Tunneling effect in regeneration reaction of vitamin e by ubiquinol

Article abstract of DOI:10.1021/jp910856m

A kinetic study of the regeneration reaction of vitamin E by ubiquinol was carried out by means of double-mixing stopped-flow spectroscopy. A substantial deuterium kinetic-isotope effect was observed on the second-order rate constant and the activation energy. In the regeneration reaction of α-tocopherol, deuteration of ubiquinol increased and decreased the activation energy and the second-order rate constant by 6.1 kJ/mol and a factor of 18.3, respectively. From this result, it is considered that proton tunneling plays an important role in the regeneration reaction of vitamin E by ubiquinol. The conditions under which the tunneling effect becomes an important factor were discussed in conjunction of our experimental results.

Full text of DOI:10.1021/jp910856m

J. Phys. Chem. B 2010, 114, 6601–6607
6601
Tunneling Effect in Regeneration Reaction of Vitamin E by Ubiquinol
Aya Ouchi,* Shin-ichi Nagaoka,† and Kazuo Mukai
Department of Chemistry, Faculty of Science, Ehime UniVersity, Matsuyama 790-8577, Japan
ReceiVed: NoVember 15, 2009; ReVised Manuscript ReceiVed: April 9, 2010
A kinetic study of the regeneration reaction of vitamin E by ubiquinol was carried out by means of double-
mixing stopped-flow spectroscopy. A substantial deuterium kinetic-isotope effect was observed on the second-
order rate constant and the activation energy. In the regeneration reaction of R-tocopherol, deuteration of
ubiquinol increased and decreased the activation energy and the second-order rate constant by 6.1 kJ/mol and
a factor of 18.3, respectively. From this result, it is considered that proton tunneling plays an important role
in the regeneration reaction of vitamin E by ubiquinol. The conditions under which the tunneling effect becomes
an important factor were discussed in conjunction of our experimental results.
1. Introduction
LOO• (prooxidant reaction, Figure 1), which is dangerous to
the biomembrane.27
It is well-known that ubiquinone has an important function
as an electron carrier in the mitochondrial respiratory chain.1-3
Mitochondria are rich in unsaturated lipid4 and must be protected
against peroxidation. In spite of recent criticism,5 the traditional
and most widely accepted view6 is that vitamin E (TocH)
prevents peroxidation in the biomembrane (reaction 1 and Figure
1) by scavenging the lipid peroxyl radical (LOO•).7-10
As mentioned above, UQH2 is the reduced form of ubiquinone.
Ubiquinone predominantly found in human cells is ubi-
quinone-10 (coenzyme Q10),28 although ubiquinone homologues
containing various isoprene units occur in nature, and some
ubiquinones having short isoprenoid-tail lengths are found in
microorganisms.29 In human plasma and various tissues except
brain and lung,2,3,30-33 ubiquinol-10 (reduced form, UQ10H2,
Figure 2) is superior to ubiquinone-10 (oxidized form) in
concentration, and so UQ10H2 is considered to prevent the lipid
peroxidation. In this paper, we focus our attention on reaction
3 for UQ10H2 (reaction 4).
TocH + LOO• f Toc• + LOOH
(1)
where Toc• and LOOH denote a tocopheroxyl radical and a
lipid hydroperoxide, respectively.
Several investigators found that the reduced form of ubi-
quinone (ubiquinol, UQH2) also has strong activity in inhibiting
the lipid peroxidation in various mammalian organisms such
as liver mitochondria, bovine heart submitochondria, rat liver,
and human low-density lipoprotein.3,11-14 It is considered that
UQH2 prevents the lipid peroxidation in biomembrane (Figure
1)2,3,15-22 by scavenging LOO• (reaction 2) and by regenerating
TocH (reaction 3) from Toc•, which has been produced through
reaction 1.
