molecules

Review
Peroxidase Activity of Human Hemoproteins:
Keeping the Fire under Control
Irina I. Vlasova 1,2
1 Federal Research and Clinical Center of Physical-Chemical Medicine, Department of Biophysics,
Malaya Pirogovskaya, 1a, Moscow 119435, Russia; [email protected]; Tel./Fax: +7-985-771-1657
2 Institute for Regenerative Medicine, Laboratory of Navigational Redox Lipidomics, Sechenov University,
8-2 Trubetskaya St., Moscow 119991, Russia




Received: 28 August 2018; Accepted: 1 October 2018; Published: 8 October 2018


Abstract: The heme in the active center of peroxidases reacts with hydrogen peroxide to form
highly reactive intermediates, which then oxidize simple substances called peroxidase substrates.
Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose
main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids
in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase
and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body,
but under the influence of external conditions they can display peroxidase-like activity. As oxidative
intermediates, these peroxidases produce not only active heme compounds, but also protein-based
tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are
considered as pseudo-peroxidases. Peroxidases play an important role in innate immunity and in
a number of physiologically important processes like apoptosis and cell signaling. Unfavorable
excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating
the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance.
Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase
activity and the biological effects of peroxidase products are specific for each hemoprotein. This review
summarizes the knowledge about the properties, activities, regulations and biological effects of true
and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and
adverse effects of this class of enzymes.

Keywords: peroxidase activity; halogenating activity; reduction potential; myeloperoxidase;

hemoglobin; Cyt c/cardiolipin complexes; cytoglobin

1. Introduction
Reactive oxygen species (ROS) are involved in many physiological processes, including signal
transduction, cell proliferation, gene expression, angiogenesis, and aging. However, excessive ROS
production initiates oxidative stress, which has been implicated in pathophysiological processes like
diabetes, cardiovascular and neurodegenerative diseases, pulmonary diseases, sepsis and cancer [1,2].
As such, a variety of special mechanisms exist in the body for controlling the concentration and activity
of molecules capable of generating oxidants and ROS.
The NADPH oxidase family of enzymes is a major source of ROS in vivo. The enzymes generate
superoxide radicals (O2 •– ) that are the precursor of many ROS [1,3]. O2 •– can be spontaneously
or catalytically (by superoxide dismutase) converted into hydrogen peroxide (H2 O2 ). The major
sources of O2 •– in plasma are NADPH oxidases on the surface of phagocytes and endothelial cells,
xanthine oxidase bound to endothelial cells, and leakage from the mitochondrial respiratory chain [4–6].

Molecules 2018, 23, 2561; doi:10.3390/molecules23102561 www.mdpi.com/journal/molecules

Molecules 2018, 23, 2561 2 of 27

A healthy physiological level of H2 O2 in the plasma is 1–5 µM, but may be as high as 50 µM in
inflammatory disease [6–8].
Hydrogen peroxide is one of the major in vivo oxidants. It is a two-electron acceptor with
reduction potential Eo (H2 O2 /H2 O) of 1.32 V. However, H2 O2 reacts poorly or not at all with most
biological molecules due the high activation energy barrier that must be overcome to release H2 O2
oxidative power [9] (see Supplementary Materials, Section S1).
The development of oxidative stress is mediated by transition metal ions, primarily with iron
ions that promote H2 O2 tunneling through the activation energy barrier. Various forms of iron react
with hydrogen peroxide to form ROS. Table 1 lists the reaction rate constants of various forms of iron
with H2 O2 and the reduction potentials of radicals and oxidants formed in the reactions. The rate
constants of the reaction of H2 O2 with free iron or free heme are low. Ligation usually stimulates Fenton
reactions. Notably, iron ions are strongly controlled in vivo. A system of ceruloplasmin-transferrin is
responsible for the transport of free iron ions in plasma; intracellular iron redox activity is arrested by
ferritin [10]. Iron chaperons guide iron delivery in cells directly to their “protein clients” thus limiting
non-enzymatic redox-cycling reactions [11]. In plasma, a special protein hemopexin (Hx) binds free
heme and inhibits its peroxidase activity [12].

