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J. Phys. Chem. B 2000, 104, 856-862
Tunneling Effect in Antioxidant, Prooxidant, and Regeneration Reactions of Vitamin E† Shin-ichi Nagaoka* Institute for Molecular Science, Okazaki 444-8585, Japan
Masayo Inoue, Chiho Nishioka, Yoshinori Nishioku, Sayuri Tsunoda, Chikage Ohguchi, Keishi Ohara, and Kazuo Mukai Department of Chemistry, Faculty of Science, Ehime UniVersity, Matsuyama 790-8577, Japan
Umpei Nagashima National Institute for AdVanced Interdisciplinary Research, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-4 Higashi, Tsukuba 305-8562, Japan ReceiVed: September 10, 1999; In Final Form: NoVember 11, 1999
Studies of the kinetics of the proton- or hydrogen-transfer reactions concerning vitamin E in solutions and in micellar dispersions by means of stopped-flow and absorption spectroscopy indicated that proton tunneling plays an important role in the antioxidant and regeneration reactions that are advantageous in vivo but not as a part of the harmful prooxidant action.
Introduction Recent studies show that one of the causes of aging is lipid peroxyl radicals (LOO•s) formed by the reactions of lipids and oxygen.1-5 The living body, however, has a way to scavenge LOO• and thus help prevent aging: it is the so-called antioxidant reaction of vitamin E (d-tocopherols, TocH). We find evidence for this function of TocH in the fact that the lifetimes of mammalian species are proportional to their plasma levels of TocH.1 Figure 1 shows the scheme of LOO• production and of a part of the antioxidant reaction of TocH.2-5 A lipid radical (L•) is first formed from lipid; the radical formation is usually caused by radicals, light, heat, metal,6 or irradiation. The L• radical reacts with oxygen to produce LOO•, and the reaction of LOO• and TocH (Figure 1) results in the production of hydroperoxide (LOOH) and the vitamin E radical (tocopheroxyl radical, Toc•). Figure 2 shows the molecular structures of natural TocHs (R-, β-, γ-, and δ-TocHs), each of which has a hydroxychroman ring and a phytyl side chain that stabilizes TocH in the cell membrane. These TocHs differ from one another only in the number and positions of the methyl groups on the aromatic ring. R-TocH, which is fully methylated, is the most biologically active of these four molecules. Toc•, which is produced from the above-mentioned reaction, combines with LOOH and again produces LOO• (Figure 1), which is harmful as described above. This is a part of the prooxidant action of TocH.7,8 To suppress the prooxidant action, TocH is regenerated by the reactions of Toc• with ubiquinol (UQH2) in the cell membrane9-11 and with vitamin C (L-ascorbic acid, AsA) at the interface of the cell membrane and the water phase12-15 (Figure 1). All of the above-mentioned reactions of TocH are essentially proton- or hydrogen-transfer reactions: † This paper is dedicated to Professor Noboru Hirota of Kyoto University on the occasion of his retirement. * To whom correspondence should be addressed.
Figure 1. Scheme of LOO• production and a part of the antioxidant, prooxidant, and regeneration reactions of TocH in the cell membrane.
