~Tryptophan is readily oxidized by various oxidizing free radicals." Except for the OH' radical which predominantly adds to the indole ring of tryptophan: free-radical oxidants such as SO4*-?Br2-,' (SCN);-,7 and N3*,8 react by one-electron transfer. The result is the tryptophan radical cation, which is in equilibrium with the neutral tryptophan radical:
R-Ind'+
R-Ind(-H)'
pK, = 4.31° The tryptophan radical was shown to oxidize tyrosine in various peptides and via inter- or intramolecular electron transfer. Because of the importance of the mechanism of freeradical damage to proteins for the understanding of free-radical-induced cell injury, the redox properties of tryptophan and tyrosine have been extensively ~ t u d i e d . ~ lIn - ~contrast ~ to tyrosine, (1) The Boris Kidric Institute of Nuclear Sciences. (2) Max-Planck-lnstitut fllr Strahlenchemie. (3) National Institute of Standards and Technology. (4) Jovanovic, S. V.; Simic, M. G. Lye Chem. Rep. 1985, 3, 124. (5) Jovanovic, S. V.; Sttcnken, S.; Simic, M. G. J . Phys. Chem. 1990, 94, 3583. (6) Redpath, J. L.;Willson, R. L. fnr. J . Radiat. Biol. 1975, 27, 389. (7) Adams, G. E.; Aldrich, J. E.; Bisby, R. H.; Cundall, R. 8.; Redpath, J. L.; Willson, R. L. Radiat. Res. 1972, 49, 278. (8) Prlltz, W. A.; Land, E.J. fnr. J . Radiat. Biol. 1979, 36, 75. (9) Armstrong, R. C.; Swallow, A. J. Radiar. Res. 1969, 40, 563. (10) (a) Posener, M. A.; Adams, G. E.; Wardman, P.; Cundall, R. B. J . Chem. Soc., Faraday Trans. I 1976, 72, 2231. (b) Jovanovic, S.V.; Simic, M. G. J. Free Radicals Biol. Med. 1985, I , 125. (1 1) Prlltz, W. A.; Land, E.J.; Sloper, R. W. J . Chem. SOC.,Faraday, Trans. I 1981, 77, 281. (12) Butler, J.; Land, E.J.; Swallow, A. J.; Priltz, W. Radiar. Phys. Chem. 1984, 23, 265. (13) Butler, J.; Land, E.J.; PrUtz, W. A.; Swallow, A. J. Biochim. Biophys. Acra 1982, 705, 150. (14) Jovanovic, S.V.; Harriman, A.; Simic, M. G. J . Phys. Chem. 1986, 90,1935. ( I S ) Butler, J.; Land, E.J.; Swallow, A. J.; PrBtz, W. A. J . Phys. Chem. 1987, 91, 3113. (16) Harriman, A. J . Phys. Chem. 1987, 91, 6102.