Table 1. Second order rate constants and oxidants produced in the reaction of different forms of
iron with H2 O2 . Standard reduction potentials (Eo ) are presented for oxidative couples: HOX/X-;
• NO /NO − ; phenoxyl radical (Ph-O• )/phenolic compound (Ph-OH); HO• /H O; HOO• /H O .
2 2 2 2 2

Form of Iron k, M−1 s−1 Reaction Products Eo , V pH 7 References

hydroxyl radical, HO• 2.31 [13,14]
* free ferrous ion, Fe2+ 76
lipid alkoxyl radical, LO• ≤1.06
Lig-Fe2+ 102 –104 HO• , LO• [15,16]
hydroperoxyl radical, HOO• 1.06 [14,17,18]
free ferric ion, Fe3+ 0.01–0.02
lipid peroxyl radical, LOO• ≤1.00
phenoxyl radicals, Ph-O• 0.4–0.94 [19,20]
Albumin-heme
lipid radical, L• (LO• , LOO• ) 0.6 [14]
hypochlorous acid, HOCl 1.28 [21,22]
hypobromous acid, HOBr 1.13
hypoiodous acid, HOI 0.78
True peroxidases (1.1–4.3) × 107
hypothiocyanous acid, HSCN 0.56
phenoxyl radicals, Ph-O• 0.4–0.94 [20]
nitrogen dioxide, • NO2 1.04 [23]
Hemoglobin 42–43.6 Ph-O• , • NO2 , L• , (LO• , LOO• ) [24,25]
Cyt c/cardiolipin ~46.4 Ph-O• , L• , (LO• , LOO• ) [26,27]
Cyt c/cardiolipin +
~5 × (103 –105 ) [27]
FFA-OOH
* Fe2+ + H2 O2 = Fe3+ + • OH + OH− —Fenton reaction; Lig—a ligand (ATP, ADP, UTP, [Fe-S] clusters); L—lipid;
FFA-OOH—free fatty acid hydroperoxides.

Another group of molecules that interact with H2 O2 are peroxidases. Peroxidases consume H2 O2
and catalyze the one- or two-electron oxidation of a diverse array of Compounds called peroxidase
substrates [1].
Heme-peroxidases are enzymes with a heme prosthetic group in the catalytic site where the iron
is bound by four coordination bonds to a protoporphyrin IX derivative [28]. The rate constants of the
interaction of heme in the active center of peroxidases with H2 O2 are several orders of magnitude
higher than that of the Fenton reaction [21] (Table 1). In the presence of H2 O2 , heme-peroxidases
are activated to highly reactive intermediates. Lipid hydroperoxides also react with the peroxidase
catalytic site. Except for cytochrome c (Cyt c)/cardiolipin (CL) complexes [27], the rate of their reaction
with heme is lower than that of H2 O2 [29–31].
Molecules 2018, 23, 2561 3 of 27

The mandatory requirements for the peroxidase activity of a heme-containing protein (i.e., its
ability to form reactive intermediates) are:

(1) The peroxidases operate having iron in ferric form (Fe(3+) or Fe(III)).
Molecules 2018, 23, x FOR PEER REVIEW 3 of 27
(2) The heme iron of a peroxidase has five coordination bonds: four with nitrogens of tetrapyrrole
ring, and theresidue
of histidine fifth heme ligand
linking theonheme
the proximal heme side
to the protein. Onisthe
a highly
distal conserved
heme side,imidazole ring ofa
the iron binds
histidine residue linking the heme to the protein. On the distal heme side,
water molecule. This H2O molecule is replaced by H2O2 upon activation of a peroxidase [28,32]. the iron binds a water
molecule. This H O molecule is replaced
(3) The peroxidases 2are characterized by the specific by H O upon activation of a peroxidase [28,32].
2 2 location of amino acids in the active center
(3) Thefor effective coordination and use of H2O2location
peroxidases are characterized by the specific . On the of amino
distalacids
heme in the active
site, center for
a conserved
effective coordination and use
histidine-arginine couple is involved of H O
2 in . On the distal heme site, a conserved
2 a hydrogen peroxide network [28,33]. histidine-arginine
couple is involved in a hydrogen peroxide network [28,33].
If a catalytic center of a hemoprotein meets the requirements listed above, H2O2 is specifically
If a catalytic
positioned in thecenter of a hemoprotein
peroxidase catalytic centermeets andthe requirements
transfers its highlisted above,
oxidation H2 O2 isto
potential specifically
the heme.
positioned
Two electronsin the areperoxidase
transferredcatalytic
from the center
enzyme andtotransfers its high
H2O2, which oxidation
is reduced potential
into water, to the heme.
whereby the
Two
heme electrons are to
is oxidized transferred
Compound from the enzyme
I (Figure to H2 O2 , Iwhich
1). Compound has twois reduced
oxidizing into water, whereby
equivalents: one inthe
the
heme is oxidized
oxyferryl heme centerto Compound
and the Isecond
(Figureis1). Compound
spread I has
over the two oxidizing
porphyrin ring as equivalents:
a porphyrin one in the
π-cation
oxyferryl
radical. heme center and the second is spread over the porphyrin ring as a porphyrin π-cation radical.