TocH + LOO• f Toc• + LOOH
(1)
LOOH + Toc• f LOO• + TocH
(2)
UQH2 + Toc• f UQH• + TocH
(3)
AsA + Toc• f AsA• + TocH
(4)
where UQH• and AsA• are respectively dehydroubiquinol and vitamin C radicals. Reaction 2 is the reversal of reaction 1. The reaction L + Toc• f L• + TocH also contributes to the prooxidant action of TocH.7,8 The proton- or hydrogen-transfer such as that in reactions 1-4 is a very simple chemical process readily subject both to accurate measurement and to quantitative theoretical analysis. It would be especially interesting to study the tunneling effect in the proton- or hydrogen-transfer. In a previous paper,16 we proposed the following mechanism for reaction 1:
10.1021/jp9932113 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/08/2000
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TocH + LOO• f [TocHδ+ - - - LOO•δ-] (partial electron transfer) f [Toc• + LOOH]
(proton tunneling)
f Toc• + LOOH
(5)
Figure 3 shows the calculated contour map of the potential surface in a model of reaction 1. The broken curve denotes the intrinsic reaction coordinate.17 In the initial stage of the reaction, TocH and LOO• approach each other and their electron clouds begin to overlap (a f b in Figure 3). Because TocH and LOO• are respectively an electron-donor and an electron-acceptor, the transition state of this reaction has the property of the chargetransfer species ([TocH+ - - - LOO•-]). In reality, however, when TocH and LOO• approach each other ([TocHδ+ - - LOO•δ-]), the proton tunneling takes place below the transition state (b f c in Figure 3). The tunneling allows the proton to cut a corner on the potential energy surface. Finally, Toc• and LOOH separate from each other (c f d in Figure 3). Both the electron transfer and the proton tunneling play important roles in reaction 1. A few questions, however, remained to be investigated further. First, although the results obtained in the previous paper16 were limited to the deuterium kinetic-isotope-effects on the second-order reaction rate constants at room temperature, it is desirable to obtain the temperature-dependence of the reaction rate constant so as to discuss the tunneling effect in further detail. Second, reactions 1, 3, and 4 are advantageous in vivo, but reaction 2 is harmful. Can the tunneling effect be observed in all of these biochemical proton- or hydrogen-transfer reactions? On the first and second points we studied the kinetics of models of reactions 1-4 by stopped-flow and absorption spectroscopy and examined the deuterium kinetic-isotope-effects on the second-order reaction rate constants and on the apparent activation energies. Third, all of our previous experiments were
Figure 3. Calculated contour map of potential surface in a model of reaction 1. In the calculation, phenol and CH3OO• were used instead of TocH and LOO•. Phenol was assumed to be planar. The computational method was AM1. The ordinate here is the distance between the phenol oxygen and the terminal oxygen of CH3OO• (O-O distance), and the abscissa is the O-H distance of the phenol hydroxyl group. The contour lines are drawn at 0.5-eV intervals. The transition state (saddle point) is located at O-H distance ) 1.198 Å and O-O distance ) 2.357 Å, and the energy is -1976.84056 eV. The broken curve denotes the intrinsic reaction coordinate.17
carried out in solution. Does the tunneling effect really play an important role under conditions such as those in the cell membrane? To answer this question we studied the kinetics of models of reactions 3 and 4 in micellar dispersions, which can be regarded as a model of the cell membrane. The work reported in this paper was presented as an invited lecture at the 3rd International Conference of Low-Temperature Chemistry.18 Experimental Section
Figure 2. Structures of molecules used in the present work.
