Archives of Biochemistry and Biophysics 525 (2012) 121–130

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Archives of Biochemistry and Biophysics

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Review

The reaction mechanisms of heme catalases: An atomistic view by ab initio

molecular dynamics
Mercedes Alfonso-Prieto a, Pietro Vidossich b, Carme Rovira c,d,⇑
a
Institute for Computational Molecular Science, Temple University, 1900 N. 12th Street, Philadelphia, PA 19122, USA
b
Unitat de Química Física, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
c
Laboratori de Simulació Computacional i Modelització (CoSMoLab), Institut de Química Teòrica i Computacional (IQTCUB), Parc Científic de Barcelona, Baldiri Reixac 4,
08028 Barcelona, Spain
d
Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08018 Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Catalases are ubiquitous enzymes that prevent cell oxidative damage by degrading hydrogen peroxide to
Available online 10 April 2012 water and oxygen (2H2O2 ? 2H2O + O2) with high efficiency. The enzyme is first oxidized to a high-valent
iron intermediate, known as Compound I (Cpd I, Por+–FeIV=O) which, at difference from other hydroper-
Keywords: oxidases, is reduced back to the resting state by further reacting with H2O2. The normal catalase activity
Ab initio molecular dynamics is reduced if Cpd I is consumed in a competing side reaction, forming a species named Cpd I⁄. In recent
Density Functional Theory years, Density Functional Theory (DFT) methods have unraveled the electronic configuration of these
Hydroperoxidases
high-valent iron species, helping to assign the intermediates trapped in the crystal structures of oxidized
Catalases
Peroxidases
catalases. It has been demonstrated that the a priori assumption that the H+/H type of mechanism for
Enzyme catalysis Cpd I reduction leads to the generation of singlet oxygen is not justified. Moreover, it has been shown
by ab initio metadynamics simulations that two pathways are operative for Cpd I reduction: a His-med-
iated mechanism (described as H/H+ + e) in which the distal His acts as an acid–base catalyst and a
direct mechanism (described as H/H) in which the distal His does not play a direct role. Independently
of the mechanism, the reaction proceeds by two one-electron transfers rather than one two-electron
transfer, as previously assumed. Electron transfer to Cpd I, regardless of whether the electron is exoge-
nous or endogenous, facilitates protonation of the oxoferryl group, to the point that formation of Cpd
I⁄ may be controlled by the easiness of protonation of reduced Cpd I.
Ó 2012 Elsevier Inc. All rights reserved.

Introduction As described in detail in other manuscripts in this issue [3],

Heme catalases are one of the most efficient enzymes known:
they are able to decompose up to one million molecules of hydro-
gen peroxide per second [1]. Structurally, they are tetrameric pro-
teins with each subunit containing a heme group [2]. The active
site contains two residues that are essential for catalysis: the prox-
imal Tyr and the distal His (Fig. 1). The former is coordinated to the
heme iron and is negatively charged, contributing to the +3 formal
oxidation state of the iron atom. The latter is essential for the for-
mation of the main reaction intermediate, Compound I (Cpd I). The
proximal arginine (hydrogen-bonded to tyrosine), together with
the distal asparagine and serine complete the list of conserved res-
idues in the active site [2].
⇑ Corresponding author at: Laboratori de Simulació Computacional i Modelització
(CoSMoLab), Institut de Química Teòrica i Computacional (IQTCUB), Parc Científic
de Barcelona, Baldiri Reixac 4, 08028 Barcelona, Spain.
E-mail address: crovira@pcb.ub.es (C. Rovira).
there are two types of monofunctional catalases depending on
the size of the subunit and the type of heme group they contain.
Small subunit catalases, such as Helicobacter pylori catalase (HPC)
(Fig. 1a), contain the most common protoporphyrin IX or heme b.
In contrast, large subunit catalases, such as Penicillium vitale cata-
lase (PVC) (Fig. 1b), contain a modified heme named heme d. In
heme d, one of the propionates is converted into a spirolactone,
formed by the cis-hydroxylation of the protoheme in a self-cata-
lyzed reaction that needs H2O2 as substrate. As a consequence of
this modification, the porphyrin of the heme loses one negative
charge and one double bond, i.e. the p-conjugation of heme d is re-
duced compared to heme b. These changes might affect the elec-
tronic properties of the reaction intermediates and thus the
reactivity of the heme, as recently investigated in our group [4–6].
The active species responsible for the decomposition of H2O2 is
a high-valent iron intermediate, known as Compound I (Cpd I) [7],
obtained by reaction of catalase with hydrogen peroxide.
EnzðPor—FeIII Þ þ H2 O2 ! Cpd IðPorþ —FeIV ¼ OÞ þ H2 O ð1Þ

