Tài liệu Báo cáo khoa học: Unraveling the catalytic mechanism of lactoperoxidase and myeloperoxidase A reflection on some controversial features Elena Ghibaudi and Enzo Laurenti docx

REVIEW ARTICLE
Unraveling the catalytic mechanism of lactoperoxidase and
myeloperoxidase
A reflection on some controversial features
Elena Ghibaudi and Enzo Laurenti
Dipartimento di Chimica I.F.M., Universita
`
di Torino, Italy
Although belonging to the widely investigated peroxidase
superfamily, lactoperoxidase (LPO) and myeloperoxidase
(MPO) share structural and functional features that make
them peculiar with respect to other enzymes of the same
group. A survey of the available literature on their catalytic
intermediates enabled us to ask some questions that
remained unanswered. These questions concern controver-
sial features of the LPO and MPO catalytic cycle, such as the
existence of Compound I and Compound II isomers and
the identification of their spectroscopic properties. After
addressing each of these questions, we formulated a hypo-
thesis that describes an integrated vision of the catalytic
mechanism of both enzymes. The main points are: (a) a
re-evaluation of the role of superoxide as a reductant in the
catalytic cycle; (b) the existence of Cpd I isomers; (c) reci-
procal interactions between catalytic intermediates and (d)
a mechanistic explanation for catalase activity in both
enzymes.
Keywords: lactoperoxidase; myeloperoxidase; aminoacid
radical; Compound I; Compound II; Compound III;
catalytic intermediates.
Introduction
The catalytic cycle of peroxidases, including lactoperoxidase

(LPO) and myeloperoxidase (MPO), is described usually as
a sequence of three consecutive reactions, according to
Scheme 1.
Compound I (Cpd I), which arises from the reaction of
the native enzyme with hydrogen peroxide (H
2
O
2
), is two
oxidizing equivalents above the resting state. It reacts with
a substrate molecule and is converted into a secondary
compound that has lost one equivalent, generally indicated
as Compound II (Cpd II). A second substrate molecule
recycles Cpd II into the resting enzyme. A large excess of
H
2
O
2
converts Cpd I into the inactive intermediate, Com-
pound III (Cpd III). The two oxidizing equivalents of
Cpd I are on an iron ion, that assumes the formal oxidation
state IV, and on the porphyrin ring, which becomes a
cationic radical. Cpd II has been shown to contain Fe
IV
¼O
[1–3], whereas Cpd III is an enzyme adduct with superoxide
[2–6]. Depending on the type of peroxidase, Cpd III
formation may be reversible, whereby it can be reconverted
into an active form of the enzyme, or irreversible, in which
case it is associated with degradation of the enzyme.

Moreover, a few peroxidases, e.g. haloperoxidases, can
oxidize halides through the bielectronic reduction of Cpd I
that is converted back to the resting state without forming
Cpd II [3,7,8].
The generally accepted definition of the three intermedi-
ates of this class of enzymes can be misleading. In fact, when
comparing different peroxidases, the same name is applied
to species with distinct electronic structures. Moreover,
several peroxidase intermediates are known where the
unpaired electron is localized onto an amino acid of the
protein scaffold [9–14] and this aspect is not taken into
account by the classical peroxidase cycle.
Within this context, we propose to re-examine some of
the literature data describing the catalytic cycle of two
mammalianperoxidases,LPOandMPO,inorderto
reconcile the apparent inconsistencies and to provide some
new insights. MPO and LPO share functional and
structural homology, reflecting their common phylogenetic
origin [15] and participate in antimicrobial host defense,
generating potent reactive species by the oxidation of
halides or pseudohalides. Based on our survey of the
experimental data concerning the reactivity of these
enzymes, we formulated four questions that are focused
on controversial features of the LPO and/or MPO
catalytic cycle: (a) is formation of Cpd I reversible (or
do mammalian peroxidases possess catalase activity); (b)
does Cpd I exist in two isomeric forms, containing the
porphyrin radical and the amino acid radical (aa
+•
),

respectively; (c) as the conversion of Cpd I fi Cpd II
occurs spontaneously in the presence of peroxide, which is
the reducing agent in this reaction step and (d) are the
optical spectra of Cpd I–[Fe
IV
¼O; aa
+•
]andCpdII
identical?
Correspondence to E. Ghibaudi, Dipartimento di Chimica I.F.M.,
Universita
`
di Torino, Via Giuria 7 – 10125 Torino, Italy.
Fax: + 39 011 670 7855, Tel.: + 39 011 670 7951,
E-mail:
Abbreviations: LPO, lactoperoxidase; MPO, myeloperoxidase;
Cpd I–III, Compound I–III.
Enzymes: lactoperoxidase (EC 1.11.1.7); myeloperoxidase
(EC 1.11.1.7).
(Received 10 April 2003, revised 18 July 2003,
accepted 23 September 2003)
Eur. J. Biochem. 270, 4403–4412 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03849.x
After addressing each of these questions, we will propose
a new hypothesis that describes an integrated vision of the
catalytic mechanism of MPO and LPO.
A brief survey of the absorption features
of MPO, LPO and their intermediates
The absorption spectrum of MPO is characterized by an
intense Soret peak at k
max

¼ 430 nm and weaker bands at
570, 620 and 690 nm, whereas at 370 nm and 496 nm, two
shoulders are evident [16–20]. The Soret molar extinction
coefficient (e)is89000
M
)1
Æcm
)1
per heme in human MPO
[4,18,20–22] and 95 000
M
)1
Æcm
)1
perhemeincanineMPO
[16].
These features change upon generation of Cpd I: it still
absorbs at 430 nm, but e is about 50% lower than in native
MPO [18,19] (Table 1); moreover, it shows bands at 572
and 625 nm [16]. Stopped-flow measurements are required
to detect Cpd I spectrum, due to its short half-life
(t
1/2
 100 ms [16]). According to Harrison et al. [16], a
40-fold excess of peroxide is required to obtain a good yield.
Other authors [18,19] claim that the amount of peroxide
required in the reaction depends on the purity of the
enzyme, as impurities in the preparation might be oxidized
and thus contribute to additional consumption of H
2

O
2
.In
the literature, there is good agreement on the fact that the
conversion of native MPO into Cpd I is a bimolecular
reaction; three k
app
values are reported and they are almost
coincident (2.3 · 10
7
[23]; 1.4 · 10
7
[19]; 1.8 · 10
7
M
)1
Æs
)1
[18]). In the presence of peroxide, Cpd I converts sponta-
neously to a secondary compound, called Cpd II. This is
relatively stable (t
1/2
 a few minutes) [18,23] and absorbs at
455 and 628 nm (Table 1). In the presence of large peroxide
excess, Cpd I yields Cpd III, an intermediate that shares
with Cpd II some spectroscopic features (e.g. bands at
450 nm and 625 nm) (Table 1), thus making it difficult to
discriminate between these two species. The absorbance at
450 nm (Soret) is useless for distinguishing between Cpd II
and Cpd III in MPO. A system for evaluating the Ôrelative

amountÕ of the two species has been proposed based on the
A
625
/A
456
value [24]. Cpd II would produce a value of 0.20
at neutral pH and 0.25 at basic pH, whereas, Cpd III gives
0.52 at neutral pH. In no case is it possible to quanti-
tate accurately Cpd III, as a residual amount of Cpd II is
always present [24].
The optical spectrum for native LPO shows a Soret band
at 412 nm (e ¼ 114 000
M
)1
Æcm
)1
), and weaker absorptions
at 501, 541, 589 and 631 nm [25]. Exposure to equimolar or
twofold excess of H
2
O
2
produces Cpd I, whose spectros-
copic features are controversial. According to reference [25],
it absorbs at 410 nm (Soret) and 562, 600, 662 nm (Table 2),
whereas, Doerge et al. [26] and Monzani et al. [27] reported
that the porphyrin-radical Cpd I is characterized by absorp-
tions at 420 and 416 nm, respectively. Such a change in k
max
with respect to the native form suggests that, in contrast with