Sample Preparation. The structures of the molecules studied in this work are shown in Figure 2. d-R-, d-β-, d-γ-, and d-δTocHs were kindly supplied by Eisai Co., Ltd. and were used without further purification. We prepared dl-tocol, 5,7-diisopropyltocopherol (di-iPr-TocH), and 2,6-di-tert-butyl-4-(4-methoxyphenyl)phenol (ArOH) as reported in a previous paper.19 Experimental details on the preparation and characterization of 5,7-dichloro-TocH (di-Cl-TocH) are available as Supporting Information. Because LOO•, Toc•, and LOOH are not stable, in the present work we instead used the stable 2,6-di-tert-butyl4-(4-methoxyphenyl)phenoxyl (ArO•), 5,7-diisopropyltocopheroxyl (di-iPr-Toc•), and alkyl hydroperoxides (ROOHs). The preparation of ArO• from ArOH and di-iPr-Toc• from di-iPrTocH was reported previously,19,20 and the ROOHs n-butyl hydroperoxide (n-BuOOH), s-butyl hydroperoxide (s-BuOOH), tert-butyl hydroperoxide (t-BuOOH), n-propyl hydroperoxide (n-PrOOH), and isopropyl hydroperoxide (iPrOOH) were prepared according to the method of Williams et al.21,22 As a representative of UQH2, ubiquinol-10 (UQ10H2) was used in the present work. It was prepared from ubiquinone-1010 kindly supplied by Kaneka Corporation. In the present study, reactions 3 and 4 were also studied in aqueous Triton X-100 micellar dispersions at pH 7.0. In aqueous solutions around pH 7, AsA exists mostly as the monoanion in which the proton at the 3-position is dissociated.23,24 Accordingly, the L-ascorbic acid sodium salt (AsAH3) was used in the present work. AsAH3
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(guaranteed reagent) was obtained from Nacalai Tesque and was used without further purification. Benzene (Bz), hexane (Hex), and methanol (MeOH) were obtained from Wako Pure Chemical Industries and were dried and purified by distillation. Ethanol-d0 (EtOH) was obtained from Nihon Alcohol and was also dried and purified by distillation. Water-d0 (H2O) was purified by ion exchange and a Millipore Q system before use. Ethanol-d1 (C2H5OD, EtOD) of 99.5% purity, methanol-d1 (CH3OD, MeOD) of 99.5% purity, and water-d2 (D2O) of 99.9% purity were purchased from Aldrich and used without purification. Triton X-100 was obtained from Wako Pure Chemical Industries and used without purification. When TocH and ROOH is each dissolved in EtOD, the hydrogen atom of the OH group is easily replaced by a deuteron to yield the deuterated molecule (TocD and ROOD). This replacement was confirmed by proton NMR. When AsAH3 is dissolved in D2O, the hydrogen atoms of the OH groups are easily replaced by deuterons to yield the deuterated molecule (AsAD3). This replacement was confirmed by the proton NMR spectrum of AsAH3 in dimethyl sulfoxide (DMSO) showing peaks due to the hydrogen atoms of the OH groups and by the disappearance of those peaks when a small amount of D2O was added. Measurements. The following model reactions were studied in the present work:
Figure 4. Change in absorption spectrum during reaction 1′. The arrows indicate the decrease in the absorbance of ArO• and the increase in the absorbance of Toc•. (a) Reaction of R-TocH and ArO• in EtOH at 25 °C. [R-TocH]t)0 ) 0.294 mM and [ArO•]t)0 ) 36.2 µM. (b) Reaction of R-TocD and ArO• in EtOD at 25 °C. [R-TocD]t)0 ) 0.293 mM and [ArO•]t)0 ) 36.2 µM.
TocH (TocD) + ArO• k1
98 Toc• + ArOH (ArOD)
(1′)
ROOH (ROOD) + di-iPr-Toc• k2
98 ROO• + di-iPr-TocH (di-iPr-TocD)
(2′)
UQ10H2 (UQ10D2) + di-iPr-Toc• k3
98 UQ10H• (UQ10D•) + di-iPr-TocH (di-iPr-TocD) (3′) AsAH3 (AsAD3) + di-iPr-Toc• k4
98 AsAH2• (AsAD2•) + di-iPr-TocH (di-iPr-TocD) (4′) The setup and experimental procedures for the measurement of the rate constants are described in detail elsewhere.10,11,16,18-20,24-27 The experimental error in the temperature was less than (0.5 °C. The observed rate constants were calculated in the usual way using a standard least-squares analysis. The kinetic data of reaction 1′ were obtained (with an Unisoku stopped-flow spectrophotometer model USP-500) by mixing equal volumes of EtOH (EtOD) solutions of TocH (TocD) and ArO• under a nitrogen atmosphere. The reactions were studied under pseudo-first-order conditions ([TocH(TocD)] . [ArO•]) and the absorption decay of ArO• was then well-characterized by a single-exponential decay. Although ArO• was stable in the absence of TocH (TocD), when an EtOH (EtOD) solution with excess TocH (TocD) was added to the ArO• solution, the ArO• absorption peak disappeared immediately. Figure 4 shows a change in absorption spectrum during reaction 1′. The pseudofirst-order rate constant (kobsd) was determined by evaluating the decrease in the absorbance of ArO•. As shown in Figure 5,
Figure 5. Dependence of kobsd on [TocH(TocD)] in the reaction of R-TocH (R-TocD) with ArO• in EtOH (EtOD). The kobsd value was obtained by monitoring the decrease in the absorbance of ArO• at 380 nm.
