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White-rot fungi are the most efficient lignin degraders in nature (USFAs) [1±7]. This indicates that the MnP/lipid/Mn(II)
and play a key role in carbon recycling on Earth. The fungi system generates unknown active oxidants with enough high
secrete three classes of extracellular ligninolytic enzymes: one oxidative potential to decompose the xenobiotic compounds.
phenol oxidase; laccase and two heme-containing peroxidases; However, the process behind the generation of active oxidants
lignin peroxidase (LiP) and manganese peroxidase (MnP). LiP by the MnP/lipid/Mn(II) system has not been elucidated. Thus
is capable of oxidizing phenolic and nonphenolic lignin far, there has been no report of detection of free radicals in the
structures directly, while the majority of the other two enzyme MnP-dependent lipid peroxidation.
classes cannot directly oxidize nonphenolic lignin substructures With regard to the lipid peroxidation by MnP, one of the most
which make up around 70±90% of the lignin in woody plants. important questions concerns the source of the primary radicals
For the catalysis of MnP, it was shown that MnP is capable of that initiate peroxidation. Kanner reported that lactoperoxidase
decomposing recalcitrant aromatic compounds such as poly- cannot initiate peroxidation of linoleic acid in the presence of
cyclic aromatic hydrocabon (PAH) and nonphenolic b-O-4 H2O2 [8]. However, addition of halide to the reaction system
lignin models in the presence of Mn(II) and unsaturated lipids 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
Correspondence to T. Watanabe, Laboratory of Biomass Conversion,
Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611-0011,
catalytic cycle of lactoperoxidase and that lipid peroxidation
Japan. Fax: 1 81 774 38 3600, E-mail: twatanab@kuwri.kyoto-u.ac.jp
was initiated indirectly by halogen radicals formed by the
Abbreviations: t-NB, tert-nitrosobutane; DMPO, 5,5-dimethyl-1-
enzymatic oxidation of the halide involved [8]. Similarly,
pyrroline-1-oxide; 4-POBN, a-4-pyridyl-1-oxide-N-tert-butylnitrone; oxidation of LDL lipids by horseradish peroxidase HRP/H2O2
DM, n-dodecyl-b-maltoside, MnP, manganese peroxidase; LiP, lignin proceeds via a-tocopheroxyl radical and there is no evidence of
peroxidase; HRP, horseradish peroxidase; 13(S )-HPODE, direct oxidation of LDL lipids by HRP/H2O2 [9].
13(S )-hydroperoxy-9Z,11E-octadecadienoic acid; SFA, saturated fatty Lipid peroxidation is a chemical process comprised of
acid; USFA, unsaturated fatty acid; PAH, polycyclic aromatic hydrocabon; three principal events: initiation (reaction 1), propagation
DPPP, diphenyl-1-pyrenylphosphine; 2,6-DMP, 2,6-dimethoxyphenol; (reactions 2 and 3) and termination (reaction 4). Peroxidation
TBARS, thiobarbituric acid reactive substances. of USFAs is initiated by hydrogen transfer from methylene of
Enzymes: manganese peroxidase (EC 1.11.1.13), lipoxygenase the 1,4-pentadienyl group in USFA to yield pentadienyl radical
(linoleate:oxygenoxidoreductase; EC 1.13.11.12). (reaction 1). The pentadienyl radical reacts with molecular
(Received 7 March 2000, revised 19 April 2000, accepted 10 May 2000) oxygen to yield peroxyl radical (reaction 2). This radical
q FEBS 2000 Initiation mechanism of lipid peroxidation by MnP (Eur. J. Biochem. 267) 4223
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
Fig. 1. 1H-NMR spectra of 13(S)-HPODE.
