Eur. J. Biochem.

267, 4222±4231 (2000) q FEBS 2000

Formation of acyl radical in lipid peroxidation of linoleic acid by

manganese-dependent peroxidase from Ceriporiopsis subvermispora
and Bjerkandera adusta
Takashi Watanabe, Shihoko Katayama, Makiko Enoki, Yoichi Honda and Masaaki Kuwahara
Laboratory of Biomass Conversion, Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto, Japan

Lipid peroxidation by managanese peroxidase (MnP) is reported to decompose recalcitrant polycyclic

aromatic hydrocabon (PAH) and nonphenolic lignin models. To elucidate the oxidative process, linoleic acid and
13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE] were reacted with MnPs from Ceriporiopsis
subvermispora and Bjerkandera adusta and the free radicals produced were analyzed by ESR. When the MnPs
were reacted with 13(S)-HPODE in the presence of Mn(II), H2O2 and tert-nitrosobutane (t-NB), the ESR
spectrum contained a sharp triplet of acyl radical (aN ˆ 0.81 mT). Formation of acyl radical was also observed in
the reactions of Mn(III)-tartrate with 13(S)-HPODE and with linoleic acid, but the latter reaction occurred
explosively after an induction period of around 30 min. Reactions of MnP with linoleic acid in the presence of
Mn(II), H2O2 and t-NB gave no spin adducts while addition of t-NB after preincubation of linoleic acid with
MnP/Mn(II)/H2O2 for 2 h gave spin adducts of carbon-centered (aN ˆ 1.53 mT, aH ˆ 0.21 mT) and acyl
(aN ˆ 0.81 mT) radicals. In contrast to linoleic acid, methyl linoleate and oleic acid were not peroxidized by
MnP and chelated Mn(III) within a few hours, indicating that structures containing both the 1,4-pentadienyl
moiety and a free carboxyl group are necessary for inducing the peroxidation in a short reaction time. These
results indicate that MnP-dependent lipid peroxidation is not initiated by direct abstraction of hydrogen from the
bis-allylic position during turnover but proceeds by a Mn(III)-dependent hydrogen abstraction from enols and
subsequent propagation reactions involving the formation of acyl radical from lipid hydroperoxide. This finding
expands the role of chelated Mn(III) from a phenol oxidant to a strong generator of free radicals from lipids and
lipid hydroperoxides in lignin biodegradation.
Keywords: manganese peroxidase; lipid peroxidation; Ceriporiopsis subvermispora; acyl radical; ESR.