kobsd was linearly dependent on the concentration of TocH (TocD). The rate equation is thus expressed as
-d[ArO•]/dt ) kobsd[ArO•] ) k1[TocH(TocD)][ArO•] (6) where k1 is the second-order rate constant for reaction 1′. It was obtained by plotting kobsd against [TocH(TocD)] (Figure 5). The second-order rate constants for reactions 3′ and 4′ (k3 and k4) were obtained in a similar way. In the experiments on reaction 2′, Bz and EtOH (EtOD) were mixed in a 20:1 volume ratio to obtain a mixed solvent Bz/ EtOH (Bz/EtOD). The kinetic data of reaction 2′ were obtained
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(with a Shimadzu UV-2100S spectrophotometer) by mixing equal volumes of Bz/EtOH (Bz/EtOD) solutions of ROOH (ROOD) and di-iPr-Toc• under a nitrogen atmosphere. These reactions were studied under pseudo-first-order conditions ([ROOH(ROOD)] . [di-iPr-Toc•]) and the absorption decay of di-iPr-Toc• was then well-characterized by a singleexponential decay. The kobsd value was determined by following the decrease in the absorbance of di-iPr-Toc• and is given by
-d[di-iPr-Toc•]/dt ) kobsd[di-iPr-Toc•]
(7)
As reported by Mahoney et al.,28 under usual conditions,20,25 the rate of the disappearance of di-iPr-Toc• is given by
-d[di-iPr-Toc•]/dt ) 2k2[ROOH(ROOD)][di-iPr-Toc•] (8) where k2 is the second-order rate constant for reaction 2′. From eqs 7 and 8, we know that kobsd is given by
kobsd ) 2k2[ROOH(ROOD)]
(9)
Accordingly, k2 can be obtained by plotting kobsd against [ROOH(ROOD)]. The value of the k2 for ArOH (ArOD) in EtOH (EtOD) was obtained in a similar way but by using the stopped-flow spectrophotometer. In the experiments on reaction 4′ in solution, MeOH (MeOD) and H2O (D2O) were mixed in a 5:1 volume ratio to obtain a mixed solvent MeOH/H2O (MeOD/D2O) because di-iPr-Toc• is alcohol-soluble and AsAH3 is water-soluble. AsAH3 was first dissolved in H2O (D2O) and then MeOH (MeOD) was added to obtain a MeOH/H2O (MeOD/D2O) solution of AsAH3 (AsAD3). di-iPr-TocH was first dissolved in MeOH (MeOD) and then H2O (D2O) was added to obtain a MeOH/H2O (MeOD/ D2O) solution of di-iPr-TocH (di-iPr-TocD). In addition, diiPr-Toc• was prepared by PbO2 oxidation of di-iPr-TocH (diiPr-TocD) in the solution under a nitrogen atmosphere. In the experiments on reactions 3′ and 4′ in micellar dispersions, we used a 5.0 wt % H2O (D2O) solution of Triton X-100. di-iPr-Toc• was prepared by ArO• oxidation of di-iPrTocH (di-iPr-TocD) in the micellar dispersion under a nitrogen atmosphere. The aqueous solution was buffered at pH 7.0, and 0.1 M KH2PO4 and 0.1 M NaHPO4 were used as the buffer. The D2O solution was similarly buffered at pD ) 7.0. pD was calculated from the pH meter reading as pD ) pH + 0.4.29,30 The mole fractions of neutral UQ10H2 and monoanionic AsA are nearly unity around pH 7.23,24,31,32 In a micellar dispersion, reaction 3′ occurs entirely in the micelles rather than throughout the dispersion. di-iPr-Toc• and UQ10H2 move freely between the micelles. Accordingly, the k3 values should be calculated for the micellar volume, which is not well-known. In addition, reaction 4′ in a micellar dispersion occurs at the interface of a micelle and the water phase rather than throughout the dispersion. di-iPr-Toc• and AsAH3 are respectively located in micelles and in the water phase. But because we calculated the k3 and k4 values in micellar dispersions as if reactions 3′ and 4′ had taken place in uniform solutions, the values obtained should be considered approximate. Results and Discussion Reaction 1′. Figure 6 shows the Arrhenius plots of the k1 values of TocH and TocD (k1H and k1D) and linear relationships between log k1 and 1/T can be seen in the plots. The average ratio of k1H to k1D (k1H/k1D) at 15-35 °C, the apparent activation energy (E1), and the apparent frequency factor (A1) are listed in Table 1, where substantial deuterium kinetic-isotope-effect
Figure 6. Arrhenius plot of k1 for the reaction of TocH (TocD) with ArO• in EtOH (EtOD).