0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.abb.2012.04.004
122 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130

(a) (b)

Ser 95
His56 Asn129 Ser 103
O Asn137
water III II His64 O
O N N IV I
water O N N
Fe
heme b Fe
N N
heme d N N
IV I
III II
O
O
O HO
Tyr339 Arg335 O heme bb
heme heme d
heme d
Tyr351 Arg347

Fig. 1. (a) Structure of native Helicobacter pylori catalase (HPC, PDB entry 2IQF). Top: Cartoon picture of the protein, with the four subunits colored in blue, red, yellow and
green, respectively. Bottom: Heme binding pocket of one of the subunits (blue) and molecular structure of heme b. (b) Same representation for Penicillium vitale catalase (PVC,
PDB entry 2IUF) and molecular structure of heme d. Reprinted with permission from reference [5]. Copyright 2009 American Chemical Society.

Cpd I is characterized to be an oxoferryl porphyrin cation radi-
cal (Por+–FeIV=O), [7] and it is formed not only in catalases but also
by other heme proteins, such as peroxidases or cytochrome P450.
The subsequent Cpd I reactivity is determined by the protein frame
in which the heme is buried and research aimed to grasp the origin
of this functional diversity is an extremely active field [8–10].
Kinetics studies [11] have shown that once catalase Cpd I forms
(Reaction (1)) [12], it rapidly reacts with another molecule of H2O2
to generate a water molecule and O2 (Reaction (2)). Isotope label-
ing studies demonstrated that both oxygen atoms of the O2 mole-
cule originate from the same H2O2 molecule [13,14].
Cpd IðPorþ —FeIV ¼ OÞ þ H2 O2 ! EnzðPor—FeIII Þ þ H2 O þ O2
Reaction (2), extremely efficient in catalases, occurs at a much
slower pace in a few other heme enzymes (e.g. chloroperoxidase
(CPO), catalase–peroxidase (KatG) and myoglobin (Mb)) [15]. The
origin of this disparity has long been sought and, even though
the catalase reaction has been known since 1940s [16] (see also
the contribution by P. Nicholls in this issue), the detailed mecha-
nism of Cpd I reduction by H2O2 remained a challenge until re-
cently [5]. Reaction (2) is a two-electron redox process (i.e. a
single H2O2 molecule provides the two electrons needed to reduce
Cpd I back to the resting state) and clarifying whether the two elec-
trons are actually transferred together in a single elementary step
would pose catalases in clear contrast with peroxidases, for which
the resting state is recovered through two consecutive one-elec-
tron reduction processes, each requiring one molecule of an organ-
ic donor [12].
Although the main activity of catalases is the decomposition of
H2O2, under certain conditions catalases can undergo undesired
side reactions that can eventually inactivate the enzyme. For in-
stance, at low hydrogen peroxide concentrations and in the pres-
ence of certain organic substrates (AH, e.g. phenols), Cpd I can
undergo a one-electron reduction to form Compound II (Cpd II).
Cpd IðPorþ —FeIV ¼ OÞ þ AH ! Cpd IIðPor—FeIV —OHÞ þ A ð3Þ
Similarly, when no substrate (electron donor) is present, the
highly reactive Cpd I can oxidize a residue of the protein (aa), typ-
ically Tyr or Trp, forming another intermediate named Compound
I⁄.
ð2Þ
Cpd IðPorþ —FeIV ¼ OÞ þ Hþ þ aa