0022-3654/91/2095-0684$02.50/0
for which the agreement between the reported oxidation potentials is reasonable (e.g. E, = 0.85 VI3J4and 0.94 VI7), the reported oxidation potentials of tryptophan at pH 7 range from 0.6414 to 1.08 V.'* In order to obtain more data on the potential of tryptophan, the electron-transfer equilibria between one-electronoxidized tryptophan and the inorganic complexes (1,4,7-triazacy~lononane)~Ni~+ and IrClb3- were studied by pulse radiolysis. The reduction potential of one-electron-oxidized tryptophan was further checked by measuring the kinetics of electron transfer to and from the organic redox standard promethazine. The kinetics and energetics of electron-transfer reactions of neutral and cation radicals of tryptophan with phenols were also investigated. Materials and Methods The compounds were of the highest purity commercially available and were used as received. Tryptophan, 4-methoxyphenol, promethazine hydrochloride, and 5-hydroxytryptophan were obtained from Sigma,20tyrosine methyl ester hydrochloride was a product of Aldrich, and sodium azide, potassium bromide, and potassium thiocyanate were obtained from Merck. Bis(1,4,7-triazacyclononane)nickel(II)perchlorate was a generous gift from Karl Wieghardt of the Ruhr University of Bochum. Water was purified through a Millipore Milli-Q system. All solutions were prepared freshly before experiment and saturated with high-purity gases (Ar or N20). The pH of the solutions was maintained by 10 mM phosphate buffer (Merck) or adjusted by HC104 (Merck). A fully computerized 3-MeV Van de Graaff instrument at the Max-Planck-Institut for Strahlenchemie2Ia and a 2-MeV Febetron 707 instrument at the Boris Kidric Institute21b were used for pulse-radiolysis experiments. Thiocyanate dosimetry22with G[(SCN),'-]. = 6.0 and a480 = 7600 M-l cm-' was used for dose determinations. Doses were adjusted to yield radical concentrations of 1-2 pM. Activation parameters were obtained from Arrhenius plots of rate data obtained for three concentrations of a reductant (e.g. (17) DeFelippis, M. R.; Murthy, C. P.; Faraggi, M.; Klapper, M. H. Biochemistry 1989, 28, 4847. (18) Merenyi, G.; Lind, J.; Shen, X . J. Phys. Chem. 1988, 92, 134. (19) Faraggi, M.; Weinraub, D.; Broitman, F.; DeFelippis, M.; Klapper, M. H. Radiar. Phys. Chem. 1988, 32, 293. (20) The mention of commercial products does not imply rec"endation or endorsement by the National Institute of Standards and Technology. nor does it imply that the products identified are necessarily the best available for the purpose. (21) (a) Trinoga, R.; Reikowski, F.; Steenken, S.; Lenk, H. To be published. (b) Markovic, V.;Nikolic, D.; Micic, 0. 1. In?.J . Radiar. Phys. Chem. 1974, 6, 227. (22) Schuler, R. H.; Hartzell, A. L.; Behar, B. J . Phys. Chem. 1981,85, 192.
0 1991 American Chemical Society
The Journal of Physical Chemistry, Vol. 95, No. 2, 1991 685
One-Electron-Transfer Reactions N20-saturated aqueous solution of 0.1 M KBr, 10 mM tryptophan, and 0.04, 0.08, and 0.16 mM 4-methoxyphenol at pH 2.81 for the reaction of the tryptophan cation radical and 0.1 M KBr, 10 mM tryptophan, and 0.6, 1.4,and 3.2 mM 4-methoxyphenol at pH 6.8 for the reaction of the neutral tryptophan radical) in the range 20-60 OC (at 20, 30, 40, 50, and 60 "C).
Results and Discussion One-Electron-Oxidation Potential. The measurement of the reduction potential of one-electron-oxidized tryptophan was attempted at pH 5.8 (where the radical is in the neutral form R-Ind(-H)') by using the hexachloroiridate(IV/III) redox couple with E = 0.892 V at p = 0.1 Ma as a reference. The tryptophan radical, R-Ind(-H)', was generated by oxidation of tryptophan with the azide radical:
+
N3* R-Ind
-
R-Ind(-H)'
+ N3- + H+
(2)