White-rot fungi are the most efficient lignin degraders in nature
and play a key role in carbon recycling on Earth. The fungi
secrete three classes of extracellular ligninolytic enzymes: one
phenol oxidase; laccase and two heme-containing peroxidases;
lignin peroxidase (LiP) and manganese peroxidase (MnP). LiP
is capable of oxidizing phenolic and nonphenolic lignin
structures directly, while the majority of the other two enzyme
classes cannot directly oxidize nonphenolic lignin substructures
which make up around 70±90% of the lignin in woody plants.
For the catalysis of MnP, it was shown that MnP is capable of
decomposing recalcitrant aromatic compounds such as poly-
cyclic aromatic hydrocabon (PAH) and nonphenolic b-O-4
lignin models in the presence of Mn(II) and unsaturated lipids
Correspondence to T. Watanabe, Laboratory of Biomass Conversion,
Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611-0011,
Japan. Fax: 1 81 774 38 3600, E-mail: twatanab@kuwri.kyoto-u.ac.jp
Abbreviations: t-NB, tert-nitrosobutane; DMPO, 5,5-dimethyl-1-
pyrroline-1-oxide; 4-POBN, a-4-pyridyl-1-oxide-N-tert-butylnitrone;
DM, n-dodecyl-b-maltoside, MnP, manganese peroxidase; LiP, lignin
peroxidase; HRP, horseradish peroxidase; 13(S )-HPODE,
13(S )-hydroperoxy-9Z,11E-octadecadienoic acid; SFA, saturated fatty
acid; USFA, unsaturated fatty acid; PAH, polycyclic aromatic hydrocabon;
DPPP, diphenyl-1-pyrenylphosphine; 2,6-DMP, 2,6-dimethoxyphenol;
TBARS, thiobarbituric acid reactive substances.
Enzymes: manganese peroxidase (EC 1.11.1.13), lipoxygenase
(linoleate:oxygenoxidoreductase; EC 1.13.11.12).
(Received 7 March 2000, revised 19 April 2000, accepted 10 May 2000)
(USFAs) [1±7]. This indicates that the MnP/lipid/Mn(II)
system generates unknown active oxidants with enough high
oxidative potential to decompose the xenobiotic compounds.
However, the process behind the generation of active oxidants
by the MnP/lipid/Mn(II) system has not been elucidated. Thus
far, there has been no report of detection of free radicals in the
MnP-dependent lipid peroxidation.
With regard to the lipid peroxidation by MnP, one of the most
important questions concerns the source of the primary radicals
that initiate peroxidation. Kanner reported that lactoperoxidase
cannot initiate peroxidation of linoleic acid in the presence of
H2O2 [8]. However, addition of halide to the reaction system
initiated peroxidation of linoleic acid depending on the
concentration of the halide. Thus, it has been shown that
hydrogen abstraction from linoleic acid is not part of the
catalytic cycle of lactoperoxidase and that lipid peroxidation
was initiated indirectly by halogen radicals formed by the
enzymatic oxidation of the halide involved [8]. Similarly,
oxidation of LDL lipids by horseradish peroxidase HRP/H2O2
proceeds via a-tocopheroxyl radical and there is no evidence of
direct oxidation of LDL lipids by HRP/H2O2 [9].
Lipid peroxidation is a chemical process comprised of
three principal events: initiation (reaction 1), propagation
(reactions 2 and 3) and termination (reaction 4). Peroxidation
of USFAs is initiated by hydrogen transfer from methylene of
the 1,4-pentadienyl group in USFA to yield pentadienyl radical
(reaction 1). The pentadienyl radical reacts with molecular
oxygen to yield peroxyl radical (reaction 2). This radical
q FEBS 2000 Initiation mechanism of lipid peroxidation by MnP (Eur. J. Biochem. 267) 4223
species abstracts hydrogen from another USFA molecule to
yield lipid hydroperoxide (reaction 3).
L ÿ H 1 X´ ! L´ 1 X ÿ H …1†
L´ 1 O2 ! LOO´ …2†
LOO´ 1 L ÿ H ! L´ 1 LOOH …3†
2LOO´ ! nonradicalproducts …4†
LOO´ 1 Mn…II† 1 H1 ! LOOH 1 Mn…III† …5†
LOOH 1 Fe…II† ! LO´ 1 OH2 1 Fe…III† …6†
The lipid hydroperoxide generates free radicals by a reaction
with transition metal complexes (e.g. reaction 6) or active
oxygen species such as hydroxyl and hydroperoxyl radicals.
LOO´ is a chain-carrying radical of the propagation cycle.
Therefore, extinction of LOO´ by radical coupling leads to a
termination process (e.g. reaction 4). In the reaction media
containing Mn(II), Mn(II) reacts with the peroxyl radicals to
terminate the chain reaction (reaction 5). The chain-breaking
antioxidative activity of Mn(II) suppresses the generation of
free radicals from USFAs [10±11]. This raises the question of
how the MnP-lipid system generates free radicals in the
presence of antioxidant, Mn(II). One explanation for this
phenomenon is regeneration of free radicals by breakdown of
LOOH by MnP or chelated Mn (III). In this paper, we present
unequivocal evidence that chelated Mn(III) and MnP can react
with LOOH to generate free radicals including acyl radical,
thereby carrying the chain reactions. Mechanisms for the
initiation of reaction via an enolic form of fatty acids by
Mn(III) are also proposed.
M AT E R I A L S A N D M E T H O D S
General methods
Manganese (II) sulfate, iron(II) sulfate, oleic acid, methyl
linoleate and 1,2,3-trimethoxybenzene were obtained from
Wako Pure Chemical Industries (Osaka, Japan). 5,5-Dimethyl-
1-pyrroline-N-oxide (DMPO) was purchased from Sigma.
a-4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN) and tert-
nitrosobutane (t-NB) were obtained from Labotech (Tokyo,
Japan). n-Dodecyl-b-maltoside (DM) was purchased from
Dojindo (Kumamoto, Japan). Linoleic acid and soybean
lipoxygenase were purchased from Nacalai Tescque (Kyoto,
Japan). The linoleic acid was purified by passing through a
Sep-PakTM CN Light cartridge (Waters, Milford, USA) after
dissolving in n-hexane. The eluent from the cartridge was
evaporated with a gentle stream of N2 gas. The purity of the
linoleic acid was confirmed by 1H-NMR spectroscopy and
HPLC on Shodex ODP-50 (6.0  250 mm, Showadenko,
Japan) using CH3CN/MeOH/H2O (75 : 11 : 14, v/v/v) as an
eluent. The elution was carried out with a Hitachi L-6200 pump
equipped with a L-4200 UV-detector at a flow rate of
0.8 mL min21 and recorded at 205 and 234 nm. All the fatty
acids, methyl linoleate and spin trapping agents were stored
under nitrogen at 2 30 8C. Milli-QTM water was used
throughout the experiments. All of the chemicals used were
of analytical reagent grade and the absence of peroxide in
organic solvents was confirmed by diphenyl-1-pyrenylpho-
sphine (DPPP) assay [12]. NMR spectra were recorded at 20 8C
on a JEOL LA-400 NMR Spectrometer (1H: 400 MHz) in
CDCl3.
Enzyme preparation
Crude MnP from Ceriporiopsis subvermispora FP-90031
[13±14] was collected from 7 day-cultures grown on a
wood medium composed of birch wood (5 g), glucose (0.7 g)
and peptone (0.7 g) at 28 8C. The culture filtrate was dialyzed
against 20 mm Na-succinate buffer (pH 4.5). The dialyzate was
concentrated by ultrafiltration and then purified by gel filtration
on Superdex 75 PG (1.6  60 cm, Amersham Pharmacia
Biotech) using 20 mm Na-succinate buffer containing 0.1 m
NaCl as an eluent. Fractions showing MnP activities were
collected, desalted with Centriprep YM-30 (cutoff, 30 000;
Millipore) and then purified by preparative IEF as described
[14] to obtain a single protein (pI 3.40, RZ value: 3.0,
1.0 U ˆ 8.75  10211 mol). Crude MnP from Bjerkandera
adusta was collected from 14 day-cultures grown on a glucose-
peptone medium at 28 8C [15]. The culture filtrate was
dialyzed against 20 mm Na/succinate buffer (pH 4.5). The
dialyzate was concentrated by ultrafiltration with Amicon
PM-10 membrane and then purified on DEAE-Sepharose
CL-6B (25  100 mm) and MONO-Q (10/10) (Amersham
Pharmacia Biotech, Sweden) columns to obtain a single protein
(pI 3.40, RZ value: 3.9, 1.0 U ˆ 4.97  10211 mol). Low-
molecular-mass compounds were removed from these two
enzyme fractions by successive washings with Milli-QTM water
with a Centricut N-10 ultrafiltration concentrator (cut off,
10 000; Kurabo, Osaka, Japan) before use.
Enzyme assay
MnP activity was measured with 2,6-dimethoxyphenol (2,6-DMP).
The reaction mixture contained 0.2 mm 2,6-DMP, 0.5 mm
MnSO4, 0.1 mm H2O2, 250 mm sodium tartrate buffer (pH 3.0)
and the enzyme solution. Reactions were started by adding
H2O2 and were quantified by monitoring the initial rate of
increase in absorbance at 470 nm in the presence and absence
of manganese. One unit (U) of enzyme activity is defined as the
amount of enzyme that oxidizes 1 mmol of 2,6-DMP in one
minute. Lipoxygenase activity was measured by O2 uptake in a
reaction system containing 1.5 mm linoleic acid, 1 mm DM and
20 mm Tris/HCl buffer (pH. 9.0). One unit (U) of enzyme
activity is defined as the amount of enzyme that absorbs
1 mmol of O2 in 1 min.
Preparation of 13(S )-hydroperoxy-9Z,11E-octadecadienoic
acid [13(S )-HPODE]
Linoleic acid (20 mm) was reacted with soybean lipoxy-
genase (20 U) at 4 8C in darkness in 66 mm Tris/HCl buffer
containing 0.33% Tween 20 for 24 h. 13(S)-HPODE was
isolated from a reaction mixture by HPLC on ChemkoPak
Nucleosil 5CN (7.5  300 mm, Chemko Scientific, Japan) as
described [16]. The elution was carried out with n-hexane/
2-propanol/trifluoroacetic acid (98 : 2 : 0.1, v/v/v) at a flow
rate of 2 mL´min21. UV absorbance was monitored at 205 nm
1
H-NMR coupling constants of J11,12 ˆ 15.2 Hz and
J9,10 ˆ 11.0 Hz supported the 9Z,11E configuration. No con-
taminating isomers of the hydroperoxide were detected (Fig. 1).
Production of 4-POBN spin adduct of pentyl radical
(aN ˆ 1.58 mT, aH ˆ 0.26 mT) was confirmed by the reaction
of the isolated 13(S)-HPODE with Cu(en)2 (data not shown).
Oxygen consumption experiments
Oxygen consumption experiments were carried out with an
Oxygen Monitor (Type FM-2000, Eyela, Japan) equipped
4224 T. Watanabe et al. (Eur. J. Biochem. 267) q FEBS 2000