! Cpd I ðPor—FeIV —OH þ aaðþÞ Þ ð4Þ
⁄
The formation of Cpd I can be detected by a change in the UV–
visible spectrum (from a green to a red colored intermediate) [16]
and in the EPR signature (from a porphyrin radical to a protein rad-
ical, aa(+) in Eq. (4)) [17]. Structurally, Cpd I⁄ typically contains an
hydroxoferryl porphyrin with a protein radical instead of the oxo-
ferryl porphyrin cation radical of Cpd I. In catalases, formation of
Cpd I⁄ is a side reaction that consumes Cpd I, decreasing the effi-
ciency of the decomposition of hydrogen peroxide. Therefore, one
would like to understand the factors that drive Cpd I⁄ formation
in order to find strategies to minimize this undesirable side reac-
tion and design more efficient catalases to be used in biosensors
and bioreactors. Interestingly, it has been observed that Cpd I⁄
 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130
formation only occurs for catalases containing heme b, like HPC,
but not for those containing heme d, such as PVC. On the other
hand, in peroxidases, it has been proposed that Cpd I⁄ is an alterna-
tive reactive intermediate in the oxidation of bulky substrates that
cannot access the heme active site [16,18]. Therefore, understand-
ing the factors leading to radical migration could also help to engi-
neer new catalytically competent protein radical sites for the
oxidation of substrates that cannot be oxidized at the heme active
site [19].
In recent years, we have investigated the molecular mecha-
nisms and electronic reorganizations associated to the above men-
tioned reactions in catalase, using Density Functional Theory (DFT)
in combination with advanced theoretical approaches (ab initio
molecular dynamics [20], QM/MM [21] and metadynamics
[22,23]). Our calculations have been performed with the BP86
exchange–correlation functional and a plane wave basis set. As dis-
cussed in reference [6], the structures and electronic configura-
tions obtained with this computational setup are in agreement
with experiments as well as with DFT/B3LYP and multireference
calculations. In particular, we investigated the electronic configu-
ration of Cpd I, II and I⁄, the electronic state of the oxygen released
by catalase, the reduction of Cpd I and the formation of Cpd I⁄. The
results obtained concerning spin distributions, energies and molec-
ular mechanisms contributed to get insight into some of the main
questions of catalase research, e.g. why heme d catalases do not
form Cpd I⁄. Our results also helped to clarify previous controver-
sies, such as whether catalase could release singlet oxygen, and
provided with an atomistic description of the mechanism of Cpd
I reduction by H2O2. We here summarize the results of our inves-
tigation on monofunctional heme catalases. Bifunctional cata-
lase–peroxidases [24,25] are not reviewed here.
Electronic configuration of the reaction intermediates: Cpd I, II
and I⁄
Cpd I is the main reaction intermediate of catalases and it is also
involved in the catalytic cycle of other heme proteins (e.g. peroxi-
dases, cytochrome P450 and nitric oxide synthase). Cpd I formation
(Reaction (1)) is formally a two-electron process. It can be viewed
as removal of two electrons from the heme resting state (Por–FeIII):
one electron is removed from the iron atom (which changes from
FeIII to FeIV), whereas the second electron is removed from the por-
phyrin, which acquires cation radical character (Por+). Several
experimental [26,27] and theoretical studies [4,6,28–30] have
shown that Cpd I bears two electrons delocalized on the Fe–O moi-
ety and one unpaired electron delocalized on the porphyrin ring, in
ferromagnetic interaction in the case of catalase. This is the elec-
tronic picture of ‘‘canonical’’ Cpd I, which can be well described
using Density Functional Theory [4,30–33].
In vitro, Cpd I is normally trapped by reaction of the native en-
zyme with peroxoacetic acid (PAA, see Fig. 2), allowing its struc-
tural characterization by X-ray crystallography (Table 1). It has
been generally assumed that Cpd I contains an oxoferryl (Fe=O)
bond, with an associated distance around 1.7 Å, according to com-
putational studies [4,31,32] . However, the long Fe–O distances
(about 1.9 Å) observed in the PAA-oxidized crystal structures of
catalases and other heme proteins (CcP [34] and KatG [35]) suggest
that a species with a single Fe–O bond has been isolated. A possible
explanation for the long Fe–O distance is the photoreduction of
Cpd I to Cpd II, as the latter bears an iron–hydroxoferryl bond
[36–38]. In fact, fine crystallographic data collection using the mul-
ticrystal approach have demonstrated that the X-ray radiation can
reduce Cpd I (in horseradish peroxidase, HRP, cytochrome c perox-
idase, CcP, ascorbate peroxidase, APX, and also catalase) [39–42]
suggesting that the species trapped during data collection might
123
Fig. 2. Possible intermediates formed upon treatment of catalase with peroxoacetic
acid (PAA).
be an average of different oxidation states. Another possible expla-
nation is Cpd I reduction by a protein residue, i.e. conversion to Cpd
I⁄, which is isoelectronic with Cpd II in the heme active site and
thus it is also expected to bear a Fe–OH group (Fig. 2). Therefore,
either photoreduction of Cpd I to Cpd II or formation of Cpd I⁄
can explain the long Fe–O distances observed in some PAA-oxi-
dized crystal structures. In other words, electron transfer to the
porphyrin, regardless of whether the electron is exogenous or
endogenous, facilitates protonation of the oxoferryl group.
The hypothesis that reduced Cpd I (either Cpd II or Cpd I⁄) is
protonated can be tested by comparing the intermediates obtained
by PAA treatment of small and large subunit catalases. Large sub-
unit catalases that have heme d (such as PVC, and Escherichia coli
hydroperoxidase II, HPII) are known to be resistant against reduc-
tion (i.e. they do not form Cpd II) [43,44]. Therefore, it is expected
that these enzymes will only exhibit short Fe–O bonds. In fact, the
only X-ray structure of an oxidized heme d catalase (PVC) shows a
Fe–O bond of 1.70 Å, much shorter than the distances observed for
heme b catalases such as HPC (1.80–1.85 Å, see Table 1). QM/MM
calculations performed for PVC and HPC considering several elec-
tronic configurations and protonation states (Table 2) [4] con-
firmed that the bond lengths observed experimentally are
compatible with the presence of a Fe=O group in the crystal struc-
ture of oxidized PVC but a Fe–OH group for HPC. Besides, the UV–
visible spectrum of PVC indicates the presence of a porphyrin rad-
ical, whereas the HPC spectrum was found to be compatible with a
reduced Cpd I. Therefore, the intermediate trapped for PVC is Cpd I,
whereas for HPC it might be Cpd II or Cpd I⁄. Since the crystals of
oxidized HPC turned red (indicating Cpd I reduction) before X-
ray exposure, photoreduction of Cpd I to Cpd II can be discarded,
thus suggesting that the intermediate trapped for HPC is Cpd I⁄ [4].
It is also worth noting that the electronic spin distribution
changes qualitatively among heme b and heme d catalases. The un-
paired spin distribution of HPC Cpd I is the canonical porphyrin
cation radical (Fig. 3a). However, the unpaired electron in PVC is
partially delocalized into the distal His (Fig. 3b) (i.e. there has been
a partial transfer of electrons from His to the porphyrin). Room
temperature QM/MM molecular dynamics simulations show that
the spin density of PVC Cpd I fluctuates between the distal His,
the porphyrin and the proximal tyrosinate. This particular spin dis-
tribution can be explained in terms of the heme modification and
the occupation of the highest energy orbitals of Cpd I (Fig. 4). For
HPC (heme b) the highest in energy porphyrin orbital is the a2u
orbital. As a consequence the a2u orbital becomes the donor of
the electron removed from the porphyrin during Cpd I formation,
124 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130