k = 4.1 x 109 M-1 s-18 In the absence of other solutes, R-Ind(-H)' decays by a second-order process, 2k = 6 X lo8 M-' s-l,' as monitored at 520 nm. Upon addition of hexachloroiridate(III), IrC163-, the decay 6f the indolyl radical changed to first order due to the following reaction: R-Ind(-H)'
+ IrC163- + H+
-
R-Ind
+ IrC162-
(3)
k = (1.6 f 0.2) X lo8 M-l s-l The oxidation of IrC163-by the tryptophan radical (reaction 3) was found to proceed to completion. Consequently, only a lower limit for the reduction potential of that radical may be derived, i.e., E6 > 0.892 V. This value is to be compared to E7 = 0.64,14 0.83,19- l , I 3 1.08,18and 1.05 Val7 In order to obtain a more precise value for the reduction potential of R-Ind(-H)', a redox standard with a higher reduction potential is required. The bis( 1,4,7-triazacyclononane)nickel(III/II), ( t a ~ n ) ~ N i ~ +redox / ~ + couple , with E7 = 0.95 V" vs NHE was hence selected as a standard. The tryptophan radical, R-Ind(-H)', was generated at pH 7 by Br;--induced oxidation of tryptophan:2s Br2'-
+ R-Ind
-
k = 7.7
R-Ind(-H)' X
+ 2Br- + H+
(4)
lo8 M-l s-I7
The tryptophan radical was found to oxidize the nickel complex, ( t a ~ n ) ~ N iaccording ~+, to the following reversible electron-transfer reaction: k
R-Ind(-H)'
+ (tacn),Ni2+ + H+ -1 .R-Ind + ( t a ~ n ) ~ N i ~ + kr
(5) However, because of the fast second-order decay of the tryptophan radicals and in order to see the establishment of electron-transfer equilibrium, it was necessary to use a low dose rate, Dlpulse 1 Gy, at which the concentration of radicals was -0.6 pM. Reaction 5 was monitored at 520 nm in an N,O-saturated aqueous solution of 0.1 M KBr, 11 mM tryptophan, 0-10 mM Ni( t a ~ n ) ~ ( C l Oand ~ ) ~10 , mM phosphate buffer at pH 7 and 20 OC. The equilibrium constant from the equilibrium absorbencies of the tryptophan radical was measured as Kabr= 10 f 3. The results of the kinetic treatment of the experimental data are shown in Figure 1. The forward and reverse rates of equilibrium 5 were obtained as the slope and intercept of Figure 1 and are k f = (4.8 f 5) X los M-' s-I and k, = (5 1) X lo4 M-'s-I, respectively. Consequently, the equilibrium constant is Kkin= kr/k, = 9.6 f 3, within experimental error, the same as Kat*. The mean equilibrium
-
*
(23) Margerum, D. W.; Chellappa, K. L.; Bow, F. P.; Burce, G. L. J. Am.
Chem. Soc. 1975, 97,6894.
(24) Wieghardt, K.; Schmidt, W.; Herrmann, W.; Ktlppers, H.-J. Inorg.
Chem. 1983, 22, 2953.
(25) The tryptophan radical can also be 8enerated by the azide radical (reaction 2).
T ]
v; , J
*-
f
I
0 L
'I
Figure 1. Kinetic treatment of reversible electron transfer between (taa&Ni2+ and the neutral tryptophan radical. Dose/pulse = 1.2 Gy in an N,O-saturated aqueous solution of 0.1 M KBr containing 10 m M phosphate buffer (pH 71, 1 1 m M tryptophan, and 0.49-10 m M (tacn)2Ni2t. The reaction was monitored at 520 nm. TABLE I: Eleetroft-Twfw Ructiolls of tk Twptopbra hdid h Aqueous Solution Measured by pulpe Radiolysis at 20 OC indolyl radical substrate, S generator k(R-Ind'+ + S )' k(R-Ind(-H)' S)' (tacn)2Ni2t Br2'7.7 x 107 4.8 x 105 1.0 x 10'b promethazine Br2'1 . 1 x 109 tyrosine methyl N,' 6.5 x 1 0 7 ~ 5.4 x lOJd ester HCI 4-methoxyphenol Br2'- or N,' 1.9 X IO9 1.2 x 107dc 5-hydroxyBr2'1.3 x 1091 2.0 x 1071 tryptophan 1,l'-ferroceneN,' n.m! 4.0 x 1078 dicarboxylate
+
"Estimated to be accurate to flO%. Reactivities of the indolyl radical cation, R-lnd'+, were measured at pH 3, whereas those of the neutral indolyl radical, R-Ind(-H)', were determined at pH 7. Units are M-' s-l. From ref 10b at pH 6. (Determined at pH 4. dFrom ref 14. cThe value of I X lo* M-' s-I at pH 7 was reported in ref 15. Our value at pH 7 is (1.2 f 0.1) X lo7 M-I s-l, whereas k = (1.0 f 0.1) X 10' M-I s-I was obtained at pH 6.5. At pH 7.7, we measured (5.5 f 0.5) X IO6 M-' s-I, which is equal to the value in ref 14. /From ref 4. Gimilar to the data in ref 19. *Not measured.