MPO, not only the transition probability (and thus e) but
the energy of the porphyrin electronic levels is affected by
Cpd I formation. The reaction is extremely favoured, as
witnessed by its high second-order constant (1.8 · 10
7
[28];
1.2Æ10
7
M
)1
Æs
)1
[29]), and is more influenced by lipophilicity
and steric factors than by pH [28]. In the presence of peroxide
only, LPO Cpd I converts spontaneously (within 200 ms)
[29] into a relatively stable species (t
1/2
 several minutes
[30]) with spectroscopic features clearly distinct from those of
thenativeenzyme(k
Soret
¼ 430 nm). The electronic structure
of such species is still uncertain. According to several authors
[26,27,31–33], this unknown species would be a porphyrin-
radical Cpd I isomer, having its electronic vacancy localized
onto an amino acid residue instead of the heme ring. Such a
compound would in turn generate conventional Cpd II that
is characterized by absorptions at 430, 535 and 567 nm
[3,26,27,30,32,34–37]. On the basis of kinetic, thus, indirect
evidence, Monzani et al. [27]suggestthatCpdIIcan

isomerize also, giving rise to a radical species with k
Soret
at
412 nm. Exposure to a slight peroxide excess rapidly
inactivates LPO and generates Cpd III (with bands at 424,
550 and 588 nm) (Table 2). In contrast to MPO, LPO
Cpd III spectral pattern is clearly distinct from that of
Cpd II and can be easily distinguished from it. LPO is much
more sensitive to peroxide inactivation than is MPO or other
members of the animal peroxidase family [4,27].
Scheme 1. The catalytic cycle of peroxidases described as a sequence of
three consecutive reactions. Peroxidases first react with H
2
O
2
, their first
substrate, and generate a highly oxidizing intermediate, indicated as
Cpd I. In the presence of peroxide excess, the intermediate Cpd III
[a superoxide-Fe(III) adduct] can be generated. Cpd I is able to oxidize
mono-electronically a second substrate, that can be either an organic
or an inorganic molecule; it is simultaneously reduced to a second
intermediate, named Cpd II – this can still oxidize a substrate mole-
cule; it can also react with peroxide and give rise to Cpd III.
Table 1. Summary of the spectroscopic properties of MPO, Compound
I, II and III.
Reference
Compound I
(nm)
Compound II
(nm)

Compound III
(nm)
[18] 430 (e ¼ 88000)
dimer
455 (e ¼ 160000) –
[4] – – 450,
626
[1] – 628 (e ¼ 18000)
at pH 7
–
635 (e ¼ 25000)
at pH 11
[24] – 454 (452–458) 450 (449–452)
628 (622–630) 625 (622–625)
[17] – 455 451
628 626
[16] 425 (e ¼ 52000) 455 –
572 (e ¼ 9700)
625 (e ¼ 9900)
[39] – 452 –
622
4404 E. Ghibaudi and E. Laurenti (Eur. J. Biochem. 270) Ó FEBS 2003
Four unanswered questions
Reversibility of Cpd I formation and catalase activity
Catalase activity in peroxidases is strictly related with the
reversibility of Cpd I formation. Although this reaction had
been long considered strictly irreversible, several forms of
evidence against this statement are now available.
In the case of MPO, reversibility of Cpd I formation was
first stated by Wever et al. [38] and subsequently confirmed

by Marquez et al. [18] and Kettle et al. [22]. As for the
presence of pseudocatalase or catalase activity, this was
suggested by several authors [16,18,38–40] and demonstra-
ted unambiguously by Kettle and Winterbourne [22]. They
showed that MPO possess a true catalase activity, whose
extent is so important, compared to other peroxidases (e.g.
horseradish and chloroperoxidase) that one may look at
MPO as a catalase/peroxidase. A first-order rate constant of
2.2 · 10
6
s
)1
has been reported for the catalatic breakdown
of Cpd I [22]. In order to explain their experimental
findings, Kettle et al. [22] proposed a reaction scheme
(Scheme 2) that unifies the previous mechanisms proposed
by Iwamoto et al. [40] and Marquez et al. [18].
According to experimental findings, H
2
O
2
either reduces
Cpd I to native MPO by a two-electron reaction or to
Cpd II by a one-electron process. The latter reaction is
about two orders of magnitude slower than the former.
Superoxide contributes to maintain the catalase activity by
preventing Cpd II accumulation and reducing this inter-
mediate to the native form [41].
Kettle’s proposal [22] does not take into account the
possibility (suggested by Marquez et al. [18]) that superoxide

act as a reductant towards Cpd I as well, thus, generating
Cpd II. This possibility has been included in Scheme 2.
Further support for the presence of catalase activity
comes from the E°¢ values of the redox couples H
2
O
2
/O
2
and Cpd I/native MPO, the former being 0.281 V [42] and
the latter 1.16 V [19,43], from which it is evident that Cpd I
can oxidize bi-electronically H
2
O
2
fi O
2
.
MPO and LPO share so many structural and functional
features that one would expect LPO to display catalase
activity as well. Nevertheless, a final word on this item has
not yet been mentioned. In fact, LPO has been proven by
several authors to possess pseudo-catalase activity
[3,30,36,44,45]. This derives from oxidation of H
2
O
2
by
hypoiodite, which in turn is generated by the two-electron
oxidation of iodide catalysed by LPO Cpd I. So, pseudo-

catalase activity is related to the ability of certain substrates
(i.e. halides and pseudo-halides) to undergo a two-electron
oxidation, thus, preventing formation of Cpd II and
converting Cpd I directly to native LPO [3,46].
As for the catalase reaction in LPO, the following
evidence is available: Huwiler et al. [35] described oxygen
release by LPO in the presence of a slight excess of peroxide,
suggesting that LPO can exhibit catalase-like behaviour.
Kohler et al. [30] report O
2
release and peroxide con-
sumption in the stoichiometric ratio 1 : 2 (typical of catalase
activity) both during the conversion of Cpd III into the
native form and in the Cpd II fi native LPO reaction.
In order to provide a mechanistic explanation for these
experimental findings, they propose catalase activity to stem
from a reaction loop involving Cpd III, Cpd II and a ferrous
form of LPO. Although,theycannot exclude the intervention
of H
2
O
2
as electron-donor. Such a reaction scheme is cited
Table 2. Summary of the spectroscopic properties of LPO, Compound I, II and III.
Reference
Compound I
p
+•
(nm)
Compound I