Table 1
Reported crystal structures of the oxidized intermediates of catalases.

Protein PDB accession No. Type of heme Resolution (Å) Fe–O length (Å) Reference
a
PMC [1MQF] Heme b 2.5 1.76 [80]
MLC [1GWF] Heme b 2.0 1.87 [81]
HPC [2IQF] Heme b 1.8 1.80/1.85b [4]
PVC [2IUF] Heme d 1.7 1.72/1.72b [4]
a
This distance is intermediate between a Fe=O double bond and a Fe–OH single bond. Unfortunately, the low resolution of 2.5 Å and use of restraints precludes drawing
firm conclusions on the nature of the Fe–O bond.
b
Values corresponding to the two independent protein subunits.

Table 2
Fe–O distance (in Å) for the possible oxidized intermediates of HPC and PVC.

Catalase Experiment QM/MM calculationsb

X-ray Cpd I(Fe=O, His) Cpd I (Fe=O, His H+) Cpd I(Fe–OH, His) Cpd I⁄/II(Fe=O, His) Cpd I⁄/II(Fe=O, His H+) Cpd I⁄/II(Fe–OH, His)
HPC 1.80/1.85a 1.68 1.71 1.78 1.69 1.72 1.79
PVC 1.72/1.72a 1.69 1.70 1.80 1.69 1.71 1.80
a
Values corresponding to the two independent protein subunits.
b
The protonation state taken for the oxoferryl unit and the distal histidine is specified in parentheses.

Fig. 3. Computed spin distributions of Cpd I (Por+–FeIV=O) for HPC (left) and PVC (right). Adapted with permission from reference [4]. Copyright 2007 American Chemical
Society.