with an Oxygen electrode (12/120 T-Type, Mettler Toredo,

Switzerland) and a reaction vessel (sample volume: 1.2 mL). A
solution containing 0.25 mm MnSO4, 20 mm Na/tartrate buffer
(pH 5.0) and 1 mm DM was initially placed into a reaction
chamber and equilibrated by constant stirring at 25 8C. The O2
concentration in a MnP reaction system was recorded from
when 1.5 mm linoleic acid was added to the solution. MnP
(240 mU) from B. adusta and 0.2 mm H2O2 was added at 10
and 40 min, respectively. Reactions with oleic acid and methyl
linoleate were carried out under the conditions described above.
Oxygen consumption experiments with a Mn(III)-tartrate
system were carried out in 20 mm Na/tartrate buffer (pH 5.0)
containing 1.5 mm linoleic acid, methyl linoleate or oleic acid.
The solution was dispersed with 1 mm DM. The reaction was
started by addition of 0.2 mm Mn(III)-tartrate which had been
prepared by dissolving Mn(III)acetate in a 0.1-m Na/tartrate
buffer (pH 5.0) in darkness just before use. Oxygen uptake
experiments for Mn (III)-tartrate in the presence of 1 mm DM,
1.5 mm linoleic acid and 0.2 mm MnSO4 were also carried out
under the conditions described above.

