Additivity rule holds in the hydrogen transfer reactivity of unsaturated fatty acids with a peroxyl radical: Mechanistic insight into lipoxygenase

Article abstract of DOI:10.1039/b515004c

A simple additivity rule holds in the hydrogen transfer reactivity of unsaturated fatty acids with cumylperoxyl radical, which is expressed by the additive contributions of the reactivity of active hydrogens from the 1,4-pentadiene subunit and those of the allylic subunit; the kinetic isotope effect on the hydrogen transfer reactions (KIE = 6.1) is significantly smaller than that observed for lipoxygenase (KIE = 81). The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b515004c

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Additivity rule holds in the hydrogen transfer reactivity of unsaturated
fatty acids with a peroxyl radical: mechanistic insight into
lipoxygenase{
Hironori Kitaguchi, Kei Ohkubo, Seiji Ogo and Shunichi Fukuzumi*
Received (in Cambridge, UK) 24th October 2005, Accepted 14th December 2005
First published as an Advance Article on the web 20th January 2006
DOI: 10.1039/b515004c
Direct measurements of the rates of hydrogen transfer from a
series of unsaturated fatty acids to cumylperoxyl radical were
performed in propionitrile (EtCN) at various temperatures by
means of ESR. The photoirradiation of an oxygen saturated EtCN
solution containing di-t-butylperoxide (ButOOBut) and cumene
with a 1000 W high-pressure mercury lamp results in formation
of cumylperoxyl radical which is readily detected by ESR.9 The
cumylperoxyl radical is formed via a radical chain process started by
the UV-photoirradiation of ButOOBut as shown in Scheme 1.10–12
In the termination step, cumylperoxyl radicals decay by a
bimolecular radical coupling reaction to yield the corresponding
peroxide [(C6H5CMe2O)2] and oxygen (Scheme 1).10,12 When the
light is cut off, the ESR signal intensity decays obeying second-order
kinetics due to the bimolecular reaction (Fig. 1a).
In the presence of linoleic acid (4.0 6 1022 mol dm23) and
arachidonic acid (4.0 6 1022 mol dm23), the decay of
cumylperoxyl radical after cutting off the light becomes much
faster than that in the absence of linoleic acid and arachidonic acid,
as shown in Fig. 1b and 1c. The decay in the presence of linoleic
acid and arachidonic acid obeys pseudo-first-order kinetics rather
than second-order kinetics (see Supplementary Information S1, S2
for the first-order plots{).13 The pseudo-first-order rate constants
(kobs) increase linearly with an increase in concentration of
unsaturated fatty acid to exhibit a first-order dependence (S3).
A simple additivity rule holds in the hydrogen transfer reactivity
of unsaturated fatty acids with cumylperoxyl radical, which is
expressed by the additive contributions of the reactivity of
active hydrogens from the 1,4-pentadiene subunit and those of
the allylic subunit; the kinetic isotope effect on the hydrogen
transfer reactions (KIE = 6.1) is significantly smaller than that
observed for lipoxygenase (KIE = 81).
Soybean lipoxygenase-1 (SLO) has received special attention,1–5
because the hydrogen atom abstraction step in SLO exhibits the
largest H/D kinetic isotope effect (KIE), of kH/kD = 81 at near
room temperature, so far reported for biological redox systems.6–8
This result provides compelling evidence for tunneling effects in the
SLO reaction. The abstraction of the pro-S hydrogen atom from
the C11 position of linoleic acid by the active site (Fe3+–OH)
species to form the substrate radical is the rate-determining step in
the catalytic mechanism.6–8 Oxygen rapidly reacts with the
substrate radical to generate the peroxyl radical which reacts with
Fe2+–OH2 to yield the hydroperoxide, 13-(S)-hydroperoxy-9,11-
(Z,E)-octadecadienoic acid (13-(S)-HPOD), accompanied by
regeneration of the active form of the enzyme, Fe3+–OH. Other
origins of the large kinetic isotope effect (KIE) on catalytic rates
such as magnetic field effects or reaction branching have been
ruled out to conclude that the large KIE reflects an intrinsic KIE
for a single hydrogen transfer step, which proceeds by a tunneling
event. In such a case it would be quite interesting to examine a
single hydrogen transfer step of unsaturated fatty acids and the
KIE with other reactive radical species in order to know the
difference in the hydrogen-abstraction step between the non-
enzymatic and enzymatic systems. However, the direct determina-
tion of hydrogen transfer rates of a series of unsaturated fatty acids
with reactive radical species has yet to be reported.
