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

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
+•
),

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

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

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)

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

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

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

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

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).

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

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

)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

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

+•
] 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
+•
]

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-

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.

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

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

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].
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