kUQ
UQ10H2 + Toc• 8 UQ10H• + TocH
(4)
It is well-known that hydrophilic vitamin C (ascorbate
monoanion, AsH-) also regenerates TocH from Toc• at the
interface between biomembrane and water phase (reaction 5,
Figure 1).34-37
UQH2 + LOO• f UQH• + LOOH
UQH2 + Toc• f UQH• + TocH
(2)
(3)
where UQH• refers to a dehydroubiquinol radical. The TocH
regenerated through reaction 3 can contribute again to reaction
1 and prevents the peroxidation in the biomembrane by
scavenging LOO• (Figure 1). In fact, TocH and UQH2 coexist
in relatively high concentrations in plasma, various tissues, and
skin.23-26 Toc•’s other than those transformed into TocH’s
through some regeneration reactions such as reaction 3 may
react with lipid (LH) to produce lipid radical (L•) leading to
* Corresponding author. Address: Department of Chemistry, Faculty
of Science, Ehime University, Matsuyama 790-8577, Japan. Tel:
+(81)-89-927-9588. Fax: +(81)-89-927-9590. E-mail: oouchi@
chem.sci.ehime-u.ac.jp.
Figure 1. Partial scheme of LOO• production and of the antioxidant,
prooxidant, and regeneration reactions of TocH in a biomembrane.
† Also at Graduate School of Science and Engineering, Ehime University.
10.1021/jp910856m  2010 American Chemical Society
Published on Web 04/23/2010

6602 J. Phys. Chem. B, Vol. 114, No. 19, 2010
Ouchi et al.
kVC
conditions. For example, why does the structure of Toc•
influence the presence (or absence) of the tunneling effect in
reaction 5 as noted above?
AsH- + Toc• 8 As-• + TocH
(5)
In the work presented here we have studied the regeneration
reaction of natural TocH by UQ10H2 (reaction 4) with a double-
mixing stopped-flow spectrophotometer, which allows us to
follow kinetics of reactions of short-lived radicals such as R-
and ꢀ-Toc•’s. We have determined the second-order rate
constants and the activation energies of reaction 4 and examined
the deuterium kinetic-isotope effects. From these results, we
have clarified the conditions under which the tunneling effect
plays an important role in the regeneration reaction of TocH.
where As-• denotes an ascorbate monoanion radical. However,
reaction 4 has a rate constant comparable, within about 1 order
of magnitude, to that of reaction 5.17,19 Furthermore, UQ10H2 is
found in relatively high concentration in human plasma and
various tissues,3,23,25,30-33 and lipophilic UQ10H2 can react with
Toc• inside the biomembrane, in contrast to hydrophilic AsH-
existing in the water phase (Figure 1). Like this, reaction 4 has
some advantages to the regeneration of TocH in vivo.
In previous studies,38,39 by using stable 5,7-diisopropyl-Toc•
(Figure 2), we carried out kinetic studies of reaction 4, and by
substituting deuterons for the hydroxy protons in UQ10H2
(UQ10D2, Figure 2) we observed a substantial deuterium kinetic-
isotope effect on the second-order rate constant and the
activation energy. This result suggested that the tunneling effect
plays an important role in reaction 4, which is a proton (or
hydrogen) transfer reaction from UQ10H2 to Toc•. In a recent
paper, Kikuta et al40 suggested that what we refer to as the
“tunneling effect” is more accurately described as a “quantum
effect.” For consistency with previous usage, however, in this
paper we use the designation “proton tunneling effect.”
Similarly to the case of reaction 4 for 5,7-diisopropyl-Toc•,
the proton tunneling effect plays an important role in reaction
5 (TocH-regeneration by AsH-) for 5,7-diisopropyl-Toc•.39
However, the tunneling does not contribute to reaction 5 for R-
and ꢀ-Toc•’s originating from natural R- and ꢀ-TocH’s (Figure
2).37 Accordingly, it is essential to study the kinetics of reaction
4 for natural Toc• and to examine whether the tunneling effect
really contributes to reaction 4 in vivo. Furthermore, it is very
important to clarify the conditions under which the tunneling
effect becomes an important factor, and it is also necessary to
elucidate the reason why the proton tunneling occurs under those
2. Experimental Methods
The structures of the molecules used in this work are shown
in Figure 2. R- and ꢀ-TocH’s were kindly supplied by Eisai
Co., Ltd. and used without further purification. UQ10H2 was
prepared by the reduction of ubiquinone-10 with sodium
hydrosulfite under a nitrogen atmosphere.22 Ubiquinone-10 was
kindly supplied by Kaneka Corporation. We prepared 2,6-di-
t-butyl-4-(4′-methoxyphenyl)phenoxyl radical (ArO•) as reported
in a previous paper.41 Ethanol (C2H5OH, EtOH) was obtained
from Wako, dried over magnesium ribbon, and purified by
distillation. Ethanol-d1 (C2H5OD, EtOD) of 99.5% purity was
purchased from Aldrich and used without purification. When
UQ10H2 or TocH was dissolved in EtOD, replacement of the
hydrogen atom(s) of the OH group(s) by (a) deuteron(s) was
easily accomplished, and the deuterated molecule (UQ10D2 or
TocD) was obtained. The deuteration was verified by proton
NMR.