We report herein the direct determination of the absolute rates
of hydrogen transfer from a series of unsaturated fatty acids to
cumylperoxyl radical by use of electron spin resonance (ESR) at
low temperatures. The absolute rates together with the direct
determination of the KIE at various temperatures provide valuable
insight into the tunneling hydrogen transfer mechanism of
lipoxygenases.
Department of Material and Life Science, Division of Advanced Science
and Biotechnology, Graduate School of Engineering, Osaka University,
SORST, Japan Science and Technology Agency (JST), Suita, Osaka
565-0871, Japan. E-mail: fukuzumi@chem.eng.osaka-u.ac.jp;
Fax: +81-6-6879-7370; Tel: +81-6-6879-7368
{ Electronic supplementary information (ESI) available: Figures and
Tables of kinetic data (S1–S5). See DOI: 10.1039/b515004c
Scheme 1
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Fig. 1 The decay profile of the ESR signal intensity due to cumylperoxyl
radical in the presence of (a) no unsaturated fatty acid, (b) linoleic acid
(4.0 6 1022 mol dm23) and (c) arachidonic acid (4.0 6 1022 mol dm23
Fig. 3 Eyring plots of kH for hydrogen transfer from oleic acid (#),
linoleic acid (n), linolenic acid (%), arachidonic acid ($) and
[11,11-2H2]linoleic acid (m) to cumylperoxyl radical in O2-saturated EtCN.
)
in an O2-saturated EtCN solution of di-t-butyl peroxide (1.0 mol dm23
)
and cumene (1.0 mol dm23) with a 1000 W high-pressure mercury lamp at
203 K.
Eyring plots of the kH and kD values are shown in Fig. 3. The
Thus, the decay process is ascribed to the hydrogen transfer from
unsaturated fatty acid to cumylperoxyl radical (Scheme 1).
The rate constants (kH) of hydrogen transfer from a series of
unsaturated fatty acids to cumylperoxyl radical were determined
from the slopes of linear plots of kobs vs. the concentration of
unsaturated fatty acid. The results are summarized in Fig. 2 (for
the kH values, see S4), where the kH values at different
temperatures are linearly correlated with the number of active
hydrogens of the 1,4-pentadiene subunits of unsaturated fatty
acids (n). Such linear correlations shown in Fig. 2 reveal that a
simple additive rule holds in the hydrogen transfer reactions as
given by eqn (1):
activation parameters are obtained from eqn (2):
ln(kH/T) = 2DH{/RT + DS{/R + ln(kB/h)
(2)
where kB is the Boltzmann constant, h is Planck’s constant and R
is the universal gas constant.15 The values obtained are
summarized in Table S5.16 There is a significant difference between
the activation enthalpies of the unsaturated fatty acids containing
1,4-pentadiene subunits (linoleic acid: 21.9 kJ mol21, linolenic acid:
20.1 kJ mol21 and arachidonic acid: 20.6 kJ mol21) and that
containing only an allylic subunit (oleic acid: 34.7 kJ mol21). This
is consistent with the calculated energy differences between
hydrogen-abstracted radicals and unsaturated fatty acids (DE),
since the DE values of the allyl-type radicals, which are nearly the
same among the unsaturated fatty acids, are larger by 50 ¡
9 kJ mol21 than those of the corresponding pentadienyl-type
radicals derived from the same unsaturated fatty acids, which are
also nearly the same among unsaturated fatty acids (see Table 1).17
kH = kH0 + nkH1
(1)
where kH1 is the hydrogen transfer rate constant of one hydrogen
of a 1,4-pentadiene subunit (linoleic acid, linolenic acid, and
arachidonic acid) and kH0 is the hydrogen transfer rate constant of
the allylic subunit (oleic acid).14
Table 1 Energy differences (DE) between hydrogen-abstracted radi-
cals and the neutral form of the unsaturated fatty acids, obtained from
DFT calculations
Position of
hydrogen- DEa/
Unsaturated
fatty acid
Structure of unsaturated
abstraction kJ mol21 fatty acid
Oleic acid
8
11
1689.6
1706.4
Linoleic acid
8
11
14
8
11
14
17
4
1695.7
1644.8
1697.1
1689.0
1638.8
1639.4
1688.6
1687.5
1641.0
1645.8
1640.7
1690.0
Linolenic acid
Arachidonic acid
7
Fig. 2 Plots of rate constants (kH) vs. the number of hydrogens of the
1,4-pentadiene subunits (n) for hydrogen transfer from unsaturated fatty
acids (the n values are given in parentheses) to cumylperoxyl radical in an
O2-saturated EtCN solution at 203 K (#), 213 K (n), 223 K (%), 233 K
($) and 243 K (m).