aa
+•
(nm)
Compound II
Ferryl (nm)
Compound II
Fe(III) aa
+•
(nm) Compound III (nm)
[29] 410 (562, 600, 662) – 433 (537, 568) – 428 (551, 590)
[32] – 430 430 – –
[34] – – 430 – 423
[35] – – 430 – 423
[36] – – 430 (535, 565) – 423 (549, 588)
[37] – – – – 423
[30] – – 430 (535, 567) – 424 (550, 588)
[4] – – – – 420 (550, 589)
[3] – – 430 (535, 567) – 424 (550, 588)
[26] 420 430 430 – –
[31] – 430 – – –
[27] 416 430 430 412 424
Scheme 2. A reaction scheme that unifies the previous mechanisms
proposed ([18,22] and [40]). Once MPO Cpd I is formed, H
2
O
2
can
reduce Cpd I either to native MPO by a two-electron reaction or to
Cpd II by a one-eletron process. The former reaction implies catalase
activity. The latter reaction is about two orders of magnitude slower

than the former and generates superoxide. This radical can either
reduce Cpd II to the resting enzyme or Cpd I to Cpd II and generate
dioxygen [18,22].
Ó FEBS 2003 Controversies on myelo- and lactoperoxidase mechanisms (Eur. J. Biochem. 270) 4405
again in reference [3] and a similar hypothesis is made by
Jenzer et al. [36]. They hypothesize that peroxide acts as a
reductant in the Cpd I fi Cpd II reaction, thus generating
superoxide: this may in turn reduce Cpd II fi native LPO,
while O
2
is released. According to this scheme, the stoichio-
metric ratio between H
2
O
2
and O
2
would be 2 : 1, which is
typical of catalases; although it seems to us that such an
activity cannot be defined as truly catalatic, as it is
superoxide-mediated. We would rather stress the fact that
Jenzer et al. [36] observed a biphasic behaviour for peroxide
consumption and O
2
release that is characterized by a quick
initial step and a slow secondary one. This is an extremely
interesting result in the light of the experimental findings by
Kettle et al. on MPO [22] who observed a burst phase for
peroxide consumption (as above). In fact, this set the stage
for an extension to LPO of the reaction scheme adopted for

MPO. Another argument relevant to catalase activity is the
Cpd I redox potential. Unlike MPO, no E°¢ values for LPO
Cpd I have been available until now; actually, its value is a
point of considerable contention. Ohlsson et al. [28,47]
reported the redox potential of LPO to be )191 mV at 25 °C
and pH 7.0; however, this value refers to the Fe(III)/Fe(II)
reduction and does not relate with the redox change
undergone by the iron ion during the catalytic cycle. To
our knowledge, no E°¢ value for the CpdI/native LPO redox
couple has been reported until now. In spite of this fact, an
estimate of the LPO Cpd I redox potential can be made, by
considering that: (a) LPO cannot oxidize chloride whereas
MPO does and (b) LPO Cpd I oxidizes phenolic substrates
characterized by redox potentials that range from 760 to
1060 mV [27]. Consequently, an E°¢ value between 1.16 and
1.06 mV seems reasonable for LPO Cpd I-[Fe
IV
¼ O; p
+•
]; a
value of  900 mV has been suggested for Cpd I-[Fe
IV
¼ O;
aa
+•
] [27,46]. Such an estimate is consistent with the presence
of catalase activity, based on similar arguments to those
offered for MPO. In conclusion, experimental evidence for
O
2

evolution concurrent to peroxide consumption are
available for LPO; the estimated value of E°¢ for LPO Cpd I
also supports the presence of catalase activity. As for the
mechanistic details, we believe that the reaction mechanism
adopted for MPO could be extended to LPO, although this
should be unequivocally demonstrated by further studies.
Isomeric forms of Cpd I
It is well known that, in the absence of cosubstrates, MPO
and LPO Cpd I decays spontaneously to a secondary
product that is generically indicated as Cpd II [7,16].
Whether this intermediate derives from a mono-electronic
reduction of Cpd I and, thus, is the actual Cpd II, or is a
Cpd I isomer that contains the same number of oxidizing
equivalents as Cpd I is matter of discussion. Several pieces
of evidence suggest the existence of two Cpd I isomers both
in MPO and LPO.
In the case of MPO the evidence is: (a) the EPR spectrum
of a trapped amino acid radical found by Lardinois et al.
[48] in at least one catalytic intermediate of MPO and (b) the
high redox potential of this intermediate, which is able to
oxidize chloride.
The E°¢ for MPO Cpd I has been found to be 1.16 V
[43,49], a high enough value to make the oxidation of an
amino acid residue on the polypeptide chain possible by
intramolecular electron-transfer process [18]. Displacement
of an oxidizing equivalent from the porphyrin ring to an
amino acid would be expected to change the redox potential
of the protein. The two Cpd I isomers are not expected to
share the same redox properties, thus, agreeing with
experimental evidence regarding the reactivity of MPO

intermediates. The species obtained by spontaneous decay
of Cpd I, which is usually called Cpd II regardless of its
electronic structure, does not react with halides, whereas
Cpd I can oxidize them [19]. On the other hand, this fact
does not represent proof of the presence of one or two
oxidizing equivalents on the decay product of Cpd I.
The EPR spectrum of a spin-trapped radical in MPO
Cpd I has been reported recently [48]. The signal, assign-
able to an amino acid radical localized on the protein
scaffold of MPO was generated only in the presence of
H
2
O
2
. The nature of such a radical has not been
ascertained, although the authors suggest that it might
be a Tyr or a Trp. There is no unambiguous evidence
implicating this radical intermediate in the catalytic
mechanism of the enzyme and it might represent an
alternative pathway, whose biological role is still unclear
[48]. The same authors [48] suggest that the protein radical
might stem from a vestigial process related to that
responsible for the autocatalytic cross-linking of the heme
groups to the protein [50]. Alternatively, they suggest that
it could be involved in the oxidation of peroxidase
substrates at the protein surface, akin to the activities of
Cytochrome c oxidase or ligninase. Finally, one cannot
exclude that this intermediate is part of a mechanism
unrelated to the physiological role of MPO but that leads
to eventual enzyme inactivation [48].

It is important to recognize that the EPR signal of the
trapped radical in MPO clearly derives from the super-
position of two different components that could correspond
to two adducts of 3,5-dibromo-4-nitroso-benzenesulfonic
acid with radicals having different mobility or to two forms
of the protein present in solution simultaneously [48].
In the case of LPO, both spectral [51,52, E. Ghibaudi and
E. Laurenti, unpublished observation] and kinetic data
[26,27,31–33,53,54] suggest that a protein radical forms
during the enzyme turnover. Lardinois et al. [51] used mass
spectrometry of tryptic digests of LPO and EPR spectros-
copy of spin-trapped species to demonstrate that there are
two radical species, each of which might be a Tyr. One
residue has been identified as the Tyr289, which is involved
in LPO dimerization and has no catalytic role. Tyr289 could
also be involved in H
2
O
2
-mediated cross-linking between
LPO and myoglobin [55]. The precise identity of the second
residue has not been determined but may play a functional
role. In contrast, Goff et al. [52] have implicated two Phe
radicals in the catalytic turnover of the enzyme. A multi-
frequency (9 and 285 GHz band) EPR investigation at
liquid helium temperature, performed by the present
authors, has shown that the reaction of LPO in a slight
excess of peroxide at 0 °C generates a protein radical, whose
identity has yet to be defined (E. Ghibaudi and E. Laurenti,
unpublished observation).