as reflected in the A2u symmetry of the spin density distribution on
the porphyrin (Fig. 3a). The loss of ring aromaticity of the porphy-
rin in PVC affects mainly the a1u porphyrin orbital which has con-
tributions from the pyrrole b carbon atoms. As a consequence, the
energy of the a1u orbital increases relative to the a2u orbital and the
second oxidation equivalent during Cpd I formation in PVC is taken
from the a1u orbital. Unlike a2u, this orbital can mix with one p
imidazole orbital and, therefore, the oxidation equivalent from
the porphyrin of heme d in PVC is removed from a mixed orbital
(a1u–pHis), as reflected in the spin density distribution (Fig. 3b).
In summary, the calculations support that oxidized PVC con-
tains the typical oxoferryl bond and porphyrin radical (with the
particularity that the radical is partially delocalized on the distal
His) while oxidized HPC contains a hydroxoferryl bond and a pro-
tein radical. The particular electron distribution of PVC Cpd I may
be probed by EPR experiments.
Electronic state of the oxygen released by catalase: triplet or
singlet?
As shown in Reaction (2) (hereafter referred to as the catalatic
reaction), H2O2 serves as a two-electron reductant in the catalatic
reaction (i.e. Cpd I reduction by H2O2 back to the resting state). For-
mally, one electron reduces the porphyrin radical, and the other
one contributes to the change of oxidation state of the iron atom
(from FeIV to FeIII). The transfer of two electrons from H2O2 is asso-
ciated with the transfer of two protons, forming O2 (the details of
the reaction pathway are presented in the next section). Neverthe-
less, for some time it was unclear whether H2O2 loses the two elec-
trons as H/H+ (transfer of a hydride ion and a proton), H/H
(double transfer of one hydrogen atom) or other mechanisms. It
was early proposed by Fita and Rossmann [45], and recently sup-
ported by Watanabe et al. [46], that the distal His is also involved
in the catalatic reaction. In view of the known ability of histidines
to mediate the transfer of protons, a H/H+ scheme was assumed,
in which H is transferred directly to the oxoferryl unit and the
transfer of H+ is mediated by the distal His (Fig. 5).
One aspect of the catalase reaction that has generated certain
controversy is the spin state of the oxygen molecule released in
the reaction. Based on spin conservation arguments, some authors
[47–50] have suggested that the H/H+ mechanism proposed in the
literature generates singlet oxygen,
H2 O2 ðS ¼ 0; "#Þ ! Hþ ðS ¼ 0Þ þ H ðS ¼ 0; "#Þ þ 1 O2 ðS ¼ 0; "#Þ ð5Þ
where S is the spin quantum number, " represent the a spin (spin-
up electron) and ; the b spin (spin-down electron). In contrast,
simultaneous uptake of two hydrogen atoms from H2O2 (i.e. a H/
H mechanism) would generate triplet oxygen:
 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130
Fig. 4. Changes on the relative energy of the a1u and a2u orbitals of the oxoferryl porphyrin species as a result of the heme modification (indicated by an arrow) and possible
mixing with other orbitals (pTyr and pHis).
H passes across species with triplet (O2), doublet (O
H
N N
N HOOH N
O H2O2 can only be a singlet, the global spin state of Cpd I + H2O2
IV
Fe
Fig. 5. Schematic representation of Cpd I reduction by H2O2 in catalase.
H2 O2 ðS ¼ 0; "#Þ ! H ðS ¼ 1=2; #Þ þ H ðS ¼ 1=2; #Þ þ 3 O2 ðS
¼ 1; Þ
In other words, a ‘‘pairwise movement of electrons’’ produces
singlet oxygen, whereas ‘‘two single electron movements’’ lead to
triplet oxygen. For instance, these arguments have been used to
propose reaction mechanisms in catalases and catalase–peroxi-
dases [48]. Therefore, a priori it might seem that the H/H+ mech-
anism involving proton transfer to the distal His generates singlet
oxygen in catalase. Indeed, some experiments have detected oxida-
tion of catalase by singlet oxygen [49,50] or chemoluminescence
attributed to 1O2 emission [47,51]. However, as pointed out by
Jakopitsch et al. [48], it would not make biological sense to release
large amounts of singlet oxygen out of the heme pocket, because
molecular oxygen in the singlet state is a very powerful oxidant.
Its damaging action in a variety of biological processes has been
well recognized [52,53] and it has been shown to inactivate
enzymes [54].
As we argued in reference [55] this apparent contradiction can
be solved by taking into consideration that oxygen is not the only
species with non-zero spin in the active site. The oxoferryl porphy-
rin cation radical of Cpd I, with three unpaired electrons in a quar-
tet spin configuration in the ground state, also contributes to the
total spin. Therefore, invoking spin conservation implies consider-
ing the spin state of both species, the heme group and the released
oxygen molecule.
Generation of O2 from H2O2 is part of the known Latimer series
[56,57] that go from O2 to H2O formation and reverse. This series
125