The kinetic data were obtained by using a Unisoku RSP-
1000-03F double-mixing stopped-flow spectrophotometer under
a nitrogen atmosphere at 15-40 °C. The error in the temperature
reading was less than 0.5 °C. First Toc• radical (R- or ꢀ-Toc•,
Figure 2) was prepared by mixing equal volumes of EtOH
(EtOD) solutions of ArO• and the TocH (TocD) (reaction 6) in
the first-mixing unit of the stopped-flow spectrophotometer.
ArO• + TocH(TocD) f ArOH(ArOD) + Toc•
(6)
The concentrations of R-TocH, R-TocD, ꢀ-TocH, and ꢀ-TocD
were 7.38 × 10-4, 6.76 × 10-4, 2.54 × 10-3, and 1.25 × 10-2
M, respectively. After 2-30 s (5 s in R-TocH, 30 s in R-TocD,
and 2 s in ꢀ-TocH and ꢀ-TocD), equal volumes of the resultant
mixture and an EtOH (EtOD) solution of UQ10H2 (UQ10D2) were
mixed [reaction 4-H (4-D)] in the second mixing unit.37
H
kUQ
UQ10H2 + Toc•
UQ10D2 + Toc•
8 UQ10H• + TocH
(4-H)
(4-D)
D
kUQ
8 UQ10D• + TocD
Reaction 4-H (4-D) was studied under pseudo-first-order
conditions ([UQ10H2(UQ10D2)] . [Toc•], where the brackets
[
]
indicate molar concentration). The prepared
[UQ10H2(UQ10D2)] was chosen so that the [Toc•] would not
largely decrease from the initial concentration within the time
required to completely mix UQ10H2 (UQ10D2) with Toc• and to
make the solution homogeneous.
Figure 2. Molecular structures of TocH’s, Toc•’s, UQ10H2, UQ10D2,
and ArO•.

Tocopherol Regeneration by Ubiquinol
J. Phys. Chem. B, Vol. 114, No. 19, 2010 6603
Figure 3. Change in absorption spectrum (at 60 ms intervals) during
reaction of R-TocH and ArO• in EtOH at 25 °C (reaction 6). The
prepared [R-TocH] was 0.738 mM. The arrows indicate a decrease and
an increase in absorbance of ArO• and R-Toc•, respectively.
Figure 5. (a) Absorbance decay of R-Toc• at 429 nm at 25 °C during
reaction 4-H in EtOH. (b) The absorbance decay during reaction 4-D
in EtOD.
Figure 4. Change in absorption spectrum (at 4 ms intervals) during
reaction of UQ10H2 and R-Toc• in EtOH at 25 °C (reaction 4-H). The
prepared [UQ10H2] was 0.237 mM. The arrow indicates a decrease in
absorbance of R-Toc•.