The indirect evidence available on this problem comes
essentially from two types of experiments: (a) titrations
of the decay product of LPO Cpd I with ferrocyanide
(a monoelectronic donor) and (b) kinetic studies on the role
4406 E. Ghibaudi and E. Laurenti (Eur. J. Biochem. 270) Ó FEBS 2003
of LPO in the frame of the thyroid hormone biosynthetic
pathway.
Courtin et al. [32,33] demonstrate, by titration with
ferrocyanide, that the decay product of Cpd I-[Fe
IV
¼O;
p
+•
], characterized by k
Soret
¼ 430 nm, is still two oxidizing
equivalents above the resting state of the enzyme and, thus,
is isoelectronic with Cpd I-[Fe
IV
¼O; p
+•
]. By analogy to
Cyt c peroxidase, they suggest that one equivalent is
localized on the protein scaffold, designated Cpd I-
[Fe
IV
¼O; aa
+•
]. At pH 6.1 and 7.3, reduction of Cpd I-
[Fe

IV
¼O; aa
+•
] by ferrocyanide requires 2 mol of oxidant
per mol of enzyme and is a biphasic process.
This might indicate that such a reaction occurs in two
mono-electronic steps, yielding first the genuine Cpd II,
which is only one oxidizing equivalent above the resting
state, and subsequently native LPO.
The second source of indirect evidence comes from
studies using LPO in place of thyroid peroxidase (TPO) in
the biogenesis of thyroid hormone. This synthetic process
occurs in two steps, tyrosine iodination followed by
coupling of iodotyrosines to produce the hormone.
Comparison of the kinetics of these two steps
[31,32,54,56] catalysed by LPO or TPO shows that the
reaction intermediate containing the porphyrin cationic
radical Cpd I-[Fe
IV
¼O; p
+•
] catalyses both steps. On the
contrary, its decay product is active only in the second
step. This indicates that the two species possess different
redox potentials, that of Cpd I-[Fe
IV
¼O; p
+•
]being
higher than its decay product. Transfer of an oxidizing

equivalent from the porphyrin ring to an amino acid
residue is likely to reduce the redox potential of the
protein [53] and, on this basis, the authors [31,32,54,56]
claim that the species responsible for iodotyrosine coup-
ling is the isomer Cpd I-[Fe
IV
¼O; aa
+•
]. A comparison
between HRP, CcP and LPO [56] supports this statement
as HRP, which can generate only Cpd I-[Fe
IV
¼O; p
+•
],
can iodinate tyrosine, whereas CcP, which forms
Cpd I-[Fe
IV
¼O; aa
+•
] only, cannot. The same argument
is used to explain the reactivity of LPO towards sulphur-
containing compounds [53], suggesting the presence of a
reaction intermediate (Cpd I-[Fe
IV
¼O; aa
+•
]) that would
be a weaker oxidant than is traditional Cpd I. In our
opinion, these arguments are valid but still do not answer

the question in a satisfactory fashion, as they do not
exclude the possibility that the coupling of Tyr residues is
mediated by Cpd II, whose oxidizing power is lower than
that of Cpd I.
Also unclear is the physiological role of the amino acid
radical that forms only in the presence of an ÔactiveÕ form of
LPO, as shown by Lardinois et al. [51]. It cannot be
generated in the presence of inhibitors and has been
demonstrated only when LPO was incubated with peroxide
in the absence of other substrates, suggesting that it may not
form under physiological conditions. Monzani et al. [27]
suggest that its formation may represent a way for the
enzyme to avoid undesired reactions and thereby reserve the
oxidizing equivalents for the oxidation of thiocyanate.
Lardinois et al. [51] posit that it is part of a Ôdead-endÕ
metabolic pathway, aiming to protect LPO from the
disrupting effect of H
2
O
2
or be part of a vestigial enzymatic
pathway, now abandoned. O’Brien suggests its role is in the
oxidation of physiologically relevant substrates, drugs and
in the etiology of some pathologies [57]. In the light of the
work by De Pillis et al. [50,58], we would not exclude that
the radical plays a role in the autocatalytic binding of heme
to the apoprotein, a post-translational modification that
is typical of mammalian peroxidases.
Cpd I fi Cpd II conversion
The remarks made in the previous section set the stage for

another question: why is Cpd I decay to Cpd II in the
presence of H
2
O
2
spontaneous, as such a reaction would
require a substrate able to reduce Cpd I?
First, we should emphasize that in this paper we assign
the term ÔCpd IIÕ to the reaction intermediate that actually
lacks an oxidizing equivalent with respect to Cpd I and
must be generated through a mono-electronic reduction.
MPO behaviour has already been described above.
Marquez et al. [18] and Kettle et al. [22] showed that, in
the absence of exogenous substrates, peroxide acts as a
mono-electronic reductant and converts MPO Cpd I fi
Cpd II; as a result of peroxide oxidation, superoxide ions
are produced. This species might be found either free in
solution or bound to the enzyme, thus, giving rise to Cpd III.
Superoxide (or Cpd III) would enter the cycle again, by
acting in turn as a reductant (Scheme 2). This model is
also supported by the fact that the kinetics of the Cpd I fi
Cpd II reaction is either monophasic or biphasic, at low or
high peroxide concentration, respectively. Consistent with
this hypothesis, experimental evidence demonstrates that
superoxide is generated during this chemical process in a
pH-dependent fashion [18]. The kinetic constant for
Cpd I fi Cpd II conversion at pH 7.0 has been reported
to be 8.2 · 10
4
M

)1
Æs
)1
, which is three to fourfold the k of the
bielectronic reduction of Cpd I fi MPO by chloride [19].
Nonetheless, we would like to emphasize that the above-
mentioned hypothesis does not exclude the possibility that
Cpd I-[Fe
IV
¼O; p
+•
] decays first to Cpd I-[Fe
IV
¼O; aa
+•
],
whichinturnisreducedtoCpdII.
LPO behaviour is less straightforward. Cpd I spontane-
ously converts to a relatively stable species [4,30,35,36] that
absorbs at 430 nm but the reducing agents responsible for
this process and the subsequent reaction step have yet to be
defined. In fact, Cpd II slowly converts back to native LPO
[30] through a mono-electronic reaction. A comparison of
the experimental behaviours of MPO and LPO in the
presence of peroxide might help to solve some of these
issues.
The mono-electronic oxidation of H
2
O
2