2 ; HO2 and
HO) and singlet (H2O, H2O2, OH and HO 2 ) ground spin states.
O2 These a priori spin-forbidden reactions can take place due to the
presence of a paramagnetic transition metal [58,59], which in-
creases the multiplicity of the overall system, thus increasing the
OH2 number of spin-allowed reactions. For instance, whereas isolated
III (i.e. the reactants of the catalase reaction) can be a doublet, quartet
Fe
or sextet, based on the possible multiplicities of the separate frag-
ments. Likewise, the overall spin state of the products (i.e. Por–
FeIII + H2O + O2) can be a doublet, quartet, sextet or octuplet.
All the possible electronic configurations of the reactants (i.e.
Cpd I + H2O2) and the products (i.e. Por–FeIII + H2O + O2) of the cat-
alatic reaction in monofunctional catalases are depicted in Fig. 6.
ð6Þ To facilitate the analysis, we have classified each species according
to the total spin (Stotal = 1/2, 3/2, 5/2 and 7/2). A detailed analysis of
possible reaction pathways from reactants to products is provided
in reference [55]. It follows from Fig. 6 that, when the spin state of
all the species involved in the reaction is taken into account (and
not only H2O2), the catalatic reaction could generate both types
of oxygen spin configurations, singlet (1O2) and triplet (3O2),
regardless of the mechanism how the two electrons and two
protons are transferred. Therefore, the a priori assumption that
the H+/H scheme leads to the generation of singlet oxygen in cata-
lases, whereas the H/H schemes leads to triplet oxygen, is not
justified.
Reactivity of Cpd I
The catalase reaction
As mentioned before, the catalase reaction consists of two
steps: Cpd I formation (Reaction (1)) and Cpd I reduction (Reaction
(2)). Several heme enzymes (e.g. catalases, peroxidases, catalase–
peroxidases and myoglobin) are able to form Cpd I (Reaction (1)),
but only catalases can perform Reaction (2) with high efficiency
[15,60]. Hence, the term ‘‘catalase reaction’’ or ‘‘catalatic reaction’’
is often used to refer to Cpd I reduction by H2O2.
Cpd I formation (Reaction (1)) has been studied for horseradish
peroxidase using QM/MM methods [61–63]. As predicted by
126 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130

Fig. 6. Possible electronic configurations of the reactants (R, i.e. Cpd I + H2O2) and products (P, i.e. Por–FeIII + H2O + O2) of the catalatic reaction in monofuntional catalases.
The number in the left upper side (2, 4, 6 or 8, i.e. doublet, quartet, sextet and octuplet) denotes the spin multiplicity and the letter in the right lower side (a, b or c) indicates
the different electronic configurations compatible with the overall spin multiplicity. The a spin (spin-up electron) is represented by " and the b spin (spin-down electron) by ;
.