Figure 1. Peroxidase and halogenation cycles of human true peroxidases [34–36]. The interaction
Figure 1. Peroxidase and halogenation cycles of human true peroxidases [34–36]. The interaction
between hydrogen peroxide and native ferric peroxidase heme leads to the reduction of H2 O2 to water
between hydrogen peroxide and native ferric peroxidase heme leads to the reduction of H2O2 to
with formation of Compound I which is oxoferryl porphyrin-π-cationic radical. Compound I can be
water with formation of Compound I which is oxoferryl porphyrin-π-cationic radical. Compound I
sequentially reduced to the native enzyme via formation of Compound II by two one-electron oxidations
can be sequentially reduced to the native enzyme via formation of Compound II by two
caused by a number of simple compounds, peroxidase substrates (peroxidase cycle). AH is a peroxidase
one-electron oxidations caused by a number of simple compounds, peroxidase substrates
substrate that is oxidized with formation of a radical (A• ). Compound I can catalyze the two-electron
(peroxidase cycle). AH is a peroxidase substrate that is oxidized with formation of a radical (A•).
oxidation of (pseudo)halides, thus completing the so-called halogenation cycle. X− stands for halide
Compound I can catalyze the two-electron oxidation of (pseudo)halides, thus completing the
ions (Cl− , Br− , I− ) and thiocyanate ions (SCN− ). HOX is the corresponding (pseudo)hypohalous acid.
so-called halogenation cycle. X− stands for halide ions (Cl−, Br−, I−) and thiocyanate ions (SCN−). HOX
is the corresponding
Compound I has very(pseudo)hypohalous acid. with a reduction potential above 1.1 V, and it can
high oxidizing ability,
oxidize many simple substances—the peroxidase substrates—through a one-electron mechanism via
Compound Compound
the intermediate I has very high oxidizing ability,
II—PorFe(4+)=O. with
At the enda reduction potential
of the reaction, abovecenter
the active 1.1 V, returns
and it can
to
oxidize many simple substances—the peroxidase
the native form, completing the peroxidase cycle. substrates—through a one-electron mechanism
via the intermediate
Participation Compound
in the metabolismII—PorFe(4+)=O. At the
of nitric oxide and end of theisreaction,
its derivatives the active
an important center
function of
returns to the native form, completing the peroxidase cycle.
hemoproteins, including those that have peroxidase activity. This topic is discussed in a number of
papersParticipation
and reviewsin[37–40].
the metabolism of nitric oxide and its derivatives is an important function of
hemoproteins, including
This review summarizes those the
that author’s
have peroxidase activity.
experience This topic
in working is discussed
with different in a number of
hemoproteins
papers and reviews [37–40].
possessing peroxidase activity. Under inflammation or cellular disorders, relatively high levels of
This review summarizes the author’s experience in working with different hemoproteins
possessing peroxidase activity. Under inflammation or cellular disorders, relatively high levels of
H2O2 can be generated, fueling peroxidase activity. Regulable production of oxidants and ROS is
important for immune defense or as meaningful signals, but if out of control, can lead to the
development of oxidative stress. Oxidative stress is implicated in the initiation and development of
various pathologies. Antioxidants (glutathione, ascorbate, uric acid, etc.) are the first line of defense
Molecules 2018, 23, 2561 4 of 27

H2 O2 can be generated, fueling peroxidase activity. Regulable production of oxidants and ROS
is important for immune defense or as meaningful signals, but if out of control, can lead to the
development of oxidative stress. Oxidative stress is implicated in the initiation and development
of various pathologies. Antioxidants (glutathione, ascorbate, uric acid, etc.) are the first line of
defense against radical species and oxidants. However, under oxidative stress, antioxidants are rapidly
depleted, and then specific mechanisms to reduce oxidant production, including mechanisms for the
inhibition of peroxidase activity, become significant. Due to differences in the structure and functions of
peroxidases, the processes through which protein activities are limited differ. A variety of mechanisms
protecting our body from excessive peroxidase activity prevent the transition of local disorders into
systemic diseases.