generates super-
oxide that can co-ordinate to the Fe(III) ion and yield
Cpd III. LPO is much more sensitive than MPO to
inactivation by peroxide [4,27] and produces Cpd III even
in the presence of slight H
2
O
2
excess, although its inacti-
vation is partially reversible [3,30,34,35,37]. By analogy to
MPO, we suggest that the superoxide ion or, more
probably, its adduct with the enzyme, that is Cpd III,
reduces both Cpd I, regardless of its electronic structure,
and Cpd II according to Scheme 3. Dioxygen release from
the conversion of Cpd II fi LPO [30,36], presumably by
the oxidation of superoxide anion, provides experimental
data to support this model. In the first step, Cpd II would be
produced, whereas native LPO would be generated in the
second (Schemes 2 and 3).
Ó FEBS 2003 Controversies on myelo- and lactoperoxidase mechanisms (Eur. J. Biochem. 270) 4407
Two groups have independently suggested that LPO
Cpd II exists in two isoelectronic forms, Cpd II-[Fe
IV
¼O]
and Cpd II-[Fe
III
;aa
+•
], whose stability would be strongly
pH-dependent [27,32,33]. The individual isomers could be

distinguished by their optical spectrum as they contained
iron in two different oxidation states. At acidic pH, Cpd I
reduction with an equimolar amount of ferrocyanide
yielded Cpd II-[Fe
III
;aa
+•
] whose optical spectrum is
identical to that of the native enzyme [31,33]. At neutral
or basic pH, the amino acid site would be preferentially
reduced, giving rise to Cpd II-[Fe
IV
¼O], which lacks
radicals and absorbs at 430 nm. This species would
reconvert into Cpd II-[Fe
III
;aa
+•
], by changing pH,
whereas the reverse reaction would not be possible.
Addition of a second ferrocyanide molecule reconverts the
enzyme to the native form. Such a mechanism, if confirmed,
is analogous to that proposed for Cyt c peroxidase that is
thought to generate two different intermediates, each being
one oxidizing equivalent above the resting enzyme [32,59].
Nevertheless, we believe that the evidence for two Cpd II
isomers is not compelling and merits additional investiga-
tion as the correlation between iron redox state and optical
spectrum of the protein is not straightforward. For example,
the conversion of MPO from the resting state, containing

Fe(III), to Cpd I, containing Fe(IV), affects e without
shifting the Soret band. Most kinetic data offered to support
this model comes from studies using different experimental
conditions [27]. All experimental evidence of radical LPO
intermediates [32,33] was obtained exclusively in the pres-
ence of H
2
O
2
alone, a non-physiological condition. We
believe that the absence of direct evidence of the radical in
the presence of cosubstrates limits the validity of extrapo-
lating the model proposed by Courtin et al. [32,33] to other
experimental conditions.
Optical spectra of Cpd I-[Fe
IV
=O; aa
+•
] and Cpd II
Once the existence of Cpd I-[Fe
I
¼O; aa
+•
] is accepted, its
optical features remain to be defined. In the light of the
experimental data available on MPO, one can think of two
possibilities (Scheme 4). First, the unstable intermediate
Cpd I-[Fe
IV
¼O; p

+•
] decays to the relatively stable Cpd II,
which is characterized by the bands at k
max
455 and 628 nm.
Were this hypothesis correct, the protein radical observed by
Lardinois et al. [48] would not participate in the traditional
catalytic cycle but rather belong to an alternative or to
Ôdead endÕ pathway. Alternatively Cpd I-[Fe
IV
¼O; p
+•
]
(t
1/2
¼ 100 ms) decays first to Cpd I-[Fe
IV
¼O; aa
+•
], whose
t
1/2
is still unknown, and subsequently to Cpd II (t
1/2
 10 min) [4]. In the former case, the optical features of
the intermediates are clearly assigned: Cpd I-[Fe
IV
¼O; p
+•
]

absorbs at 430 nm and its decay to Cpd II is followed by a
shift of the Soret band towards 455 nm. Such a band is stable
for several minutes, a feature consistent with Cpd II’s t
1/2
.In
the latter case, the peak absorption at 455 nm has yet to be
assigned: it might be associated with either Cpd I-[Fe
IV
¼O;
aa
+•
]orCpdII[Fe
IV
¼O] or with both of them.
It seems reasonable to speculate that the optical spectra of
both intermediates Cpd I-[Fe
IV
¼O; aa
+•
] and Cpd II-
[Fe
IV
¼O] are extremely similar, if not identical. In fact,
the two species differ only in the presence of a protein
radical aa
+•
, a species not likely to dramatically modify the
spectrum relative to that of the holoprotein. Neither the Fe
redox state nor the electronic configuration of the porphyrin
ring are expected to change upon conversion of one

intermediate into the other. It is reasonable to assume that
the transient species Cpd I-[Fe
IV
¼O; aa
+•
] shows an
absorption at 455 nm and that its decay to Cpd II leaves
the spectrum unaltered.
Scheme 3. LPO reacts with peroxide to generate a porphyrin-radical intermediate that can isomerize spontaneously. By analogy to MPO, we suggest
that the superoxide ion or, more likely, its adduct with the enzyme (Cpd III) reduces Cpd I and Cpd II, by losing an oxidising equivalent and gives
rise to dioxygen, according to evidence available in the literature [30,36]. Some authors suggest that Cpd II isomerize as well [27].
Scheme 4. Two hypothesis for explaining the
optical changes observed in MPO solutions
upon addition of peroxide. Does the spectrum
observed upon decay of the porphyrin radical
Cpd I (430, 572 and 625 nm bands) corres-
pond to the real Cpd II? In such a case, the
optical features of Cpd I isomer have to be
defined. The second hypothesis assigns the 455
and 625 nm bands to this isomer. In such a
case, the spectral properties of Cpd II remains
undefined.
4408 E. Ghibaudi and E. Laurenti (Eur. J. Biochem. 270) Ó FEBS 2003
The observed effect of pH on the band at 628 nm of
Cpd II [1,24] might reflect protonation of an amino acid
found in the heme crevice and involve Fe
IV
¼O. As this
group should be present in Cpd I as well, one would expect
similar pH dependence for the spectrum of such inter-

mediate. Unfortunately, no data are currently available,
partly due to the short t
1/2
of Cpd I.
As for LPO, the decay product of Cpd I absorbs at
430 nm [probably due to the presence of Fe(IV)] and is stable
for several minutes [26,30,34–36] and is not associated with
an EPR signal assignable to a radical species. On the basis of
these facts, the spectrum is unlikely to correspond to Cpd I-
[Fe
IV
¼O; aa
+•
], which is highly unstable at room tempera-
ture and can only be detected at low temperature after rapid
freezing of the sample (E. Ghibaudi and E. Laurenti,
unpublished observation). The same arguments offered
earlier for MPO pertain to LPO. Cpd I-[Fe
IV
¼O; aa
+•
]
and Cpd II-[Fe
IV
¼O] should form sequentially and their
spectra are likely identical as these two intermediates differ
only for the presence of the radical aa
+•
in the former.
Based on the evidence presented above, it should be clear

that the catalytic cycles of both LPO and MPO have
unusual features with respect to the classical peroxidase
mechanism and interactions with H
2
O
2
. In the light of the
experimental data presented above, we offer the following
conclusions: (a) both enzymes are claimed to possess
catalase activity [16,18,19,22,26]. In the case of MPO, this
wasproventobeassociatedwithCpdIreconversiontothe
resting state [22]. As for the catalase function in LPO, it is
supported by stoichiometric oxygen release measured
during catalytic cycling in the absence of cosubstrates and
by structural resemblance to MPO. As catalase activity
recycles Cpd I into native LPO, one should expect accu-
mulation of the resting enzyme to take place in the exclusive
presence of peroxide, unless the extent of catalase vs.
peroxidase activity is low. This seems to be the case for
LPO, where the competition of catalase- vs. peroxidase-
activity is weaker than in MPO and shifts all catalytic
equilibria towards accumulation of Cpd II rather then
native LPO. This should also explain the higher sensitivity
of LPO towards peroxide inactivation with respect to MPO.
Unanswered is the question: which factors are discrimi-
nant in determining a peroxidase to possess catalase
activity? Although the individual enzyme-peroxide adducts
are characterized by different redox potentials, this fact
alone does not explain the wide catalase activity-spectrum
among members of the animal peroxidase protein family.