ESR experiments
ESR spectral recordings were made in a flat cell on a JEOL
FR-30 X-band ESR spectrometer operating at room tem-
perature with a modulation amplitude of 0.079 mT, time
constant of 0.10 s, scanning time of 2 min and microwave
power of 4 mW. Gains for Fig. 2A,B were 10 and 200,
respectively. Gains for the other experiments were 320. In the
reaction systems of t-NB, the spin trapping agent was dissolved
in 75 mL of EtOH and immediately used for each measurement.

R E S U LT S

Preparation of MnPs
For the analysis of MnP-dependent lipid peroxidation, MnP
isozyme from C. subvermispora and that from B. adusta were
isolated. Before analyzing the free radical reactions of the
enzymes, we checked side reactions catalyzed by H2O2 and low
molecular mass compounds contaminating in the enzyme
solutions because ´OH formed by the Fenton type reaction is
a strong initiator of lipid peroxidation as shown in Fig. 2A. In
the presence of Cl2 and O2´2, ´OH is also produced by way of a
HOCl formation if peroxidase has myeloperoxidase-like
activity [17]. Becuase enzymes purified by ion-exchange
chromatography and preparative IEF contain buffer salts and
a trace amount of transition metals, particular attention should
be paid to the side reactions caused by the low molecular mass
substances and H2O2.
Without removal of the low molecular mass contaminants,
we found that the MnPs generated free radicals from linoleic
acid even in the absence of Mn(II). On removal of low
molecular mass salts by successive washing with Milli-QTM
water, the enzymes produced no free radicals from linoleic acid
in the absence of Mn(II) (data not shown). Suppression of
Mn(II)-independent free radical generation by the pretreatment
was also confirmed by spin trapping with DMPO (data not
shown). Throughout this study, the purified MnPs without low
molecular mass contaminants were used for evaluating
initiation activity of the enzymes.
Reactions of MnP with linoleic acid
To analyze the initiation activity of MnP by ESR, linoleic acid
was reacted with the purified MnPs in the presence of a spin
Fig. 1. 1H-NMR spectra of 13(S)-HPODE.
trap, t-NB. It is known that t-NB can trap carbon-centered
radicals initially produced by hydrogen abstraction from the
bis-allylic position [18,19]. As shown in Fig. 3, signals from
free radicals were not observed when C. subvermispora MnP
was reacted with linoleic acid in the presence of Mn(II), H2O2
and t-NB. However, addition of t-NB after preincubation for
2 h without the spin trap generated ESR signals of t-NB spin
adducts composed of a sharp triplet (aN ˆ 0.81 mT) and sextet
(aN ˆ 1.53 mT, aH ˆ 0.21 mT) lines (Fig. 3C). The small
hyperfine splitting of the triplet due to nitrogen of the nitroxyl
group was characteristic to a t-NB spin adduct of acyl radical
[20±22]. The hfcc of sextet lines is identical to a t-NB spin
adduct of carbon-centered radical observed in peroxidation of
linoleic acid by lipoxygenase [23]. Thus, MnP initiated lipid
peroxidation but the consecutive peroxidation reactions were
observed only after the slow induction reactions. Addition of
t-NB inhibited the slow induction reactions.
Figure 2C shows the ESR spectra of 4-POBN spin adducts
formed by the reaction of purified MnP from C. subvermispora
with linoleic acid in sodium tartrate buffer. In the reaction with
4-POBN, weak signals from O2´2 adduct (aN ˆ 1.42 mT,
aH ˆ 0.17 mT) [24] were observed, but no other free radicals
were detected. The O2´2 adduct was produced without linoleic
acid (Fig. 2C 0 ) and originates from the reaction of MnP with an
excess H2O2 as reported before [25]. Thus, ESR spectra
indicated that direct hydrogen abstraction from bis-allylic
position during turnover is not involved in the catalysis of MnP.
q FEBS 2000 Initiation mechanism of lipid peroxidation by MnP (Eur. J. Biochem. 267) 4225

[27]. However, the rate of oxygen uptake in the linoleic acid

system gradually increased and reached a propagation phase
around 30 min after addition of H2O2 [28] (5.8 nmol´min21)
(Fig. 4).
In the reaction of linoleic acid with Mn(III)-tartrate,
51 nmol´min21 of oxygen was taken up in 5 min after the
addition of Mn(III)-tartrate. Around 8 min after the reaction,
the O2 concentration showed a slight increase (Fig. 5). Addition
of an equimolar amount of Mn(II) to this reaction system