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 trap, t-NB. It is known that t-NB can trap carbon-centered
molecular mass compounds contaminating in the enzyme radicals initially produced by hydrogen abstraction from the
solutions because ´OH formed by the Fenton type reaction is bis-allylic position [18,19]. As shown in Fig. 3, signals from
a strong initiator of lipid peroxidation as shown in Fig. 2A. In free radicals were not observed when C. subvermispora MnP
the presence of Cl2 and O2´2, ´OH is also produced by way of a was reacted with linoleic acid in the presence of Mn(II), H2O2
HOCl formation if peroxidase has myeloperoxidase-like and t-NB. However, addition of t-NB after preincubation for
activity [17]. Becuase enzymes purified by ion-exchange 2 h without the spin trap generated ESR signals of t-NB spin
chromatography and preparative IEF contain buffer salts and adducts composed of a sharp triplet (aN 0.81 mT) and sextet
a trace amount of transition metals, particular attention should (aN 1.53 mT, aH 0.21 mT) lines (Fig. 3C). The small
be paid to the side reactions caused by the low molecular mass hyperfine splitting of the triplet due to nitrogen of the nitroxyl
substances and H2O2. group was characteristic to a t-NB spin adduct of acyl radical
Without removal of the low molecular mass contaminants, [20±22]. The hfcc of sextet lines is identical to a t-NB spin
we found that the MnPs generated free radicals from linoleic adduct of carbon-centered radical observed in peroxidation of
acid even in the absence of Mn(II). On removal of low linoleic acid by lipoxygenase [23]. Thus, MnP initiated lipid
molecular mass salts by successive washing with Milli-QTM peroxidation but the consecutive peroxidation reactions were
water, the enzymes produced no free radicals from linoleic acid observed only after the slow induction reactions. Addition of
in the absence of Mn(II) (data not shown). Suppression of t-NB inhibited the slow induction reactions.
Mn(II)-independent free radical generation by the pretreatment Figure 2C shows the ESR spectra of 4-POBN spin adducts
was also confirmed by spin trapping with DMPO (data not formed by the reaction of purified MnP from C. subvermispora
shown). Throughout this study, the purified MnPs without low with linoleic acid in sodium tartrate buffer. In the reaction with
molecular mass contaminants were used for evaluating 4-POBN, weak signals from O2´2 adduct (aN 1.42 mT,
initiation activity of the enzymes. aH 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
Reactions of MnP with linoleic acid
excess H2O2 as reported before [25]. Thus, ESR spectra
To analyze the initiation activity of MnP by ESR, linoleic acid indicated that direct hydrogen abstraction from bis-allylic
was reacted with the purified MnPs in the presence of a spin 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
DISCUSSION
In this study, free radicals produced by MnP were analyzed by Fig. 7. ESR spectrum of t-NB spin adducts formed by the reaction of
ESR to understand the unique peroxidation processes that occur linoleic acid with chelated Mn(III). The spectra were recorded at 0.5, 5,
in the presence of antioxidant Mn(II). To discuss on the 10, 15, 30, 60 and 90 min. The reaction was initiated by addition of 2.5 mm
possible roles of the MnP-dependent lipid peroxidation in Mn(III)-tartrate to a solution containing 7.5 mm linoleic acid and
lignolysis of the selective white rot fungus C. subvermispora, a 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- observed (Fig. 3A) although t-NB can trap a wide range of free
mediate, four different mechanisms can be proposed. The first radicals from linoleic acid including conjugated and non-
is the formation of alkoxyl radical from lipid hydroperoxide conjugated carbon-centered radicals at position 9, 10, 12 and 13
with concomitant oxidation of Mn(II) as reported in Fe(II)- [18,19,29]. Experiments with 4-POBN also gave no spin
dependent decomposition of lipid hydroperoxides (Fig. 9A). adducts of free radicals from linoleic acid (Fig. 2-C). These
We demonstrated that this reaction cannot take place (Fig. 6B). results indicate that the direct hydrogen abstraction from the
Formation of peroxyl radical from lipid hydroperoxide with bis-allylic position during turnover is not involved in the
coreduction of Mn(III) is another possible route (Fig. 9B). The catalysis of MnP.