Structural factors (such as the presence of a sulphonium
linkage in MPO, hydrophobicity of the substrate access
channel, distorsion of the heme plane and size of the
catalytic pocket) must be crucial in playing on kinetic
equilibria of these enzymes and determining whether
the peroxidase or catalase function has to prevail. The role
of water ÔsittingÕ in the active site has also to be taken
into account, as suggested by a recent work by Peter
Jones [60].
(b) Both LPO and MPO have been shown to generate an
aminoacid radical as decay product of Cpd I-[Fe
IV
¼O;
p
+•
], whose identity has not yet been ascertained. Its
contribution to the catalytic cycle of both enzymes is not
clear. Ferrocyanide titration on LPO intermediates showed
that it is isoelectronic with Cpd I-[Fe
IV
¼O; p
+•
] and can be
reduced to a secondary species that has lost an oxidizing
equivalent (Cpd II). The fact that such a product does not
react with halides indicates that its redox potential is lower
than that of Cpd I, although it cannot be correlated with the
number of oxidizing equivalents localized on it. Of note, all
detections of the amino acid radical were made in the
absence of cosubstrates, far from the conditions in which the

enzyme displays its physiological catalytic activity. These
arguments suggest that the radical may not have a
functional role.
(c) The model proposed for MPO by Marquez et al. [18]
and Kettle et al. [22] concerning the spontaneous reduction of
Cpd I-[Fe
IV
¼O; p
+•
] in the presence of peroxide stresses the
role of superoxide in the catalytic cycle of peroxidases and
may be extended to LPO. Accordingly, the one electron
oxidation of peroxide by Cpd I yields superoxide. This
superoxide anion could either be free in solution or, more
likely, bound to the enzyme and give rise to Cpd III, which is
responsible for a reversible inactivation mechanism. Cpd III
might in turn participate to the reduction of Cpd I fi Cpd II
and to the subsequent step Cpd II fi native enzyme,
generating molecular oxygen, consistent with the experi-
mental data reported by Jenzer et al. [36] (Scheme 5). The
involvement of superoxide in the reduction of Cpd I, through
a ÔfeedbackÕ cycle, provides a mechanism for the mono-
phasic/biphasic kinetics observed by Marquez et al. [18].
Scheme 5. The catalytic cycle of LPO and
MPO. In this scheme, the role of H
2
O
2
as an
oxidant is shown in steps 1 and 2; catalase

activity is displayed in step 1;CpdIisomeri-
zation is taken into account (step 2); the
reducing effect of superoxide ions, in the form
of Cpd III, is shown in steps 5, 6 and 7 and
goes along with dioxygen release. The optical
absorption peaks of each intermediate are
shown on top of the scheme for both enzymes.
Ó FEBS 2003 Controversies on myelo- and lactoperoxidase mechanisms (Eur. J. Biochem. 270) 4409
(d) The optical absorption data relative to the species
obtained by spontaneous decay of Cpd I can be explained
by positing that Cpd I-[Fe
IV
¼O; aa
+•
]andCpd IIformsin
turn and share the same UV-Vis spectroscopic features.
The radical intermediate is short-lived and can be
detected by EPR only after stabilization with a spin-trap
at room temperature or after rapid freezing at liquid helium
temperature. The radical, as a transient species, quickly
evolves towards a secondary species that corresponds to
Cpd II and remains for several minutes at RT.
Conclusions
A summary of LPO and MPO catalytic features is found in
Scheme 5. This model assumes that the enzymes share the
same behaviour: a reasonable assumption, in the light of the
common phylogenetic origin of LPO and MPO and of their
similar physiological role. It also provides a partial answer
to the four questions addressed in this paper.
According to Scheme 5, the native enzyme, once incuba-

tedwithH
2
O
2
, gives rise to Cpd I-[Fe
IV
¼O; p
+•
], which is
able to oxidize peroxide to molecular oxygen, thereby
exhibiting catalase activity (step 1). Cpd I-[Fe
IV
¼O; p
+•
]
may also isomerize to Cpd I-[Fe
IV
¼O; aa
+•
](step2).Inthe
absence of other substrates, the conversion of
Cpd I fi Cpd II is still mediated by peroxide, which acts
as a mono-electronic reductant and generates superoxide
(step 3). This ion is able to co-ordinate to the Fe(III) of
native enzyme and to form Cpd III (step 4), which can still
act as a reductant. Cpd III may either display a feedback
action, supporting the Cpd I fi Cpd II conversion (steps
5 and 6), or induce Cpd II reduction to the resting form.
Both reaction steps are mono-electronic and are associated
with release of molecular oxygen (step 7). This hypothesis

would explain both the reversible nature of Cpd III-
mediated inactivation and the evolution of small quantities
of dioxygen, reported by several authors.
It’s worth noting that accumulation of a specific
intermediate is never observed in LPO or MPO: this
suggests that k values for each reaction step are comparable,
thus, preventing a single species to convert quantitatively
into the following one. As a consequence, several chemical
intermediates coexist and sometimes interact mutually, thus,
justifying the complexity of the scheme.
Despite their common mechanism, significantly different
catalytic features are found in LPO and MPO. They dealwith
the the major importance of catalase activity in MPO with
respect to LPO, the different sensitivity of the enzymes to
Cpd III inactivation and the redox potential of the chemical
intermediates. This means that a distinct balance is estab-
lished between catalytic steps of MPO and LPO cycles. This
is due to differences in the kinetic constant values and likely
reflects distinct structural features of the two heme pockets.
A survey of the experimental data available in the
literature allowed us to highlight some unresolved questions
concerning the catalytic behaviour of LPO and MPO. A
critical examination of these data resulted in a model that
offer a solution to some of these opened questions. It
re-evaluates the role of superoxide ion as a reductant in the
cycle; it includes Cpd I isomers in it; it highlights the presence
of mutual chemical interactions between enzyme intermedi-
ates and provides and explanation for catalase activity.
Other questions, such as the localization of the amino
acid radical in Cpd I, its role and spectroscopical properties