TABLE 1: knH/knD, En, and log An (n ) 1-4) solvent
radical
knH/knD
En (kJ/mol)
log Ana
18.0 ( 0.8 22.2 ( 2.1 20.5 ( 1.3 28.5 ( 3.8 20.9 ( 2.5 28.9 ( 1.7 23.8 ( 0.4 34.3 ( 3.8 26.8 ( 1.3 41.8 ( 4.6
6.8 ( 0.2 6.2 ( 0.4 7.0 ( 0.2 7.2 ( 0.7 7.1 ( 0.4 7.3 ( 0.3 7.2 ( 0.1 8.0 ( 0.6 7.3 ( 0.2 9.1 ( 0.8
R-TocH R-TocD β-TocH β-TocD γ-TocH γ-TocD δ-TocH δ-TocD tocol tocol-d
EtOH EtOD EtOH EtOD EtOH EtOD EtOH EtOD EtOH EtOD
Reaction 1′ ArO• 22.5 ArO• ArO• 15.3 ArO• ArO• 14.6 ArO• ArO• 10.5 ArO• ArO• 8.0 ArO•
n-BuOOH n-BuOOD s-BuOOH s-BuOOD t-BuOOH t-BuOOD n-PrOOH n-PrOOD iPrOOH iPrOOD
Bz/EtOH Bz/EtOD Bz/EtOH Bz/EtOD Bz/EtOH Bz/EtOD Bz/EtOH Bz/EtOD Bz/EtOH Bz/EtOD
Reaction 2′ di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc• di-iPr-Toc•
3.4 80.3 ( 7.0 86.2 ( 6.0 3.6 55.6 ( 0.5 58.2 ( 3.2 4.7 44.8 ( 2.0 52.7 ( 2.0 3.6 86.2 ( 5.3 87.0 ( 3.8 3.4 71.1 ( 3.1 73.2 ( 1.9
12.4 ( 1.3 12.9 ( 0.9 8.4 ( 0.1 8.3 ( 0.6 6.8 ( 0.4 7.5 ( 0.3 13.1 ( 0.9 12.6 ( 0.7 10.9 ( 0.6 10.8 ( 0.3
ArOH ArOD
EtOH EtOD
Reaction 2′′ di-iPr-Toc• 3.7 13.7 ( 1.3 di-iPr-Toc• 15.1 ( 5.1
5.4 ( 0.2 5.0 ( 0.9
UQ10H2 UQ10H2
Hex micelle
Reaction 3′ di-iPr-Toc• di-iPr-Toc• >18
11.4 ( 0.6
7.4 ( 0.1
AsAH3 AsAD3 AsAH3
Reaction 4′ MeOH/H2O di-iPr-Toc• 12.5 11.1 ( 0.4 MeOD/D2O di-iPr-Toc• 24.2 ( 0.7 micelle di-iPr-Toc• 12.1 29.7 ( 2.9
7.1 ( 0.1 8.3 ( 0.1 8.6 ( 0.5
a Because log A was obtained by extrapolating the linear log k vs n n 1/T plot in limited 1/T range around room temperature to the intercept, these values have a large uncertainty.