10
13
16
a
Determined by ROHF formalism with B3LYP/3-21G basis set.
980 | Chem. Commun., 2006, 979–981
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Table 2 Rate constants (kH1a and kD1b) and the kinetic isotope effect
(kH1/kD1) for hydrogen transfer from linoleic acid ([11,11-2H2]linoleic
acid) to cumylperoxyl radical
Notes and references
1 A. Kohen and J. P. Klinman, Acc. Chem. Res., 1998, 31, 397.
2 N. Lehnert and E. I. Solomon, JBIC, J. Biol. Inorg. Chem., 2003, 8, 294.
3 (a) Y. Zhang, Q.-F. Gan, E. G. Pavel, E. Sigal and E. I. Solomon, J. Am.
Chem. Soc., 1995, 117, 7422; (b) E. I. Solomon, J. Zhou, F. Neese and
E. G. Pavel, Chem. Biol., 1997, 4, 795.
4 M. H. M. Olsson, P. E. M. Siegbahn and A. Warshel, J. Am. Chem.
Soc., 2004, 126, 2820.
5 (a) T. Borowski and E. Broclawik, J. Phys. Chem. B, 2003, 107, 4639;
(b) I. Tejero, L. A. Eriksson, A. Gonzalez-Lafont, J. Marquet and
J. M. Lluch, J. Phys. Chem. B, 2004, 108, 13831.
T/K
kH1a/mol21 dm3 s21
kD1b/mol21 dm3 s21
kH1/kD1
203 K
213 K
223 K
233 K
243 K
a
(4.5 ¡ 0.3) 6 1021
(8.1 ¡ 0.2) 6 1021
1.5 ¡ 0.1
2.3 ¡ 0.2
3.9 ¡ 0.1
(8.7 ¡ 0.1) 6 1022
(1.2 ¡ 0.1) 6 1021
(2.3 ¡ 0.2) 6 1021
(3.7 ¡ 0.5) 6 1021
(6.5 ¡ 0.5) 6 1021
5.2 ¡ 0.4
6.8 ¡ 0.6
6.5 ¡ 0.6
6.2 ¡ 1.0
6.0 ¡ 0.5
b
kH1 = kH 2 kH0
.
kD1 = kD 2 kH0
.
6 (a) M. H. Glickman, J. S. Wiseman and J. P. Klinman, J. Am. Chem.
Soc., 1994, 116, 793; (b) M. H. Glickman and J. P. Klinman,
Biochemistry, 1995, 34, 14077; (c) M. J. Knapp and J. P. Klinman, Eur.
J. Biochem., 2002, 269, 3113.
When linoleic acid is replaced by [11,11-2H2]linoleic acid,18 the
kD value becomes smaller than that of linoleic acid (S4). The KIE
values of one active hydrogen of a pentadiene subunit are
determined as 6.1 ¡ 3 from the kH, kD and kH0 values using the
relation, kH1/kD1 = (kH 2 kH0)/(kD 2 kH0); see Table 2. The
kH1/kD1 value is significantly smaller than that observed for SLO
(KIE = 81), exhibiting no significant tunneling effect in hydrogen
transfer from linoleic acid to cumylperoxyl radical. In addition, the
hydrogen transfer reactivity of oleic acid is only 5 times smaller
than that of linoleic acid at 243 K (S4), whereas the SLO-catalyzed
oxygenation rate of oleic acid is 105 times slower than that of
linoleic acid.19 On the other hand, the hydrogen transfer rate of
arachidonic acid in the SLO-catalyzed oxygenation has been
reported to be similar to that of linoleic acid.20 No additivity rules
hold in the hydrogen transfer reactivity of unsaturated fatty acids
in the enzymatic reactions. Thus, there are significant differences
with respect to the hydrogen-abstraction reactivity and the KIE of
unsaturated fatty acids between the non-enzymatic and enzymatic
systems.
7 (a) T. Jonsson, M. H. Glickman, S. Sun and J. P. Klinman, J. Am.