need further investigations to be solved.
Acknowledgements
The authors thank Prof. Ivano Bertini, Prof. Bill Nauseef and Prof.
R.P. Ferrari for the very helpful comments and remarks and for
critically reading the paper. This work was supported by Italian
ÔMinistero per lÕUniversita
`
e la Ricerca scientifica e Tecnologica’
(MURST) [PRIN 2000 – prot. MM03185591–005].
References
1. Oertling, W.A., Hoogland, H., Babcock, G.T. & Wever, R. (1988)
Identification and properties of an oxoferryl structure in Myelo-
peroxidase Compound II. Biochemistry 27, 5395–5400.
2. Chance, B., Powers, L., Ching, Y., Poulos, T., Schonbaum, G.R.
& Yamazaki, I. (1984) X-ray absorption studies of intermediates
in peroxidase activity. Arch. Biochem. Biophys. 235, 596–611.
3. Kohler, H. & Jenzer, H. (1989) Interaction of lactoperoxidase with
hydrogen peroxide. Free Rad. Biol. Med. 6, 323–339.
4. Metodiewa, D. & Dunford, B. (1989) The reactions of horseradish
peroxidase, lactoperoxidase and myeloperoxidase with enzi-
matically generated superoxide. Arch. Biochem. Biophys. 272, 245–
253.
5. Hu, S. & Kincaid, J.R. (1991) Resonance Raman structural
characterization and the mechanism of formation of lactoper-
oxidase Compound III. J. Am. Chem. Soc. 113, 7189–7194.
6. Kettle, A.J., Sangster, D.F., Gebicki, J.M. & Winterbourne, C.C.
(1988) A pulse radiolysis investigation of the reactions of myelo-
peroxidase with superoxide and hydrogen peroxide. Biochim.
Biophys. Acta 956, 58–62.
7. Dunford, H.B. (1999) Heme Peroxidases, Wiley-VCH Publishers,

New York.
8. Fenna, R.E. (2001) Handbook of Metalloproteins (Messersch-
midt,A.,Huber,R.,Poulos,T.,&Wieghardt,K.,eds),Vol.1p.
219. Wiley-VCH Publishers, New York.
9. Stubbe, J. & Van der Donk, W.A. (1998) Protein radicals in
enzyme catalysis. Chem. Rev. 98, 705–762.
10. Ivancich, A., Mazza, G. & Desbois, A. (2001) Comparative EPR
study of radical intermediates in turnip peroxidase isoenzymes.
Biochemistry 40, 6860–6866.
11. Miller, V.P., Goodin, D.B., Friedman, A.E., Hartmann, C. &
Ortiz de Montellano, P.R. (1995) Horseradish peroxidase Phe172/
Tyr mutant. J. Biol. Chem. 270, 18413–18419.
12. Rutter, R. & Haber, L.P. (1982) The detection of two electron
paramagnetic resonance radical signals associated with chloro-
peroxidase compound I. J. Biol. Chem. 257, 7958–7961.
13. Fishel, L.A., Farnum, M.F., Mauro, J.M., Miller, M.A. & Kraut,
J. (1991) Compound I radical in site directed mutants of CCP as
probed by EPR and ENDOR. Biochemistry 30, 1986–96.
14.Erman,J.E.,Vitello,L.B.,Mauro,J.M.&Kraut,J.(1989)
Detection of an oxoferryl porphyrin cation radical intermediate in
the reaction between H
2
O
2
and a mutant yeast cytochrome c
peroxidase. Biochemistry 28, 7992–7995.
15. De Gioia, L., Ghibaudi, E.M., Laurenti, E., Salmona, M. &
Ferrari, R.P. (1996) A theoretical three-dimensional model for
LPO and eosinophil peroxidase built on the scaffold of the MPO
X-ray structure. J. Biol. Inorg. Chem. 1, 476–485.

16. Harrison, J.E., Araiso, T., Palcic, M. & Dunford, B. (1980)
Compound I of MPO. Biochem. Biophys. Res. Comm. 94, 34–40.
17. Svensson, B.E., Domeij, K., Lindvall, S. & Rydell, G. (1987)
Peroxidase and peroxidase-oxidase activities of isolated human
myeloperoxidase. Biochem. J. 242, 673–680.
4410 E. Ghibaudi and E. Laurenti (Eur. J. Biochem. 270) Ó FEBS 2003
18. Marquez, L.A., Huang, J.T. & Dunford, H.B. (1994) Spectral and
kinetic studies on the formation of MPO Compound I and II: roles
of hydrogen peroxide and superoxide. Biochemistry 33, 1447–
1454.
19. Furtmuller, P.G., Burner, U. & Obinger, C. (1998) Reaction of
myeloperoxidase Compound I with chloride, bromide, iodide and
thiocyanate. Biochemistry 27, 17923–17930.
20. Odajima, T. & Yamazaki, I. (1972) Myeloperoxidase of the leu-
kocyte of normal blood. V. The spectral conversion of myelo-
peroxidase to a cytochrome oxidase like derivative. Biochim.
Biophys. Acta 284, 368–374.
21. Bakkenist, A.R.J., Wever, R., Vulsma, T., Plat, H. & Van Gelder,
B.F. (1978) Isolation procedure and some properties of myelo-
peroxidase from human leucocytes. Biochim. Biophys. Acta 524,
45–54.
22. Kettle, A.J. & Winterbourn, C.C. (2001) A kinetic analysis of the
catalase activity of myeloperoxidase. Biochemistry 40, 10204–
10212.
23. Floris, R. & Wever, R. (1992) Reaction of myeloperoxidase with
its product HOCl. Eur. J. Biochem. 207, 697–702.
24. Hoogland, H., van Kuilenburg, A., van Riel, C., Muijsers, A.O. &
Wever, R. (1987) Spectral properties of myeloperoxidase Com-
pounds II and III. Biochim. Biophys. Acta 916, 76–82.
25. Ohtaki, S., Nakagawa, H., Nakamura, M. & Yamazaki, I. (1982)

Reaction of purified hog thyroid peroxidase with H
2
O
2
,tyrosine
and methylmercaptoimidazole in comparison with bovine lacto-
peroxidase. J. Biol. Chem. 257, 761–766.
26. Doerge, D.R., Taurog, A. & Dorris, M. (1994) Evidence for a
radical mechanism in peroxidase-catalysed coupling: single turn-
over experiments with HRP. Arch. Biochem. Biophys. 315, 90–99.
27. Monzani, E., Gatti, A.L., Profumo, A., Casella, L. & Gullotti, M.
(1997) Oxidation of phenolic compounds by lactoperoxidase.
Evidence for the presence of a low-potential compound II during
catalytic turnover. Biochemistry 36, 1918–1926.
28. Ohlsson, P.I., Paul, K.G. & Wold, S. (1984) The formation of
compound I of lactoperoxidase and horseradish peroxidase. A
comparison. Acta Chem. Scand. B38, 853–859.
29. Kimura, S. & Yamazaki, I. (1979) Comparisons between hog
intestinal peroxidase and bovine LPO-compound I formation and
inhibition by benzohydroxamic acid. Arch. Biochem. Biophys. 198,
580–588.
30. Kohler, H., Taurog, A. & Dunford, H.B. (1988) Spectral studies
with lactoperoxidase and thyroid peroxidase: Interconversions
between native enzyme, compound II, and compound III. Arch.
Biochim. Biophys. 264, 438–449.
31. Taurog, A., Dorris, M.L. & Doerge, D.R. (1996) Mechanism of
simultaneous iodination and coupling catalysed by thyroid per-
oxidase. Arch. Biochem. Biophys. 330, 24–32.
32. Courtin, F., Deme, D., Virion, A., Michot, J.L., Pommier, J. &
Nunez, J. (1982) The role of LPO-H