Fig. 2. ESR spectrum of 4-POBN spin adducts formed in the

reaction of linoleic acid with (A) Fe(II)/H2O2 (B) lipoxygenase and
(C) C. subvermispora MnP/Mn(II)/H2O2. The spectra were recorded at
10 min. (A) H2O2 (0.5 mm) was added to a solution containing 6 mm
linoleic acid, 100 mm FeSO4 and 100 mm 4-POBN. (B) Soybean
lipoxygenase (100 mU) was reacted with 6 mm linoleic acid in 50 mm
Tris/HCl buffer (pH 9.0) containing 100 mm 4-POBN. (C) C. sub-
vermispora MnP was reacted with 6 mm linoleic acid in 12.5 mm Na/
tartrate buffer (pH 5.0) containing 0.5 mm MnSO4, 0.25 mm H2O2 and
100 mm 4-POBN. (C 0 ) As in (A), but without linoleic acid. The
spectrum was recorded at 10 min. Relative receiver gains for (A), (B),
(C, C 0 ) were 1 : 20 : 32.

This is in stark contrast to lipid peroxidation by soybean

lipoxygenase in which bis-allylic hydrogen is abstracted during
turnover to produce a 4-POBN spin adduct of pentyl radical
(aN ˆ 1.58 mT, aN ˆ 0.26 mT) (Fig. 2B) [26].

Oxygen consumption during reactions of MnP and

Mn(III)-tartrate with methyl linoleate, oleic and
linoleic acids
Oxygen uptake experiments were carried out using linoleic
acid, methyl linoleate and oleic acid (Fig. 4). In the reaction of
methyl linoleate and oleic acid, generation of O2 by the
catalatic activity of MnP was observed after addition of H2O2
but no decrease in the O2 concentration below the starting level
was observed. In the reaction of linoleic acid, 1.7 nmol´min21
of O2 was taken up in the 15 min after the O2 concentration
reached a maximum by the catalatic decomposition of H2O2
Fig. 3. ESR spectrum of t-NB spin adducts formed in the reaction of
linoleic acid with C. subvermispora MnP. Spectra were assigned to
adducts of carbon-centered (L) and acyl (P) radicals. The spectra were
recorded at 0.5, 5 and 10 min. (A) The reaction was initiated by addition of
MnP to a solution containing 7.5 mm linoleic acid, 0.5 mm MnSO4,
0.25 mm H2O2 and 40 mm t-NB in 12.5 mm Na/tartrate buffer (pH 5.0). (B)
As in (A), but t-NB was added 1 h after reaction without the spin trap. (C)
As in (A), but t-NB was added 2 h after reaction without the spin trap.
4226 T. Watanabe et al. (Eur. J. Biochem. 267) q FEBS 2000

Reaction of Mn(III) with 13(S )-HPODE and

linoleic acid
Reactions of 13(S)-HPODE with Mn(III)-tartrate were
analyzed by ESR using t-NB as a spin trapping agent.
When 13(S)-HPODE was reacted with Mn(III)-tartrate com-
plex, intensive triplet signals with aN ˆ 0.81 mT were
produced (Fig. 6A). The narrow hfcc with no splitting of
b-proton was identical to that of acyl radical. The formation
of acyl radical continued for over 60 min. Acyl radicals were
not found when Mn(III) or 13(S)-HPODE were omitted from
the complete reaction system (Fig. 6A). In contrast to the
reaction with Mn(III), no ESR signal was observed by the
reaction of Mn(II) with 13(S)-HPODE (Fig. 6B). The formation
of acyl radical was also observed on mixing 2.5 mm Mn(III)-
tartrate with 7.5 mm linoleic acid after an induction period of
around 30 min (Fig. 7).

Reaction of MnP with 13(S )-HPODE

Reaction of 13(S)-HPODE with MnP/Mn(II) in sodium tartrate
buffer gave no ESR signals (Fig. 8B) but addition of H2O2 to
this reaction system produced spin adducts of acyl radical
(Fig. 8A). The formation of acyl radical continued for over
60 min
No distinct difference was observed between MnPs
from C. subvermispora and B. adusta (Fig. 8C). The MnP
reactions in acetate buffer also produced the t-NB adduct of
acyl radical (Fig. 8D). Thus, chelator for Mn(II) and
Mn(III) is not essential for producing free radicals from
13(S)-HPODE in MnP/Mn(II)/H2O2 system. Acyl radicals were
not found when any of the reactants were omitted from the
complete reaction system.
Fig. 4. Oxygen uptake during the reaction of MnP with (A) linoleic acid
(B) methyl linoleate and (C) oleic acid. (A) Monitoring of the oxygen
concentration was started with a solution containing 1.5 mm linleic acid,
1 mm DM and 0.25 mm MnSO4. After 10 and 20 min, 200 mU MnP from
B. adusta and 0.2 mm H2O2 was added, respectively. (B) As in (A), but
methyl linoleate was used instead of linoleic acid. (C) As in (A), but oleic
acid was used instead of linoleic acid.