The pseudo-first-order rate constant (kobsd) of reaction 4 was
evaluated by using a standard linear least-squares analysis for
each of the single-exponential absorbance decay-curves of R-
and ꢀ-Toc•’s. The experimental equation is given as
3. Results and Discussion
The change in absorption spectrum measured during reaction
6 for R-TocH is shown in Figure 3. Although ArO• was stable
in the absence of R-TocH, when an EtOH solution with excess
R-TocH was added to the ArO• solution, the ArO• absorption
peak disappeared immediately, an R-Toc• peak appeared, and
the isosbestic points were observed. The change in absorption
spectrum measured during reaction 4-H for R-Toc• is shown in
Figure 4, where the R-Toc• absorption peak disappears over
time. UQ10H• is so short-lived that the peak is not seen in the
spectrum. Figures 5a and 5b show the absorbance decay of
R-Toc• during reactions 4-H and 4-D in EtOH and EtOD,
respectively. The absorbance decay of R-Toc• was well-
characterized as a single-exponential decay, indicating that the
bimolecular reaction of R-Toc•42 is negligibly slower than
reactions 4-H and 4-D. The absorbance decay was accelerated
as the prepared [UQ10H2(UQ10D2)] increased, and the decay of
reaction 4-H (Figure 5a) was much faster than that of reaction
4-D (Figure 5b) when [UQ10H2] = [UQ10D2]. Similar results
were also obtained for ꢀ-Toc•. γ- and δ-Toc•’s were so short-
lived that their absorbance decays were not observed under our
experimental conditions.
-d[Toc•]/dt ) kobsd[Toc•]
(7)
The rate equation of reaction 4 is expressed as
-d[Toc•]/dt ) (kO + kUQ)[UQ10H2(UQ10D2)][Toc•]
(8)
where k0 stands for the first-order rate constant for the natural
decay of Toc• in EtOH (EtOD), and kUQ is the second-order
rate constant of reaction 4. kobsd in eq 7 is given by
kobsd ) k0 + kUQ[UQ10H2(UQ10D2)]
(9)
where k0 was much less than kUQ [UQ10H2(UQ10D2)] under our
experimental conditions. Since [UQ10H2(UQ10D2)] was nearly
constant during reaction 4 under pseudo-first-order conditions
([UQ10H2(UQ10D2)] . [Toc•]), kUQ was evaluated from the slope

6604 J. Phys. Chem. B, Vol. 114, No. 19, 2010
Ouchi et al.
Figure 7. Arrhenius plots of kUQ’s for reactions of UQ10H2 and R-Toc•
in EtOH (open circles), of UQ10D2 and R-Toc• in EtOD (filled circles),
of UQ10H2 and ꢀ-Toc• in EtOH (open squares), and of UQ10D2 and
ꢀ-Toc• in EtOD (filled squares) (reaction 4).
Figure 6. Dependence of kobsd on prepared [UQ10H2(UQ10D2)] at 25
°C in reactions of UQ10H2 and R-Toc• in EtOH (open circles), of
UQ10D2 and R-Toc• in EtOD (filled circles), of UQ10H2 and ꢀ-Toc• in
EtOH (open squares), and of UQ10D2 and ꢀ-Toc• in EtOD (filled
squares) (reaction 4).
(EH and ED) and the frequency factors (AH and AD) can be
evaluated according to eq 10.
TABLE 1: kUQ Values of Reaction 4 at 15, 20, 25, 30, 35,
and 40 °C
10-4kUQ (M-1 s-1
)
H(D)
UQ
log k
) -0.434EH(D)/RT + log AH(D)
(10)
15 °C
20 °C
25 °C
30 °C
35 °C
40 °C
reaction 4
UH10H2 + R-Toc•
UH10D2 + R-Toc•
UH10H2 + ꢀ-Toc•
UH10D2 + ꢀ-Toc•
19.9
0.93
20.6
1.03
20.6
1.09
24.0
1.37
21.2
1.16
29.5
1.85
21.7
1.25
36.0
2.75
22.5
1.30
42.5
3.70
23.4
1.41
52.4
4.73
H
D
where R denotes the gas constant. The ratio of kUQ to kUQ
(kUQH/kUQD) at 25 °C, EH, ED, their difference (ED - EH), log
AH, and log AD are listed in Table 2. In general, when the proton
tunneling effect plays an important role, a substantial deuterium
kinetic-isotope effect appears, and large values of kUQH/kUQD and
ED - EH are obtained.47,48 Additionally, for reaction 4, a
of the plot of kobsd versus the prepared [UQ10H2(UQ10D2)]
(Figure 6). The kUQ values of reaction 4 for R- and ꢀ-Toc•’s
are listed in Table 1.