on k1 and E1 is illustrated. The k1H/k1D values exceed the maximum semiclassical ratio.6-8,33,34 The E1 difference between TocH and TocD also exceeds the maximum semiclassical difference (1.3-4.2 kJ/mol).33,34 These results clearly show that the tunneling effect plays an important role in reaction 1′ in vitro. Accordingly, one might expect the tunneling effect to contribute to the TocH inhibition of lipid peroxidation in vivo. It is very interesting that the microscopic quantum-mechanical tunneling effect could manifest itself in a macroscopic vital
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ArOH (ArOD) + di-iPr-Toc• k2
98 ArO• + di-iPr-TocH (di-iPr-TocD)
Figure 7. Arrhenius plot of k2 for the reaction of ROOH (ROOD) with di-iPr-Toc• in Bz/EtOH (Bz/EtOD). It should be noted that as indicated from the comparison with the results reported in ref 20, k2 decreases as the proportion of EtOH (EtOD) in the solvent increases.
function. Proton tunneling was previously shown to contribute also to the other vital functions such as the lipoxygenase reaction,35 the reaction of long-lived radicals and AsA,36,37 and the methylmalonyl-CoA mutase catalyzed reaction.38 Because linear relationships between log k1 and 1/T can be seen in Figure 6 and the tunneling effect plays an important role in this reaction, the temperature region in the present experiments is likely to correspond to region II in Figure 3 of ref 35. We critically examined the other possibilities to explain the above-mentioned deuterium kinetic-isotope-effect on k1 and E1. Steric hindrance may play a role in the deuterium kinetic-isotope effects39-42 observed between TocHs and ArO•. In fact, kH/kD of about 10 has been found for the reactions of peroxyl radicals with sterically hindered phenols such as 2,6-di-tert-butyl-4methylphenol above room temperature.40,41 However, their activation energy differences can be explained on the basis of a simple zero-point energy effect from the O-H and O-D fundamental stretching frequencies; that is, those do not exceed the maximum semiclassical difference.40,42 In contrast, the E1 difference between TocH and TocD exceeds the maximum semiclassical difference as described above. Furthermore, for the steric hindrance, as the steric factor decreases, the magnitude of the deuterium kinetic-isotope effect will decrease. The steric factor of TocHs used in the present work is much less than that of 2,6-di-tert-butyl-4-methylphenol. Thus, it is considered that the steric hindrance does not play a major role in the deuterium kinetic-isotope-effect on k1 and E1. Reaction 2′. Figure 7 shows the Arrhenius plots of the k2 values of TocH and TocD (k2H and k2D). The average ratio of k2H to k2D (k2H/k2D) at 15-35 °C, the activation energy (E2), and the frequency factor (A2) are listed in Table 1, where it can be seen that the deuterium kinetic-isotope-effect on k2 and E2 is much less obvious than that on k1 and E1. The k2H/k2D values do not exceed the maximum semiclassical ratio.6-8,33,34 Most of the E2 differences between ROOH and ROOD do not exceed the maximum semiclassical difference (1.3-4.2 kJ/mol).33,34 These results show that, in contrast to what we found for reaction 1′, the tunneling effect does not play a major role in reaction 2′. Although we suggested in a previous paper20 that the tunneling effect plays an important role in reaction 2′, that suggestion should be reconsidered. Furthermore, the tunneling effect does not play a major role in the following reaction, either (Table 1):
(2′′)
which is exactly the reversal of reaction 1′. Judging from the results summarized in Table 1, one might think that the tunneling effect does not play an important role in reaction 2 in vivo. As noted in the Introduction, reaction 2 (an endothermic reaction) is the reversal of reaction 1 (an exothermic reaction). It seems at first strange that the tunneling effect plays an important role in reaction 1 but not in the reverse reaction, because the reaction path on the potential surface is usually the same for the forward and reverse reactions. In the present case, from some reason, the reaction path of reaction 2 on the potential surface may be different from that of reaction 1. Further investigations on this point