constant K = 9.8 was used to calculate the reduction potential difference between the couples R-Ind(-H)',H+/R-Ind and ( t a ~ n ) ~ N i ' + /from ~ + the Nernst equation AE = 0.059 log K = 0.059 log 10 = 0.059 V (6) Consequently, the reduction potential of tryptophan at pH 7 is determined as E7 = 0.95 0.059 V = 1.01 V. The reduction potential of the neutral tryptophan radical was also determined against promethazine (Pz), for which E = 0.98 V was recently reported.5 In an unbuffered N20-saturated aqueous solution of 0.1 M KBr, 1 1 mM tryptophan, and 0 4 . 8 4 mM promethazine at pH 6.0, the following electron-transfer equilibrium was observed:
+
k
R-Ind(-H)'
+ Pz & R-Ind(-H)- + Pz'+ kr
(7)
The equilibrium constant Kab = 182 was derived from the absorbencies of the radicals at equilibrium. From the kinetics of the formation of the promethazine radical cation monitored at 515 nm, kf = (1.1 f 0.05) X lo8 M-I s-I and k, = (6.2 f 1) X los M-I s-l were obtained. Consequently, the equilibrium constant from kinetics is Kkin= 177. By use of the Nernst equation (eq 6), the redox potential difference between the couples R-Ind(-H)'/R-Ind and Pz'+/Pz is calculated from the mean equilibrium constant as 0.13 V. The reduction potential of one-electron-oxidized tryptophan is therefore E6 = 0.98 + 0.13 = 1.11 v.
686 The Journal of Physical Chemistry, Vol. 95, No. 2, 1991
Jovanovic et al.
TABLE 11: Activation Panmetera for the Reactions of Tryptophan Neutral and Cation Radicals R-Ind(-H)' + S substrate, S P Eb SC log A P I,l'-ferrocenedicarbxylate 4.0 X lo7 2.8 f 1 -16 f 2 9.7 0.4 f 2 -27 f 4 4-methoxyphenol 1.2 X IO' 7.4 1.9 x 109 0.7 f 1 -24 f 4 8 1.3 X lo9 5-hydroxytryptophan 2.0 X IO'
R-In@+
+S
Eb
S'
log A
nmd 5.5 1 5.2 f 1
1f2
13.5
-1 f 2
*
13
ORate constants in M-'s-I measured at 20 O C (see also Table I). bArrhenius activation energies in kcal mo1-I obtained from the temperature dependence of the rate constants from 20 to 60 O C . The error limits are based on the standard deviations in the Arrhenius plots. 'Entropies in cal mol-' K-I. dNot measured because of the relative insolubility of ferrocenedicarboxylate in acidic media.