Poulos and Kraut [64], the distal His acts as an acid–base catalyst.
In addition, the calculations show that a water molecule is essen-
tial for the reaction to proceed with a low barrier [61,63]. It is ex-
pected that the same mechanism is operative in catalases, although
without the participation of water, as the catalase active site is not
solvent exposed [2].
Although there had been preliminary suggestions from kinetic
data [54], Cpd I reduction was first considered at a molecular level
in 1985 by Fita and Rossmann [45]. On the basis of the crystal
structure of beef liver catalase, Fita and Rossmann proposed that
the two hydrogens of H2O2 are sequentially transferred to the oxo-
ferryl unit of Cpd I, with the distal His playing an active role in the
reaction. Nevertheless, the precise mechanism how the two pro-
tons and two electrons of H2O2 are transferred to Cpd I was not dis-
cussed. Recently, the group of Watanabe, by means of a detailed
kinetic study, was able to disentangle the rate constants of Cpd I
formation and reduction for Micrococcus lysodeikticus catalase
(MLC) and a series of Mb mutants [46,65]. Two different kinetic
behaviors were observed in H2O and D2O for Reaction (2), which
were interpreted as two different mechanisms. Namely, it was pro-
posed that the reduction of Cpd I by H2O2 in native catalase, as well
as in the F43H/H64L Mb mutant, involves the transfer of a hydride
ion from H2O2 to Cpd I and the transfer of a proton mediated by the
distal His (Fig. 7a, hereafter named as the His-mediated mecha-
nism). This mechanism thus follows the Fita–Rossmann model
[45], with the distal His acting as an acid–base catalyst. Besides,
for certain Mb mutants lacking a distal residue that could act as
acid–base catalyst, an alternative mechanism was proposed
[46,65], in which two hydrogen atoms of H2O2 are directly trans-
ferred to the oxoferryl group (Fig. 7b, from now on referred to as
the direct mechanism).
As a follow-up to our experimental and computational study of
HPC and PVC Cpd I [4], we recently applied QM/MM metadynamics
simulations to investigate the reduction of Cpd I in catalase. The
calculations show that the H2O2 spontaneously transfers one
hydrogen atom to the oxoferryl of Cpd I, forming a Cpd II-like spe-
cies. To complete the reaction, two mechanisms may be operative:
a His-mediated or a direct mechanism. The former involves the dis-
tal His as an acid–base catalyst, mediating the transfer of a proton
associated with an electron transfer (pathway A in Fig. 8). By con-
trast, a hydrogen atom transfer occurs in the direct mechanism
(pathway B). Therefore, the catalatic reaction may take place by
 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130
(a)
H H
N N
HOOH
N NH
O O
FeIV
+
(b) H H
N N
HOOH
N N
O O
FeIV +
Fig. 7. (a) The His-mediated mechanism of Cpd I reduction (Fita–Rossmann) (b) The direct mechanism (Watanabe).
two different mechanisms, which can be described as H/ET + H+
(A) or H/H (B), respectively (ET = electron transfer). Independently
of the mechanism, the reaction proceeds by two one-electron
transfers, rather than one two-electron transfer, as has long been
assumed. It is also worth noting that the steps with the highest en-
ergy barrier along each pathway (steps identified by an asterisk in
Fig. 8) do not correspond to hydrogen atom transfer processes, but
rather to changes of the hydrogen bond pattern. This is consistent
with the small kinetic isotope effect (KIE) determined for catalase
[46]. Interestingly, the distal Asn changes conformation gradually
(from the Cpd II-like configuration to the products), allowing the
release of the product oxygen toward the main channel. Finally,
our results also confirmed that the oxygen molecule released by
catalase is in the triplet state [5].
Additional calculations on an in silico mutant of the distal histi-
dine (H56G) for HPC show that the hydrogen-bond network at the
distal site plays a key role in positioning the peroxide such that the
reaction can proceed with a low barrier. For the H56G HPC mutant,
the first hydrogen atom transfer becomes rate-limiting, explaining
the large KIE observed for the Mb mutants lacking the distal His
[46,65]. Therefore, in line with previous investigations [15,66],
Cpd I reactivity depends on the shape and the nature of the distal
pocket (i.e. the interactions between H2O2 and the distal residues).
Formation of Cpd I⁄
As mentioned before, in the absence of either hydrogen perox-
ide or external electron donors, an endogenous protein residue
(aa), usually Tyr or Trp, can reduce Cpd I (Reaction (4)). This intra-
molecular electron transfer, often referred to as ‘‘migration of the
porphyrin radical into the protein’’, is normally concomitant with
protonation of the oxoferryl bond [4,36–38,67]. In fact, radical
migration in catalases [4] and other heme proteins [34,35] corre-
lates with the observation of a lengthening of the Fe–O distance
(from 1.6–1.7 Å to 1.8–1.9 Å), indicative of a change from oxoferryl,
Fe=O, to hydroxoferryl, Fe–OH. The most likely proton donor was
identified in reference [4] to be the (protonated) distal His.
Whereas for some heme proteins (e.g. in CcP [34], KatG [35] or
lactoperoxidase [68]), formation of a protein radical (i.e. Cpd I⁄) is
necessary for mediating the electron transfer between the heme
and the substrate, radical migration in catalases [17,69] and P450
[70] is considered an undesirable side reaction that competes with
the main activity of the enzyme. Nevertheless, Reaction (4) has
127
H
N
O2 O2
N
H H H
O
FeIII FeIII
H
N
HOO O2
N
H H H
O
FeIV FeIII
been suggested to be the main catalase mechanism for Cpd I reduc-
tion in cells with low H2O2 concentrations [71,72]. Under these
conditions, Cpd I reduction by hydrogen peroxide is slow and the
alternative reduction by protein residues could avoid a prolonged
or frustrated Cpd I state that would result in an irreversible inacti-
vation of the enzyme. Besides, in peroxidases, it has been proposed
that Cpd I⁄ is an alternative reactive intermediate in the oxidation
of bulky substrates that cannot access the heme active site
[16,18,19]. Therefore, understanding the factors leading to radical
migration could help to engineer new catalytically competent pro-
tein radical sites for the oxidation of substrates that cannot be oxi-
dized at the heme active site [19].
By combining QM/MM calculations, UV–visible spectroscopy
and X-ray crystallography, we previously suggested that HPC
forms a protein radical, in contrast to PVC, where the radical re-
mains on the heme [4]. The different type of intermediate that is
formed (Cpd I⁄ for HPC and Cpd I for PVC) could be related to the
type of heme present, heme b in HPC and heme d in PVC (Fig. 1).
However, other heme b catalases different from HPC do form
canonical Cpd I upon PAA oxidation [26,27]. Therefore, other fac-
tors different than the type of heme are likely to influence radical
migration in catalases. A possible influencing factor is the pH. For
instance, EPR, UV–visible and Resonance Raman experiments have
shown that the heme b-containing bovine liver catalase (BLC)
yields a tyrosyl radical at acidic pH [17,26,73], but a short-lived
Cpd I at basic pH [74,75]. Another factor may be the availability
of electron-donating residues close to the heme b. E.g. wild-type
Proteus mirabilis catalase (PMC) forms a stable Cpd I upon PAA
treatment, whereas its F194Y mutant forms a tyrosyl radical.
Knowledge of the reduction potentials of the catalase Cpd I
intermediates of HPC and PVC would help to clarify why heme d
catalases behave different from heme b catalases. Unfortunately,
these are not available in an experiment due to the short lifetime
of the catalase redox intermediates. Computational methods,
where the redox state is controlled, can provide insight into this is-
sue. To this aim, we recently investigated the easiness of reduction
of HPC compared to PVC Cpd I [6]. In particular, we determined the
free energy of one-electron reduction of Cpd I (by means of the en-
ergy gap method [76–78] based on Marcus theory [79] for electron
transfer reactions), as well as the free energy of protonation of re-
duced Cpd I (using metadynamics [22,23]) of both catalases. We
found that the difference in free energy for pure one-electron
reduction of Cpd I (‘‘pure’’ means without coupled proton transfer)
128 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130