are clearly needed. Reaction 3′ in Solution. We previously studied the kinetics of reaction 3′ in EtOH and EtOD and found a substantial deuterium kinetic-isotope-effect on k3,26 indicating that the tunneling effect plays an important role in reaction 3′ in solution. In that study we also found a deviation from a linear relationship in the Arrhenius plot.26 To determine if the deviation is a general phenomenon, we studied the kinetics of reaction 3′ in the nonpolar solvent Hex. Figure 8 shows the Arrhenius plot of k3 in Hex. In contrast to what we found for the reaction in EtOH,26 we do not see a deviation from a linear relationship and can evaluate the apparent activation energy (E3) and frequency factor (A3) as listed in Table 1. Furthermore, the deviation from the Arrhenius equation in reaction 3′ in EtOH and EtOD26 cannot be simulated well with the modified Arrhenius equation43-46 that ties the rate constants obtained at room temperature and at liquid helium temperature. Because the equation is useless for the experimental data obtained in a narrow temperature range around room temperature,46 we cannot make an unambiguous explanation at present. However, it might be suggested from the above results that the deviation in EtOH and EtOD26 might originate from some cause, such as solvation, other than the tunneling effect. It is clear, however, from the substantial deuterium kinetic-isotope-effect26 that the tunneling effect plays an important role in reaction 3′. Reaction 4′ in Solution. Figure 9a shows the Arrhenius plots of the k4 values of AsAH3 and AsAD3 (k4H and k4D) in MeOH/ H2O (MeOD/D2O). The average ratio of k4H to k4D (k4H/k4D) at 15-35 °C, the apparent activation energy (E4), and the apparent frequency factor (A4) are listed in Table 1, where a substantial deuterium kinetic-isotope-effect on k4 and E4 is illustrated. The k4H/k4D value exceeds the maximum semiclassical ratio.6-8,33,34 The E4 difference between AsAH3 and AsAD3 also exceeds the maximum semiclassical difference (1.3-4.2 kJ/mol).33,34 These results clearly show that the tunneling effect plays an important role in reaction 4′ in solution. Reactions 3′ and 4′ in Micellar Dispersion. The k3 and k4 values of UQ10H2, UQ10D2, AsAH3, and AsAD3 (respectively k3H, k3D, k4H, and k4D) in H2O and D2O solutions of Triton X-100 at room temperature are available as Supporting Information. Figure 9b shows the Arrhenius plots of the k4H values in the micellar dispersion. The ratios of k3H to k3D (k3H/k3D) and k4H to k4D (k4H/k4D) at 25 °C, the apparent activation energy (E4) for k4H, and the apparent frequency factor (A4) for k4H are listed in Table 1, where a substantial deuterium kinetic-isotope-effect on k3 and k4 is illustrated. The k3H/k3D and k4H/k4D values exceed the maximum semiclassical ratio.6-8,33,34 These results clearly show that the tunneling effect also plays an important role in reactions 3′ and 4′ in micellar dispersions; that is, in a model of the cell membrane. Accordingly, one might expect the
Tunneling Effect in Reactions of Vitamin E
Figure 8. Arrhenius plot of k3 for reaction of UQ10H2 with di-iPrToc• in Hex.
J. Phys. Chem. B, Vol. 104, No. 4, 2000 861 - 1/r (where n and r respectively denote refractive index and dielectric constant), and the k1 values of di-Cl-TocH (di-ClTocD) in EtOH (EtOD) are available as Supporting Information. The plot of k1 vs 1/n2-1/r is consistent with electron transfer actually playing an important role in reaction 1 and with it corresponding to the normal regime in Marcus theory.48-50 The k1 of di-Cl-TocH (di-Cl-TocD), which has two electronwithdrawing chlorine atoms, is about 1/200 the k1 of R-TocH (R-TocD), which has three electron-donating methyl groups. This result is also consistent with electron-transfer playing an important role in reaction 1. For reaction 1, the electron transfer and the tunneling effect could be different sides of the same coin. Conclusions These studies on the kinetics of reactions 1′-4′ in solutions and in micellar dispersions, carried out by means of stoppedflow and absorption spectroscopy, have confirmed that proton tunneling plays an important role in reactions 1, 3, and 4sreactions that are advantageous in vivosbut not in the harmful reaction 2.