transfer of two electrons from lipid hydroperoxide to native In oxygen uptake experiments with methyl linoleate and
MnP may produce free radicals if it is accompanied by a decay oleic acid (Fig. 4), no decrease in O2 concentration below the
of their alkenyl chain (Fig. 9C1). One electron reduction of starting level was observed. The low reactivity of methyl
comp I or comp II by lipid hydroperoxide (Fig. 9C2,3) is linoleate supported that direct hydrogen abstraction from the
another possible pathway for generating free radicals from lipid bis-allylic position is not involved in the catalysis of MnP. In
hydroperoxide. contrast to these reactions, the rate of O2 uptake in the linoleic
If MnP could abstract hydrogen directly from the bis-allylic acid system gradually increased and reached to a propagation
position during turnover as in the peroxidation of linoleic acid phase around 30 min after addition of H2O2. The clear
with lipoxygenase, generation of free radicals followed by difference in reactivity among these substrates were also
oxygen consumption should be observed immediately after obtained with the Mn(III)-tartrate complex (Fig. 5). It is
addition of MnP according to Fig. 9,I. Therefore, spin trapping known that rate of the initiation and propagation of linoleic
experiments were carried out using t-NB and 4-POBN. When acid in autoxidation is almost equal in extent to those of methyl
MnPs from C. subvermispora were reacted with linoleic acid in linoleate [30]. Therefore, the difference in reactivity between
the presence of MnSO4 and H2O2, no spin adducts of t-NB were 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
energy of bis-allylic hydrogen. Heibe and Bush reported a free acid by the MnP/Mn(II)/H2O2 system has been reported [5]. To
radical addition of enolizable compounds to olefins by Mn(III) demonstrate the generation of free radicals from lipid
[31±33]. In the free radical reaction they reported, Mn(III) hydroperoxide, a stereochemically pure lipid hydroperoxide,
oxidized the enolic form of carboxylic acids to the corres- 13(S)-HPODE (Fig. 1) was isolated and then reacted with MnP
ponding carboxylalkyl radical by hydrogen abstraction at the and Mn(III)-tartrate in the presence of t-NB. When 13(S)-
position vicinal to the carboxylic group [31±35]. In the media HPODE was reacted with Mn(II), no ESR signals were
containing unsaturated fatty acids having a 1,4-pentadienyl observed (Fig. 6B), indicating that the Mn(II)-dependent
moiety, hydrogen abstraction from the bis-allylic position may reactions in Fig. 9A cannot have taken place. However,
occur by direct reaction with the carboxylalkyl radical or by reactions of 13(S)-HPODE with Mn(III)-tartrate complex
secondary radicals produced by addition of olefins or oxygen to gave intensive triplet signals from acyl radical (Fig. 6A).
the carboxylalkyl radical. This explains why the oxygen Since acyl radical is produced by abstraction of hydrogen from
consumption was observed only with the compound to have aldehyde by free radicals with high oxidation potential [36], it
both the 1,4-pentadienyl moiety and a free carboxyl group. is suggested that the reaction starts by initial peroxy raidal
Addition of Mn(II) to this reaction system strongly inhibited the formation by Mn(III) (Fig. 9B) and subsequent consecutive
oxidation (Fig. 5). These observations highlight the role of MnP electron transfer reactions involving aldehyde formation.
in converting the antioxidant, Mn(II), to the initiator, Mn(III), The reaction of native MnP with 13(S)-HPODE in the
in lipid peroxidation. absence of Mn(II) gave weak sextet ESR signals with
The two-phase O2 uptake profile of linoleic acid (Fig. 4) and aN 1.53 mT, aH 0.21 mT (data not shown). However,
an induction period observed in the ESR spectra (Figs 3 and 7) addition of Mn(II) to this reaction system extinguished the ESR
can be explained by the slow induction to form lipid signals. Thus, formation of free radicals were not observed in
hydroperoxides by Mn(III) and subsequent consecutive free the reaction of MnP/Mn(II)/13(S)-HPODE (Fig. 8B). Addition
radical formation from the lipid hydroperoxides by Mn(III) or of H2O2 to this reaction system produced strong ESR signals of
MnP. In fact, formation of lipid hydroperoxides from linoleic acyl radical for over 60 min (Fig. 8A). Thus, a supply of H2O2
4230 T. Watanabe et al. (Eur. J. Biochem. 267) q FEBS 2000
is necessary for a rate of turnover high enough to suppress the 3. Moen, M.A. & Hammel, K.E. (1994) Lipid peroxidation by the
antioxidative activity of Mn(II). We also observed on addition manganese peroxidase of Phanerochaete chrysosporium is the basis
of equimolar 13(S)-HPODE to native MnP, no shift in the for phenanthrene oxidation by the intact fungus. Appl. Environ.