lengthened the lag time of initiation and decreased the oxygen

uptake rate from 51 to 12.7 nmol´min21 (Fig. 5). The decrease
in O2 concentration in the reactions with oleic acid was less
than 0.1 nmol´min21. It has been reported that fluorene was
decomposed by MnP, Mn(II) and oleate, but the peroxidation
started after a lag of 10±20 h [4]. Therefore, the presence of
1,4-pentadienyl moiety is not a prerequisite for MnP-dependent
lipid peroxidation but the low dissociation energy of bis-allylic
hydrogen (75±80 kCal´mol21) compared with that of allylic
hydrogen (88 kCal´mol21) [28] greatly facilitates the induction
of peroxidation as is obvious from the reaction rate between
linoleic and oleic acid. The decrease in O2 concentration in
reactions with methyl linoleate was less than 0.4 nmol´min21, Fig. 5. Oxygen uptake during the reaction of Mn(III) with linoleic acid,
indicating that esterification of the carboxyl group markedly methyl linoleate and oleic acid. (A) Monitoring of the oxygen
decreased the susceptibility of lipid peroxidiation by chelated concentration was started with a solution containing 1.5 mm linleic acid
Mn(III). Thus, a high rate of oxygen consumption by MnP and and 1 mm DM. After 10 min, 0.2 mm Mn(III)-tartrate was added. (B) As in
Mn(III) within a few hours was observed only in the compound (A), but 0.2 mm MnSO4 was added before the reaction. (C) As in (A), but
having both the 1,4-pentadienyl structure and a free carboxyl methyl linoleate was used instead of linoleic acid. (D) As in (A), but oleic
group. acid was used instead of linoleic acid.
q FEBS 2000 Initiation mechanism of lipid peroxidation by MnP (Eur. J. Biochem. 267) 4227
Fig. 6. ESR spectrum of t-NB spin adducts formed by the reaction of
13(S)-HPODE with Mn(III) and Mn(II). The spectra of (A) were recorded
at 0.5, 5, 10, 15, 30 and 60 min. The spectra of (A 0 ), (A 00 ) and (B) were
recorded at 10 min. (A) The reaction was initiated by addition of 2.5 mm
Mn(III)-tartrate to a solution containing 2.5 mm 13(S)-HPODE and 40 mm
t-NB. (A 0 ) As in (A), but 13(S)-HPODE was omitted. (A 0 ) As in (A), but
Mn(III)-tartrate was omitted. (B) The reaction was initiated by addition of
MnSO4 to a solution containing 2.5 mm 13(S)-HPODE, and 40 mm t-NB.
DISCUSSION
In this study, free radicals produced by MnP were analyzed by
ESR to understand the unique peroxidation processes that occur
in the presence of antioxidant Mn(II). To discuss on the
possible roles of the MnP-dependent lipid peroxidation in
lignolysis of the selective white rot fungus C. subvermispora, a
major isozyme produced by this fungus on wood cultures [13]
was isolated and used for the experiments, together with a
purified MnP from B. adusta.
There are several possible pathways to generate free radicals
from unsaturated lipid. Proposed mechanisms of initiation by
MnP are summarized in Fig. 9. The first possible mechanism is
that MnP directly abstracts hydrogen from the bis-allylic
position of unsaturated lipid (Fig. 9,I). The second is the
generation of free radicals from lipid hydroperoxides produced
by autoxidation (Fig. 9,II) or by hydrogen abstraction from the
enolic form of carboxylic acids by Mn(III) (Fig. 9,III). In the
Fig. 7. ESR spectrum of t-NB spin adducts formed by the reaction of
linoleic acid with chelated Mn(III). The spectra were recorded at 0.5, 5,
10, 15, 30, 60 and 90 min. The reaction was initiated by addition of 2.5 mm
Mn(III)-tartrate to a solution containing 7.5 mm linoleic acid and
40 mm t-NB.
4228 T. Watanabe et al. (Eur. J. Biochem. 267) q FEBS 2000

Fig. 8. ESR spectrum of t-NB spin adducts formed in the reactions of 13(S)-HPODE with MnPs from (A, B, D) C. subvermispora and (C) B. adusta.
The spectra of (A) were recorded at 0.5, 5, 10, 15, 30 and 60 min. The spectra of (B±D) were recorded at 30 min (A) The reaction was initiated by addition of
C. subvermispora MnP (100 mU) to a solution containing 2.5 mm 13(S)-HPODE, 0.5 mm MnSO4, 0.25 mm H2O2 and 40 mm t-NB in 12.5 mm Na/tartrate
buffer (pH 5.0). (B) As in (A), but 0.25 mm H2O2 was omitted. (C) As in (A), but B. adusta MnP (100 mU) was used instead of C. subvermispora MnP.
(D) As in (A), but 12.5 mm Na-acetate buffer (pH 5.0) was used instead of 12.5 mm Na/tartrate buffer (pH 5.0).