From the average of E7 = 1.01 V obtained in the measurement with the nickel complex and E7 = 1.05 V converted26from the pH 6 measurement with promethazine, the mean reduction potential of the neutral tryptophan radical is E7 = 1.03 V. This value is in satisfactory agreement with the values E7 = 1.0818and 1.05 VI7 obtained in experiments in which C102'/C102-18and Os( t e r ~ y ) ~ ~ +Irc162-'3-, /~+, C102'/C10~, NO2'/N0C, and Fe( b ~ y ) , ~ +I /7 ~were + used as redox standards, respectively. A comment may be made on the determination of the oxidation potentials of some indoles and phenol derivatives by cyclic voltammetry (CV) with glassy-carbon electrodes.16 E7 = 1.015 VI6 obtained for tryptophan by CV is indeed similar to E7 = 1.03 V measured in this study. However, on the basis of the voltammetric measurements,I6 the oxidation potentials of indole derivatives with different side-chains differ by as much as -0.18 V (e.g. E7 = 1.015 V for tryptophan (a-aminoindole-3-propionicacid) is 0.175 V higher than E7 = 0.84 VI6 for indole-3-acetic acid), for which there is no reasonable explanation. The potential for indole-3acetic acid (E7 = 0.84 V)I6 is lower than E7= 0.92 VI6 for tyrosine. On this basis, it would be expected that indole-3-acetic acid is oxidizable by the tyrosine phenoxy radical. In contrast to this expectation, the electron transfer is in the opposite direction; i.e., the indolyl radical;00CCH21nd(-H)', generated by the azide radical induced oxidation of indole-3-acetic acid at pH 7.5,was found to oxidize tyrosine methyl ester, CH300C(NH3+)CHCH2PhOH, according to the following reaction:
+
-
-OOCCH21nd(-H)* CH300C(NH3+)CHCH2Ph0H -OOCCH21nd CH300C(NH3+)CHCH2Ph0' (8)
+
k = (4 f 0.8)
Hence, the oxidation potential of indole-3-acetic acid at pH 7.5 must be higher than that of tyrosine and is probably similar to the potentials of other indole derivatives. The discrepancy between the pulse-radiolysis data and those from CV can be understood if it is assumed that with the latter the reversibility of the electrode processes is not always fully established. Electron-TransferReactions of the Ttyptophan Radical. Since the tryptophan radical can exist in two protonation states (equilibrium 1), distinctly different redox and kinetic properties are expected and observed at corresponding pH's.% For example, emethoxyphenol is oxidized at substantially different rates at pH 7.5 R-Ind(-H)'
+ 4-(CH30)PhOH
-
R-Ind
+ 4-(CH30)PhO' (9)
k = 5.5 X lo6 M-Is-I
and at pH 3 R-Ind'+
+ 4-(CH30)PhOH
-+
R-Ind
k = 1.9
X
4-(CH30)PhO'
lo9 M-I
+ H+ (10)
s-I
(26) The pH dependence of the reduction potential of one-electron-oxidized tryptophan may be derived from the formula EpH= Eo + 0.059 log ([H+]/(K, + (H ])I where pK, = 4.37*10is the dissociation constant of the tryptophan radical. From the value E, = 1.03 V measured in this study, the calculated reduction potentials at selected pH's are as follows: standard potential Eo = 1.19 V; E, = 1.19 V a t pH 3; E6 1.09 V a t pH 6 .
-
R-Ind
* R-Ind(-H)- + H+
(11)
pK, = 16.8227
-
the anion will be immediately protonated by water: R-Ind(-H)-
IO5 M-l s-l
X
Other representative rate constants of electron-transfer reactions of the cation radical at pH 3 and the neutral radical at pH 7.5 are summarized in Table I. As seen in Table I, the reaction rates of the radical cation with the electron donors are 1-2 orders of magnitude higher than those of the neutral tryptophan radical. The increased reactivity of the radical-cation form with phenols in general is unlikely to be due only to its higher reduction potential (E,(cation) = 1.19 V)26 compared to the potential of the neutral indolyl radical (E7,s(neutral)= 1.00 V). If the reduction potential difference was the only factor determining reactivity, then in the case of phenols as reductants, where [E7.5(Trp)- E,,s(phenol)] > [E3(Trp) - E3(phenol)] by 0.08 V, for most phenols the rates at pH 7.5 should be higher than at pH 3, which is contrary to our measurements, i.e. k(pH 7.5)