H
substantial deuterium kinetic-isotope effect on kUQ (kUQ and
kUQD) and the activation energy (EH and ED) is illustrated in
Tables 1 and 2. The kUQH/kUQD values for R- and ꢀ-Toc•’s (18.3
and 15.9, respectively) exceed the maximum semiclassical ratio
(6-8).47,48 The ED - EH values for R- and ꢀ-Toc•’s (6.1 and
18.6 kJ/mol, respectively) also exceed the maximum semiclas-
sical difference (1.3-4.2 kJ/mol).47,48 These results clearly show
that the proton tunneling effect plays an important role in
reaction 4 for Toc• originating from natural TocH in vitro as
well as in reaction 4 for 5,7-diisopropyl-Toc•38,39 and in various
antioxidant reactions.39,43,44,49,50 The proton tunneling was previ-
ously shown to also contribute to various vital functions such
as mutation-suppression by vitamin C.44,51-60 It is very interest-
ing that the microscopic quantum-mechanical tunneling effect
could manifest itself in macroscopic vital functions.
In connection with our experimental results, the computational
study on the reaction between UQH2 and phenoxyl radical
(Ph-O•) by Yamamoto and Kato61 is interesting. Since the
moiety of phenoxyl radical contained in Toc• plays a key role
in reaction 4, we can interpret our experimental results on the
basis of their computational results. They thought that reaction
4 can proceed by proton tunneling below the transition state,
and our experimental results for reaction 4 are supported by
their computational results. In the later part of this paper, we
will discuss our present and previous experimental results in
conjunction with their computational results.
It should be noted that the [UQ10H2] range employed in the
evaluation of kUQ was lower than the [UQ10D2] range (Figure
6). The kUQ value for UQ10H2 (kUQH) is much larger than that
for UQ10D2 (kUQD) (Table 1). So, if relatively high [UQ10H2]
(e.g., > 4 × 10-4 M as in UQ10D2) had been employed, the
kobsd value would have become very large (eq 9), and the [Toc•]
would largely have decreased from the initial concentration (eq
7) within the time required to completely mix UQ10H2 with Toc•
and to make the solution homogeneous. In order to evaluate
kobsd and kUQ accurately, it is desirable to analyze the main decay
profile of [Toc•] rather than the tail portion of the time-profile.
Accordingly, we employed some relatively low [UQ10H2]’s in
the evaluation of kUQH, whose value thus obtained for R-Toc•
at 25 °C was consistent with those reported previously.17,19
The kUQ value of reaction 4 for R-Toc•, having three electron-
donating methyl groups, is less than the corresponding value
for ꢀ-Toc•, having two methyl groups (Table 1). When the
electron-donating property of the substituent in Toc• is weak,
the kUQ value is large. This result suggests that an electron-
transfer plays a role in reaction 4. A similar result was previously
obtained for reaction 5.37 An electron-transfer in reaction 1 was
also suggested by the results of conventional single-mixing
stopped-flow spectroscopy39,43,44 and femtosecond spectroscopy45
as well as of time-dependent density functional calculation.46
It is interesting that ꢀ-TocH is more effective in antioxidant
regeneration (reactions 4 and 5) than R-TocH, which is the most
biologically active of natural TocH’s.
As mentioned in the introduction, the tunneling effect does
not contribute to the regeneration reaction of natural TocH (R-
and ꢀ-TocH’s) by AsH- (reaction 5),37 judging from the fact
that the ratio kVCH/kVCD corresponding to kUQH/kUQD (see reactions
5-H and 5-D) does not exceed the maximum semiclassical ratio
(6-8).47,48
Figure 7 shows Arrhenius plots of the kUQH and kUQD values,
H
and linear relationships with negative slopes between log kUQ
(log kUQD) and the reciprocal of the absolute temperature (1/T)
can be seen in the plots, from which the activation energies

Tocopherol Regeneration by Ubiquinol
J. Phys. Chem. B, Vol. 114, No. 19, 2010 6605
H
kVC
AsH- + Toc•
8 As-• + TocH
(5-H)
(5-D)
D
kVC
AsD- + Toc•
8 As-• + TocD
where AsD- denotes a deuterated ascorbate monoanion. This
previous result for reaction 5 contrasts sharply with the present
result for reaction 4 in which the tunneling effect plays an
important role. Thus, whether or not the tunneling effect plays
an important role depends on the antioxidant reagent (UQ10H2
or AsH-) employed in the regeneration reaction of natural TocH.