2
O
2
compounds in the cata-
lysis of thyroglobulin iodination and thyroid hormone synthesis.
Eur. J. Biochem. 124, 603–609.
33. Courtin,F.,Michot,J.L.,Virion,A.,Pommier,J.&Deme,D.
(1984) Reduction of LPO-H
2
O
2
compounds by ferrocyanide:
indirect evidence of an apoprotein site for one of the two oxidising
equivalents. Biochem. Biophys. Res. Comm. 121, 463–470.
34. Jenzer, H. & Kohler, H. (1986) The role of superoxide radicals in
LPO-catalysed H
2
O
2
-metabolism and in irreversible enzyme
inactivation. Biochem. Biophys. Res. Comm. 139, 327–332.
35. Huwiler, M., Jenzer, H. & Kohler, H. (1986) The role of com-
pound III in reversible and irreversible inactivation of LPO. Eur.
J. Biochem. 158, 609–614.
36. Jenzer, H., Jones, W. & Kohler, H. (1986) On the molecular
mechanism of LPO-catalysed H
2
O
2
metabolism and irreversible

enzyme inactivation. J. Biol. Chem. 261, 15550–15556.
37. Jenzer,H.,Kohler,H.&Broger,C.(1987)Theroleofhydroxyl
radicals in irreversible inactivation of lactoperoxidase by excess
H
2
O
2
. Arch. Biochem. Biophys. 258, 381–390.
38. Bolscher, B.G.J.M. & Wever, R. (1984) A kinetic study of the
reaction between human myeloperoxidase, hydroperoxidase and
cyanide. Biochim. Biophys. Acta 788, 1–10.
39. Agner, K. (1963) Studies on myeloperoxidase activity: spectro-
photometry of the MPO- H
2
O
2
compound. Acta Chem. Scand. 17,
S332–S338.
40. Iwamoto, H., Kobayashi, T., Hasegawa, E. & Morita, Y. (1987)
Reaction of human myeloperoxidase with hydrogen peroxide and
its true catalase activity. J. Biochem. 101, 1407–1412.
41. Kettle, A.J. & Winterbourne, C.C. (1988) Superoxide modulates
the activity of myeloperoxidase and optimizes the production of
hypochlorous acid. Biochem. J. 252, 529–536.
42. Sawyer, D.T. (1988) Oxygen Complexes and Oxygen Activation by
Transition Metals. (Martell, A.E. & Sawyer, D.T., eds), p. 131.
Plenum Publishing Co, New York.
43. Arnhold, J., Furtmuller, P.G., Regelsberger, G. & Obinger, C.
(2001) Redox properties of the couple compound I/native enzyme
of myeloperoxidase and eosinophil peroxidase. Eur. J. Biochem.

268, 5142–5148.
44. Huwiler, M. & Kohler, H. (1984) Pseudo-catalytic degradation of
hydrogen peroxide in the lactoperoxidase/ H
2
O
2
/iodide system.
Eur. J. Biochem. 141, 69–74.
45. Magnusson, P.R., Taurog, A. & Dorris, M.L. (1984) Mechanism
of thyroid peroxidase- and lactoperoxidase-catalysed reactions
involving iodide. J. Biol. Chem. 259, 13783–13790.
46. Furtmuller, P.G., Jantschko, W., Regelsberger, G., Jakopitsch, C.,
Arnhold, J. & Obinger, C. (2002) Reaction of lactoperoxidase
compound I with halides and thiocyanate. Biochemistry 41,
11895–11900.
47. Ohlsson, P.I. & Paul, K.G. (1983) The reduction potential of
lactoperoxidase. Acta Chem. Scand. B37, 917–921.
48. Lardinois, O.M. & Ortiz de Montellano, P. (2000) EPR spin-
trapping of a myeloperoxidase protein radical. Biochem. Biophys.
Res. Comm. 270, 199–202.
49.Furtmuller,P.G.,Arnhold,J.,Jantschko,W.,Pichler,H.&
Obinger, C. (2003) Redox properties of the couples compound I/
compound II and compound II/native enzyme of human
myeloperoxidase. Biochem. Biophys. Res. Commun. 301, 551–557.
50. De Pillis, G.D., Ozaki, S., Kuo, J.M., Maltby, D.A. & Ortiz de
Montellano, P.R. (1997) Autocatalytic processing of heme by
lactoperoxidase produces the native protein bound prosthetic
group. J. Biol. Chem. 272, 8857–8860.
51. Lardinois, O.M., Medzihradszky, K.F. & Ortiz de Montellano,
P.R. (1999) Spin-trapping and protein cross-linking of the lacto-

peroxidase protein radical. J. Biol. Chem. 274, 35441–35448.
52. Goff, H.M. & Chen, K. (1999) Protein radicals and formation and
reactivity of the lactoperoxidase heme prosthetic group. J. Inorg
Biochem. 74,143.
53. Doerge, R.D. (1986) Oxigenation of organosulphur compounds
by peroxidases: evidence of an electron transfer mechanism for
lactoperoxidase. Arch. Biochem. Biophys. 244, 678–685.
54. Taurog,A.,Dorris,M.&Doerge,D.R.(1994)Evidencefora
radical mechanism in peroxidase-catalysed coupling steady-state
experiments with various peroxidases. Arch. Biochem. Biophys.
315, 82–89.
55. Lardinois, O. & Ortiz de Montellano, P.R. (2001) H
2
O
2
-mediated
cross-linking between lactoperoxidase and myoglobin. J. Biol.
Chem. 276, 23186–23191.
56. Deme, D., Virion, A., Michot, J.L. & Pommier, J. (1985) Thyroid
Hormone Synthesis and Thyroglobulin Iodination related to the
peroxidase localization of oxidizing equivalents. Studies with Cyt c
eHRP.Arch. Biochem. Biophys. 236, 559–566.
Ó FEBS 2003 Controversies on myelo- and lactoperoxidase mechanisms (Eur. J. Biochem. 270) 4411
57. O’Brien, P.J. (2000) Peroxidases. Chem. Biol. Interact. 129,
113–139.
58. Colas, C., Kuo, J.M. & Ortiz de Montellano, P.R. (2002) Asp-225
and Glu-375 in autocatalytic attachment of the prosthetic heme
group of lactoperoxidase. J. Biol. Chem. 277, 7191–7200.
59. Coulson, A.F.W., Erman, J.E. & Yonetani, T. (1971) Studies on
cytochrome c peroxidase. J. Biol. Chem. 246, 917–924.

60. Jones, P. (2001) Roles of water in heme peroxidase and catalase
mechanisms. J. Biol. Chem. 276, 13791–13796.
4412 E. Ghibaudi and E. Laurenti (Eur. J. Biochem. 270) Ó FEBS 2003