Figure 9. Arrhenius plot of k4. (a) Reaction of AsAH3 (AsAD3) with di-iPr-Toc• in MeOH/H2O (MeOD/D2O). (b) Reaction of AsAH3 with di-iPr-Toc• in an aqueous Triton X-100 micellar dispersion at pH 7.0.
tunneling effect to contribute to the in vivo regeneration of TocH from Toc• by AsA and UQH2. As shown in Figure 9 and Supporting Information, the k4H and k4D values obtained in the micellar dispersions are much less than those obtained in MeOH/H2O (MeOD/D2O). The reason for this can be explained in the following way. Although reaction 4′ in MeOH/H2O (MeOD/D2O) occurs throughout the uniform solution, in a micellar dispersion it occurs at the interface of micelles and the water phase. Because di-iPr-Toc• is in the micelles and AsAH3 is in the water phase, the probability of collisions between di-iPr-Toc• and AsAH3 is much smaller in the micellar dispersion than in the uniform solution. Electron Transfer. This paper is mainly concerned with the tunneling effect in the reactions of TocH, but electron transfer also plays an important role in reaction 1 (see reaction 5). In previous papers,16,47 we showed the importance of electron transfer by means of stopped-flow spectroscopy, cyclic voltammetry technique, ab initio method, and femtosecond spectroscopy. The electron transfer in reaction 1 corresponds to the socalled normal-regime16 in Marcus theory48-50 and Rehm-Weller equation.51-53 To confirm this in the present work, we investigated the effects of solvents and substituents on k1. The k1 values of R-TocH in various solvents, the plot of the k1 vs 1/n2
Acknowledgment. We thank Eisai Co. Ltd. for the generous gift of d-R-, d-β-, d-γ-, and d-δ-TocHs and thank Kaneka Corporation for generously giving us the ubiquinone-10. We also thank the Computer Center of the Institute for Molecular Science for the use of the IBM SP2, NEC SX-3/34R, SX-5, and HPC computers and the Library Program MOPAC Ver. 7.00 in the preparation of Figure 3. S.N. thanks Professor Shin Sato of Tokyo Institute of Technology (now professor emeritus) for his valuable discussion on the deviation from the linear relationship in the Arrhenius plot for the reaction of UQ10H2 in EtOH. S.N. and C.O. thank Professor Hidemitsu Uno of Ehime University for his help in the synthesis of di-Cl-TocH. S.N. and K.M. thank Professor L. Ross C. Barclay of Mt. Allison University for his valuable discussion on the solvent effects on reaction 1′, Mr. Taisyo Murakami of Ehime University for his help in the preparation of Figure 3, and Miss Aiko Tokunaga of Ehime University for her help in the preparation of Figures 4 and 5. This work was partly supported by Grants-in-Aid for Scientific Research on Priority Area “Quantum Tunneling of Group of Atoms as Systems with Many Degrees of Freedom” (Area Nos. 271/08240231, 09226229, and 10120225) from the Ministry of Education, Science, Sports and Culture of Japan. Supporting Information Available: Experimental details on the preparation and characterization of di-Cl-TocH, the k2 values of ArOH and ArOD, the k3 values of UQ10H2 (UQ10D2) and the k4 values of AsAH3 (AsAD3) at 25 °C in a 5.0 wt % H2O (D2O) solution of Triton X-100 buffered at pH (pD) 7.0, the k1 values of R-TocH at 25 °C in various solvents and the k1 values of di-Cl-TocH (di-Cl-TocD) at 25 °C in EtOH (EtOD), and a plot of k1 vs 1/n2 - 1/r for R-TocH in various solvents. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Cutler, R. G. In Free Radicals in Biology VI; Pryor, W. A., Ed.; Academic Press: Orlando, FL, 1984; pp 371-428. (2) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194. (3) Niki, E. Chem. Phys. Lipids 1987, 44, 227. (4) Niki, E. Yuki Gosei Kagaku Kyokaishi 1989, 47, 902. (5) Vitamin E, Biochemistry and Health Implications; Diplock, A. T., Machlin, L. J., Packer, L., Pryor, W. A., Eds.; Annals of the New York Academy of Sciences, Vol. 570; New York Academy of Sciences: New York, 1989.
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