The reason for the contrast between reactions 4 and 5 for
Toc• originating from natural TocH can be explained in the
following way. First, let us examine the case in which reactions
4 and 5 proceed semiclassically. Since reactions 4-H and 5-H
are transfers of protium (1H), the possibility of proton tunneling
is present. Accordingly, we will examine reactions 4-D and 5-D
for R-Toc•; that is, we will compare the deuterium transfer from
UQ10D2 to R-Toc• (reaction 4-D for R-Toc•) and the one from
AsD- (reaction 5-D for R-Toc•). Since the tunneling effect does
not have a very large influence on the deuterium transfers
compared with protium (1H) transfers in a simple physical
picture, the activation energies and rate constants in reactions
4-D and 5-D approximately reflect the real reaction potential-
barriers and the rates of the semiclassical jumps over the barriers,
respectively. In these deuterium transfers, the rate constant of
reaction 5-D at 25 °C (4.87 × 105 M-1 s-1 37
)
is much larger
that of reaction 4-D (1.16 × 104 M-1 s-1, Table 1), and the
activation energy of reaction 5-D (6.4 kJ/mol)37 is considerably
less than that of reaction 4-D (10.6 kJ/mol, Table 2). Thus, when
reactions 4 and 5 proceed semiclassically, reaction 5 is more
favorable to the regeneration of natural TocH than reaction 4
under our experimental conditions.62
Figure 8. (a) Schematic potential-energy curve of reaction 5 for R-
and ꢀ-Toc•’s. The reaction potential barrier is low. (b) Schematic
potential-energy curve of reaction 4, whose barrier is high. The tunnel
for protium (1H) transfer is shown. This scheme is also applicable to
reaction 5 for 5,7-diisopropyl-Toc•.
Next we will take the possibility of proton tunneling into
account. Although reaction 5-D has a much lower potential-
barrier than reaction 4-D as mentioned above,62 the potential
barrier of reaction 5-H for R-Toc• (4.0 kJ/mol) is still less than
that of reaction 5-D (6.4 kJ/mol),37 because the zero point energy
of the OH vibration in AsH- is larger than that of the OD
vibration in AsD-. Accordingly, reaction 5-H prefers to proceed
by jumping semiclassically over the low potential barrier rather
above-mentioned computational results by Yamamoto and
Kato,61 the computational ratio corresponding to kUQH/kUQ
D
decreases as the potential barrier decreases,63 and their results
are consistent with our above-mentioned view (Figure 8).