Chem. Soc., 1996, 118, 10319; (b) M. J. Knapp, F. P. Seebeck and
J. P. Klinman, J. Am. Chem. Soc., 2001, 123, 2931; (c) M. J. Knapp,
K. Rickert and J. P. Klinman, J. Am. Chem. Soc., 2002, 124, 3865; (d)
M. J. Knapp and J. P. Klinman, Biochemistry, 2003, 42, 11466.
8 S. Peng and W. A. van der Donk, J. Am. Chem. Soc., 2003, 125, 8988.
9 A quartz ESR tube (internal diameter: 1.5 mm) containing a sample
solution was irradiated in the cavity of the ESR spectrometer with the
focused light of a 1000 W high-pressure Hg lamp (Ushio-USH1005D)
through an aqueous filter. The ESR spectra were recorded under non-
saturating microwave power conditions on a JEOL JES-FA100 ESR
spectrometer.
10 S. Fukuzumi, K. Shimoosako, T. Suenobu and Y. Watanabe, J. Am.
Chem. Soc., 2003, 125, 9074.
11 (a) J. K. Kochi, P. J. Krusic and D. R. Eaton, J. Am. Chem. Soc., 1969,
91, 1877; (b) J. K. Kochi and P. J. Krusic, J. Am. Chem. Soc., 1969, 91,
3944.
12 J. A. Howard, Adv. Free-Radical Chem., 1972, 4, 49.
13 The decay of cumylperoxyl radical in the presence of oleic acid
(0.04 mol dm23) is clearly faster than that in the absence of oleic acid.
The pseudo-first-order rate constant (kobs) can be accurately determined
from the first-order plots at prolonged reaction time (S1, S2). It was
comfirmed that the kobs value increases linearly with increasing
concentrations of unsaturated fatty acids (S3).
In conclusion, we have successfully determined the absolute
rates of hydrogen transfer from a series of unsaturated fatty acids
to cumylperoxyl radical by the use of ESR at low temperatures. A
simple additivity rule holds in the hydrogen transfer reactivity of
unsaturated fatty acids, which is expressed by the additive
contributions of the reactivity of active hydrogens from the 1,4-
pentadiene subunit and those of allylic subunit.19 Significant
differences between the relative reactivities and the KIE values of
unsaturated fatty acids in hydrogen transfer reactions with
cumylperoxyl radical and those of lipoxygenases indicate that
simple hydrogen transfer from unsaturated fatty acids to radical
species is quite different from the tunneling hydrogen transfer
(proton-coupled electron transfer) in lipoxygenases, where an
electron and a proton may be transferred at the same time but
separately to the Fe3+ site and the OH site, respectively.21
This work was partially supported by a Grant-in-Aid (Nos.
16205020 and 17550058) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan
14 Such a simple additivity rule has so far been reported for equivalent
hydrogens of hydrogen donors; see: (a) K. U. Ingold and G. A. Russel,
in Free Radicals, ed. J. K. Kochi, Wiley, New York, 1973, pp. 283–293;
(b) C. R. Goldsmith, R. T. Jonas and T. D. P. Stack, J. Am. Chem. Soc.,
2002, 124, 83.
15 (a) S. Glasstone, K. J. Laidler and H. Eyring, The Theory of Rate
Processes, McGraw-Hill, New York, 1941; (b) J. E. Espenson, Chemical
Kinetics and Reaction Mechanisms, McGraw-Hill Book Company, New
York, 1981.
16 Negative activation entropies have been commonly observed for
hydrogen-abstraction reactions, see: C. R. Goldsmith, A. P. Cole and
T. D. P. Stack, J. Am. Chem. Soc., 2005, 127, 9904.
17 The restricted open shell Hartree–Fock (ROHF) formalism was used to
calculate the DE values using the DFT method with B3LYP/3-21G basis
set.
18 [11,11-2H2]Linoleic acid was synthesized according to the literature:
W. P. Tucker, S. B. Tove and C. R. Kepler, J. Labelled Compd., 1971, 7,
11.
19 C. H. Clapp, S. E. Senchak, T. J. Stover, T. C. Potter, P. M. Findeis and
M. J. Novak, J. Am. Chem. Soc., 2001, 123, 747.
20 S. Peng and W. A. van der Donk, J. Am. Chem. Soc., 2003, 125, 8988.
21 E. Hatcher, A. V. Soudackov and S. Hammes-Schiffer, J. Am. Chem.
Soc., 2004, 126, 5763.
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