free radical formation from the lipid hydroperoxide inter-
mediate, four different mechanisms can be proposed. The first
is the formation of alkoxyl radical from lipid hydroperoxide
with concomitant oxidation of Mn(II) as reported in Fe(II)-
dependent decomposition of lipid hydroperoxides (Fig. 9A).
We demonstrated that this reaction cannot take place (Fig. 6B).
Formation of peroxyl radical from lipid hydroperoxide with
coreduction of Mn(III) is another possible route (Fig. 9B). The
transfer of two electrons from lipid hydroperoxide to native
MnP may produce free radicals if it is accompanied by a decay
of their alkenyl chain (Fig. 9C1). One electron reduction of
comp I or comp II by lipid hydroperoxide (Fig. 9C2,3) is
another possible pathway for generating free radicals from lipid
hydroperoxide.
If MnP could abstract hydrogen directly from the bis-allylic
position during turnover as in the peroxidation of linoleic acid
with lipoxygenase, generation of free radicals followed by
oxygen consumption should be observed immediately after
addition of MnP according to Fig. 9,I. Therefore, spin trapping
experiments were carried out using t-NB and 4-POBN. When
MnPs from C. subvermispora were reacted with linoleic acid in
the presence of MnSO4 and H2O2, no spin adducts of t-NB were
observed (Fig. 3A) although t-NB can trap a wide range of free
radicals from linoleic acid including conjugated and non-
conjugated carbon-centered radicals at position 9, 10, 12 and 13
[18,19,29]. Experiments with 4-POBN also gave no spin
adducts of free radicals from linoleic acid (Fig. 2-C). These
results indicate that the direct hydrogen abstraction from the
bis-allylic position during turnover is not involved in the
catalysis of MnP.
In oxygen uptake experiments with methyl linoleate and
oleic acid (Fig. 4), no decrease in O2 concentration below the
starting level was observed. The low reactivity of methyl
linoleate supported that direct hydrogen abstraction from the
bis-allylic position is not involved in the catalysis of MnP. In
contrast to these reactions, the rate of O2 uptake in the linoleic
acid system gradually increased and reached to a propagation
phase around 30 min after addition of H2O2. The clear
difference in reactivity among these substrates were also
obtained with the Mn(III)-tartrate complex (Fig. 5). It is
known that rate of the initiation and propagation of linoleic
acid in autoxidation is almost equal in extent to those of methyl
linoleate [30]. Therefore, the difference in reactivity between
linoleic acid and methyl linoleate is not due to the dissociation
q FEBS 2000 Initiation mechanism of lipid peroxidation by MnP (Eur. J. Biochem. 267) 4229

Fig. 9. Scheme for the proposed initiation

mechanism of lipid peroxidation by MnP.
Pathways indicated by thick arrows are major
routes for the initiation. (I) Hydrogen abstraction
at the bis-allylic position of lipid by (I) MnP
or Mn(III); (II) hydrogen abstraction at the
bis-allylic position by autoxidation;
(III) hydrogen abstraction from the enolic
form of lipid by Mn(III) and subsequent attack
of the resultant radicals to the bis-allylic position.
This route is a main pathway for inducing
lipid peroxidation of pure linoleic acid,
although the reaction was slow at room
temperature; (A) homolytic cleavage of OÐO
bond of lipid hydroperoxide by Mn(II).
This study demonstrated that the reaction of
13(S)HPODE with Mn(II) cannot take place;
(B) homolytic cleavage of O-H bond of lipid
hydroperoxide by Mn(III) generated acyl
radical for over 60 min; (C) free radical
generation during electron transfer between
MnP and lipid hydroperoxide.