As mentioned in the preceding three paragraphs and the
Introduction, the tunneling effect does not contribute to reaction
5 for R- and ꢀ-Toc•’s.37 However, as mentioned in the
Introduction, the tunneling effect plays an important role in
reaction 5 for 5,7-diisopropyl-Toc•.39 Thus, whether or not the
tunneling effect contributes to reaction 5 depends on the
structure of Toc•. Although the reason for the contrast between
the reactions for these Toc•’s had not yet been elucidated, we
can now explain it in terms of the difference in reaction potential
barrier shown in Figure 8. When 5,7-diisopropyl-Toc• is used
as Toc• in reaction 5, the steric hindrance due to the two
isopropyl groups at the 5- and 7-positions prevents access of
AsH- to the O• at the 6-position of the Toc•. As a result, a
high potential barrier appears in reaction 5 for 5,7-diisopropyl-
than to proceed by proton tunneling through the barrier (Figure
H
8a). As a result, the deuterium kinetic-isotope effect on kVC
/
kVCD and the activation energy is small,37 because both reactions
5-H and 5-D for R-Toc• proceed semiclassically.47,48 By contrast,
since the potential barrier of reaction 4 is high, the semiclassical
jump over the barrier is difficult, and the proton tunneling takes
place below the transition state in reaction 4-H (Figure 8b). As
H
a result, a substantial deuterium kinetic-isotope effect on kUQ
/
kUQD and ED - EH appears, because reactions 4-H and 4-D for
R-Toc• proceed basically through different mechanisms (proton
tunneling and semiclassical jump, respectively).47,48 Also, in the
TABLE 2: kUQH/kUQD, EH, ED, ED - EH, log AH, and log AD Values of Reaction 4
EH or ED (kJ/mol)
ED - EH (kJ/mol)
log AH or log AD a
D
reaction 4
kUQH/kUQ at 25 °C
UH10H2 + R-Toc•
UH10D2 + R-Toc•
UH10H2 + ꢀ-Toc•
UH10D2 + ꢀ-Toc•
18.3
4.5 ( 0.1
10.6 ( 0.7
27.9 ( 0.8
46.5 ( 1.4
6.1
6.1
5.9
10.4
12.6
15.9
18.6
H
a Because log AH (log AD) was obtained by extrapolating the linear log kUQ (log kUQD) versus 1/T plot in a limited 1/T range around room
temperature to the intercept, these values have a large uncertainty.

6606 J. Phys. Chem. B, Vol. 114, No. 19, 2010
Ouchi et al.
Toc•, and so the proton tunneling plays an important role as
shown in Figure 8b. In contrast, since such large steric hindrance
is absent in R- and ꢀ-Toc•’s, the potential barrier of reaction 5
is low, and so the proton tunneling does not occur as shown in
Figure 8a. Yamamoto and Kato also suggested in the above-
mentioned paper61 that the deuterium kinetic-isotope effect is
not large in the absence of any steric hindrance preventing
approach of two reactants, and their suggestion is consistent
with our view. The reaction potential barrier of the regeneration
of TocH would be higher in vivo than in a uniform solution,
because the environment around the reactants in vivo prevents
their approach. Accordingly, the proton tunneling could be
induced in vivo according to the scheme given in Figure 8b.
On the basis of our experimental results mentioned above,
we propose the following mechanism for reaction 3:
series of related reactions results in EH (ED) being proportional
to log AH (log AD).
Yamamoto and Kato mentioned, in the introduction of their
paper,61 an inverse deuterium kinetic-isotope effect (a slight
decrease of kUQH/kUQD with a decrease in temperature) given in
our previous paper on reaction 4 for 5,7-diisopropyl-Toc•.38
However, the effect might originate from solvation as explained
in our subsequent paper.39
As mentioned above, when reactions 4 and 5 proceed
semiclassically, reaction 5 is more favorable to the regeneration
of natural TocH than reaction 4 under our experimental
conditions. Since the semiclassical routes on the potential-energy
curves of reactions 4 and 5 are the electron transfer from the
antioxidant reagent to Toc•, it is natural that reaction 5 for AsH-
having lower oxidation potential is semiclassically more favor-
able than reaction 4 for UQ10H2 having higher oxidation
potential.43 Then, the ratio of the rate constant of reaction 537
f [UQH2 - --Toc•] (halfway to
UQH2 + Toc•
D
to that of reaction 4 (Table 1) is very large (kVCD/kUQ ) 42.0
electron transfer)
for R-Toc• at 25 °C). However, when the proton tunneling plays
f [UQH• - --TocH] (proton tunneling)
f [UQH• - --TocH]
H
a role, the corresponding ratio is reduced (kVCH/kUQ ) 12.9).
Thus, the proton tunneling relatively accelerates reaction 4
compared with reaction 5, and the tunneling effect would give
reaction 4 an advantage to the regeneration of TocH in vivo
besides the advantages mentioned in the Introduction.
(11)
In the initial state of the reaction, UQH2 and Toc• approach
each other, and their electron clouds begin to overlap. Because
UQH2 and Toc• are respectively an electron-donor and an
electron-acceptor, the transition state of reaction 3 has the
property of an electron-transfer transition-state. In reality,
however, when UQH2 and Toc• approach each other to some
extent ([UQH2 --- Toc•]), the proton tunneling takes place below
the transition state. The proton tunneling allows the reaction to
proceed by cutting a corner on the potential energy surface.