Fig. 8. Computed mechanism of Cpd I reduction in HPC.

is indeed very small and unlikely to be the reason for the different
redox behavior of HPC and PVC. However, the subsequent proton
transfer step from the distal histidine residue to the ferryl oxygen
is much more exothermic for HPC compared to PVC (see Fig. 9). This
larger free energy released by proton transfer in HPC is enough to
compensate for the energy cost of removing an electron from the
protein residue (most likely a tyrosine), as opposed to PVC. Accord-
ingly, protein radical migration in HPC is ‘‘thermodynamically
driven’’ by proton transfer to the oxoferryl. This prediction may
be probed by performing EPR experiments at different pHs and
the location of the tyrosyl radical confirmed by combining EPR with
ENDOR and mutagenesis studies. In summary, our results indicate
that the major driving force for radical migration is the proton
transfer to reduced Cpd I, supporting the former suggestion that
HPC forms a protein radical as opposed to PVC. Moreover, we ratio-
nalized the different basicity of the ferryl oxygen in the two
 M. Alfonso-Prieto et al. / Archives of Biochemistry and Biophysics 525 (2012) 121–130
Fig. 9. Proton transfer from the distal histidine to the oxyferryl in one-electron reduced Cpd I (i.e. Cpd II). For HPC, the Fe–OH form is more stable by 15 kcal/mol, whereas the
energy gain is much smaller (4.5 kcal/mol) for PVC. Adapted with permission from reference [6]. Copyright 2011 American Chemical Society.
catalases using the different porphyrin orbital ordering in heme b
(HPC) and heme d (PVC, see Fig. 4). This confirms the view that
heme b containing catalases, such as HPC, are more prone to under-
go radical migration than heme d containing catalases, such as PVC.
Nevertheless, besides the type of heme, the protein environment
may also influence the propensity to form Cpd I⁄, by tuning the
pKa of the distal His and/or by controlling the oxidation of the elec-
tron-donating Tyr, explaining the differences among heme b cata-
lases (see above). In summary, our results suggest that oxoferryl
protonation is a key factor regulating radical migration in catalase
and possibly also in other hydroperoxidases forming Cpd I⁄.
Acknowledgments
The authors thank the Spanish Ministry of Science and Innova-
tion (MICINN) (grant CTQ2011-25871) and the Generalitat de
Catalunya (grant 2009SGR-1309) for its financial assistance. M.
A.-P. acknowledges the Generalitat de Catalunya for a postdoctoral
fellowship (Beatriu de Pinós). We acknowledge the computer sup-
port, technical expertise, and assistance provided by the Barcelona
Supercomputing Center-Centro Nacional de Supercomputación
(BSC-CNS).
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