energy of bis-allylic hydrogen. Heibe and Bush reported a free
radical addition of enolizable compounds to olefins by Mn(III)
[31±33]. In the free radical reaction they reported, Mn(III)
oxidized the enolic form of carboxylic acids to the corres-
ponding carboxylalkyl radical by hydrogen abstraction at the
position vicinal to the carboxylic group [31±35]. In the media
containing unsaturated fatty acids having a 1,4-pentadienyl
moiety, hydrogen abstraction from the bis-allylic position may
occur by direct reaction with the carboxylalkyl radical or by
secondary radicals produced by addition of olefins or oxygen to
the carboxylalkyl radical. This explains why the oxygen
consumption was observed only with the compound to have
both the 1,4-pentadienyl moiety and a free carboxyl group.
Addition of Mn(II) to this reaction system strongly inhibited the
oxidation (Fig. 5). These observations highlight the role of MnP
in converting the antioxidant, Mn(II), to the initiator, Mn(III),
in lipid peroxidation.
The two-phase O2 uptake profile of linoleic acid (Fig. 4) and
an induction period observed in the ESR spectra (Figs 3 and 7)
can be explained by the slow induction to form lipid
hydroperoxides by Mn(III) and subsequent consecutive free
radical formation from the lipid hydroperoxides by Mn(III) or
MnP. In fact, formation of lipid hydroperoxides from linoleic
acid by the MnP/Mn(II)/H2O2 system has been reported [5]. To
demonstrate the generation of free radicals from lipid
hydroperoxide, a stereochemically pure lipid hydroperoxide,
13(S)-HPODE (Fig. 1) was isolated and then reacted with MnP
and Mn(III)-tartrate in the presence of t-NB. When 13(S)-
HPODE was reacted with Mn(II), no ESR signals were
observed (Fig. 6B), indicating that the Mn(II)-dependent
reactions in Fig. 9A cannot have taken place. However,
reactions of 13(S)-HPODE with Mn(III)-tartrate complex
gave intensive triplet signals from acyl radical (Fig. 6A).
Since acyl radical is produced by abstraction of hydrogen from
aldehyde by free radicals with high oxidation potential [36], it
is suggested that the reaction starts by initial peroxy raidal
formation by Mn(III) (Fig. 9B) and subsequent consecutive
electron transfer reactions involving aldehyde formation.
The reaction of native MnP with 13(S)-HPODE in the
absence of Mn(II) gave weak sextet ESR signals with
aN ˆ 1.53 mT, aH ˆ 0.21 mT (data not shown). However,
addition of Mn(II) to this reaction system extinguished the ESR
signals. Thus, formation of free radicals were not observed in
the reaction of MnP/Mn(II)/13(S)-HPODE (Fig. 8B). Addition
of H2O2 to this reaction system produced strong ESR signals of
acyl radical for over 60 min (Fig. 8A). Thus, a supply of H2O2
4230 T. Watanabe et al. (Eur. J. Biochem. 267)
is necessary for a rate of turnover high enough to suppress the
antioxidative activity of Mn(II). We also observed on addition
of equimolar 13(S)-HPODE to native MnP, no shift in the
absorption maxima at 407, 502 and 626 nm due to ferric
protoporphyrin complex (data not shown). Therefore, it can
be concluded that 13(S)-HPODE can react with native
MnP but this peroxide species does not serve as a highly
reactive electron acceptor for MnP in aqueous media. In
the MnP-dependent peroxidation of unsaturated fatty acid,
H2O2 is supplied by disproportionation of O2´2 which is
produced by reduction of molecular oxygen with lipid free
radicals. This accounts for the observation that decomposition
of lignin model compounds by MnP/linoleic acid was
suppressed by catalase [7].
The reaction of MnP with 13(S)-HPODE in the presence of
Mn(II), H2O2 and t-NB in acetate buffer also produced acyl
radical (Fig. 8D), indicating that chelator for Mn(II) and
Mn(III) is not essential for producing free radicals from
13(S)-HPODE. Throughout this study, no differences were
observed between MnPs from C. subvermispora and B. adusta.
In conclusion, the generation of free radicals by the
MnP/linoleic acid/Mn(II)/H2O2 system proceeds mainly by the
abstraction of hydrogen from enol by Mn(III). Carboxylalkyl
radical produced by this slow reaction induces hydrogen
abstraction from the bis-allylic position to yield lipid hydro-
peroxides. The hydroperoxides then react with Mn(III) or
oxidized MnP to generate free radicals including carbon-
centered and acyl radicals.
In relation to the wood decaying mechanisms of white-rot
fungi, it has been proposed that lipid peroxidation is involved in
lignin degradation by C. subvermispora which decomposes
nonphenolic lignin without secretion of lignin peroxidase
[1,37]. This fungus produced SFAs and USFAs including
linoleic acid predominantly at the initial stage of cultivation,
and these fatty acids were consumed during cultivation with
concomitant formation of lipid hydroperoxides and thiobarbi-
turic acid reactive substances (TBARS) [38,39]. However, the
role of MnP in the lipid peroxidation process was not made
clear. In wood decay by C. subvermispora, lignin degradation
is observed without penetration by enzymes of wall regions
[40±42]. This indicates that lignin degradation of wall regions
and middle lamelae is not catalyzed by the free radicals
enzymatically generated in cell lumina because these radicals
inevitably react with the first substrates encountered and the life
span of highly reactive radicals is too short for them to
penetrate deep inside the cell wall. Therefore, low molecular
mass metal complexes have been proposed to be generators of
free radicals from lipid hydroperoxides in situ [43]. The
reactions of chelated Mn(III) with 13(S)-HPODE and linoleic
acid indicate that Mn(III) complexes take part in the generation
of free radicals from diffusible lipid hydorperoxides and USFAs
even at a site far from the enzymes. This accounts for the in situ
peroxidation of lipid by selective white-rot fungi in the
presence of antioxidant, Mn(II).
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