absorption maxima at 407, 502 and 626 nm due to ferric Microbiol. 60, 1956±1961.
protoporphyrin complex (data not shown). Therefore, it can 4. Bogan, B.W., Lamar, R.T. & Hammel, K.E. (1996) Fluorene oxidation
be concluded that 13(S)-HPODE can react with native in vivo by Phanerochaete chrysosporium and in vitro during
manganese peroxidase-dependent lipid peroxidation. Appl. Environ.
MnP but this peroxide species does not serve as a highly
Microbiol. 62, 1788±1792.
reactive electron acceptor for MnP in aqueous media. In
5. BoÈhmer, S., Messner, K. & Srebotnik, E. (1998) Oxidation of
the MnP-dependent peroxidation of unsaturated fatty acid,
phenathrene by a fungal laccase in the presence of 1-hydroxybenzo-
H2O2 is supplied by disproportionation of O2´2 which is triazole and unsaturated lipids. Biochem. Biophys. Res. Commun.
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radicals. This accounts for the observation that decomposition 6. Kapitch, A., Hoftrichter, M., Vares, T. & Hatakka, A. (1999) Coupling
of lignin model compounds by MnP/linoleic acid was of manganese peroixidase-mediated lipid peroxidation with
suppressed by catalase [7]. destruction of nonphenolic lingin model compounds and 14C-labeled
The reaction of MnP with 13(S)-HPODE in the presence of lignins. Biochem. Biophys. Res. Commun. 259, 212±219.
Mn(II), H2O2 and t-NB in acetate buffer also produced acyl 7. Kapitch, A.N., Jensen, K.A. & Hammel, K.A. (1999) Peroxyl
radical (Fig. 8D), indicating that chelator for Mn(II) and radicals are potential agents of lignin biodegradation. FEBS Lett. 461,
Mn(III) is not essential for producing free radicals from 115±119.
13(S)-HPODE. Throughout this study, no differences were 8. Kanner, J. & Kinsella, J.E. (1983) Initiation of lipid peroxidation by a
observed between MnPs from C. subvermispora and B. adusta. peroxidase/hydrogen peroixide/halide system. Lipids 18, 204±210.
In conclusion, the generation of free radicals by the 9. Witting, P.K., Upston, J.M. & Stocker, R. (1997) Role of a-toco-
MnP/linoleic acid/Mn(II)/H2O2 system proceeds mainly by the pheroxyl radical in the initiation of lipid peroxidation in human low-
abstraction of hydrogen from enol by Mn(III). Carboxylalkyl density lipoprotein exposed to horse radish peroxidase. Biochemistry
radical produced by this slow reaction induces hydrogen 36, 1251±1258.
abstraction from the bis-allylic position to yield lipid hydro- 10. SzireÂki, I., Mohanakumar, K.P., Rauhara, P., Kim, H.G., Yeh, K.J. &
peroxides. The hydroperoxides then react with Mn(III) or Chiueh, C.C. (1998) Manganese: a transition metal protects
oxidized MnP to generate free radicals including carbon- nigrostriatal neurons from oxidative stress in the iron-induced animal
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linoleic acid predominantly at the initial stage of cultivation,
13. Lobos, S., Larrin, J., Sales, L., Cullen, D. & VicunÄa, R. (1994)
and these fatty acids were consumed during cultivation with Isozymes of manganese-dependent peroxidase and laccase produced
concomitant formation of lipid hydroperoxides and thiobarbi- by the lignin-degrading basidiomycete Ceriporiopsis subvermispora.
turic acid reactive substances (TBARS) [38,39]. However, the Microbiology 140, 2691±2698.
role of MnP in the lipid peroxidation process was not made 14. Urzua, U., Larrondo, L.F., Lobos, S., Larrain, J. & VicunÄa, R. (1995)
clear. In wood decay by C. subvermispora, lignin degradation Oxidation reactions catalyzed by manganese peroxidase isozymes
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