Finally, UQH• and TocH separate from each other. To avoid
misleading, it should be noted that mechanism 11 does not refer
to a stepwise pathway involving mechanically distinct electron
transfer and proton tunneling but to a single chemical reaction
involving electron transfer in concert with proton tunneling. The
electron and proton move in one kinetic step, although, strictly
speaking, the light electron begins to move before the heavy
proton moves. The concerted reaction can proceed more
preferentially than any stepwise reaction, because a stepwise
reaction goes through an energetic intermediate.64,65 Also, in
the above-mentioned paper by Yamamoto and Kato,61 the
reaction of UQH2 was interpreted as a proton-coupled electron
transfer.64,65 At the transition state, the calculated dipole moment
reaches a maximum, and its orientation is reversed,61 indicating
that the transition state has the property of an electron-transfer
transition state. Their result is also consistent with the above-
mentioned experimental result that the kUQ value is large when
the electron-donating property of the substituent in Toc• is weak.
They also thought that the proton tunneling took place under
the transition state, as mentioned above.61 Their computational
results and interpretation thus support our mechanism 11.
Next we will critically examine our results and discussion.
In a previous paragraph, we mentioned that reaction 5 has a
much lower potential-barrier than reaction 4. However, EH of
reaction 4-H for R-Toc• (4.5 kJ/mol, Table 2) is similar to the
potential barrier of reaction 5-H for R-Toc• (4.0 kJ/mol).37 The
reason for this is that EH of reaction 4-H does not refer to
the real potential barrier but to the energy difference between
the tunnel and the reactants (Figure 8b).
In our previous studies,38,39 we used non-natural 5,7-diiso-
propyl-Toc• as Toc• in reactions 4 and 5, because UQ10H•, As-•,
and natural Toc• are so short-lived that the examinations of the
deuterium kinetic-isotope effects in reactions 4 and 5 through
detection of these radicals were difficult with conventional
single-mixing stopped-flow spectroscopy. The use of stable 5,7-
diisopropyl-Toc• allowed us to study the deuterium kinetic-
isotope effects with the conventional method. However, since
the above-mentioned steric hindrance due to the two isopropyl
groups prevents access of the antioxidant reagent to 5,7-
diisopropyl-Toc•, its kinetics is often different from that for
natural Toc• having little such steric hindrance. Accordingly,
in our present and recent37 works we have studied reactions 4
and 5 for R- and ꢀ-Toc•’s by using a double-mixing stopped-
flow spectrophotometer, which has allowed us to examine the
deuterium kinetic-isotope effect in the regeneration reaction of
natural TocH through detection of the short-lived Toc•. Thus,
the double-mixing stopped-flow spectrophotometer is a powerful
tool in detailed studies of the regeneration reaction.
4. Conclusions
A kinetic study of reaction 4 was carried out by means of
double-mixing stopped-flow spectroscopy. The proton tunneling
effect plays an important role in reaction 3 that is advantageous
to living organisms. When the reaction potential barrier is high
and/or the steric hindrance prevents access of the antioxidant
reagent to Toc•, the tunneling effect contributes to the regenera-
tion of TocH.
Acknowledgment. We express our sincere thanks to Profes-
sors Shigeki Kato and Takeshi Yamamoto of Kyoto University
for their valuable discussion. Professor Kato died of cancer on
March 31, 2010 at the age of 61. A.O. thanks Dr. Umpei
Nagashima of the National Institute of Advanced Industrial
Science and Technology for his continuous encouragement. We
thank Eisai Co., Ltd. and Kaneka Corporation for the generous
gifts of TocH’s and ubiquinone-10, respectively.
The EH (ED) and log AH (log AD) values for ꢀ-Toc• are much
larger than those of R-Toc• (Table 2). The reason for this would
be the isokinetic relationship (compensation effect)66,67 as
pointed out in our previous paper.37 In the isokinetic relationship,
the correlation between the enthalpy and entropy changes in a
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JP910856M