2. Diversity of Human Hemoproteins with Peroxidase Activity

A wide variety of peroxidases exist, but most of them are plant, fungi, or bacteria peroxidases:
horseradish peroxidase, ascorbate peroxidase, yeast cytochrome c peroxidase, lignin peroxidase
etc. [41–43]. In the human body, fewer proteins display significant peroxidase activity. These enzymes
can be divided into two groups: enzymes that are peroxidases by nature (true peroxidases),
and enzymes that are converted into peroxidase under the influence of external conditions
(pseudo-peroxidases) [21,25,26].

2.1. True Peroxidases

True peroxidases are enzymes whose main function is to the generate oxidants and ROS.
Myeloperoxidase (MPO), eosinophyl peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase
(TPO) are homologous members of the mammalian peroxidase family. With the exception of TPO,
the major physiological function of these enzymes is to contribute to innate immunity [21].
Compound I of the true peroxidases can be reduced by halide ions (Cl− , Br− , I− ) or by
thiocyanate (SCN− ) via a two-electron mechanism (Figure 1). Reduction potentials of in vivo relative
oxidants produced by peroxidases are presented in Table 1 (Supplementary Materials, Section S1).
The capacity of human peroxidases to generate cytotoxic hypohalous (HOX, X = Cl− , Br− , and I− ) and
hypothiocyanous (HOSCN) acids accounts for their antimicrobial activity [33,44,45].
Although plasma concentration of SCN− is equal to that of Br− and 1000-fold lower than Cl−
(20–120 µM, 20–100 µM, and 100–140 mM, respectively), the relative abundance of SCN− in biological
fluids and its better electron donor capacity make it one of the main substrate for Compound I of
peroxidases [22,33]. HOCl and HOBr are extremely powerful oxidants that react with biomolecules at
diffusion-controlled rates. As any powerful oxidant (reduction potential >0.9 V; Table 1), they diffuse
only a short distance and nonspecifically react with different biomolecules, causing considerable
damage within a small radius of their production site [25]. Due to its lower reduction potential,
HOSCN is a relatively long-lived oxidant that reacts specifically with SH groups. It can transfer
oxidative potential of a peroxidase Compound I for longer distances with less harm to host tissues [46].
Bacteria proteins are sensitive to SH group oxidation [47].
Due to the low iodide concentration in vivo (<1 µM) with the exception of the thyroid gland,
iodide oxidation by peroxidases is of little importance [33,36].
MPO, EPO and LPO are specifically structured for the synthesis of strong oxidants. Their active
center is composed so that the protein moiety is not substantially damaged by local fire in the form of
Compounds (Figure 1) and its oxidizing ability is directed toward the oxidation of substrates:
(1) The heme of the peroxidases is attached to protein moiety by at least two covalent bonds.
The covalent linkages stabilize the position of the heme moiety, which is important for improving
the redox ability of the heme and for the proper structural architecture of the substrate binding
site [48].
(2) True peroxidases do not have oxidizable amino acids in close proximity to heme that are able to
compete with external substrates for Compound I (except for cyclooxygenase).
Molecules 2018, 23, 2561 5 of 27

The activity of the enzyme toward guaiacol, a hardly oxidizable phenolic compound, can serve
as a test for the participation of peroxidase Compounds but not protein-derived radicals in substrate
oxidation [49].
The substrate binding site of peroxidases is connected to the surface through a hydrophobic
channel [28,50]. These enzymes prefer small anionic molecules as electron donors, such as halides,
thiocyanate and small peroxidase substrates. Though the main function of true human peroxidases is
the production of hypohalous acids and HOSCN, these enzymes can oxidize peroxidase substrates
at neutral pH in the plasma. Numerous small molecular substrates, including phenolic compounds,
aromatic amino acids, urate, ascorbate, nitrite, nitric oxide, serotonin, and others, are oxidized in the
peroxidase cycle into radicals (Table S1) [51–56]. The oxidation of nitrite to nitrogen dioxide (• NO2 )
and of tyrosine to tyrosyl radical (Tyr-O• ) forms potent oxidants that can participate in immune defense
and simultaneously contribute to damaging host tissues (Table 1) [22].
The other two members of the true peroxidase family, namely cyclooxygenase (COX) and
peroxidasins, are characterized by a multidomain structure and highly targeted catalytic activity.
The major function of these enzymes is the oxidation of specific molecules.
COX (or prostaglandin H synthase) plays a key role in controlling the biosynthesis of various
physiologically important prostaglandins. Contrary to other true peroxidases, the protein-derived
(tyrosyl) radical is formed in the peroxidase cycle of COX. As a part of the COX catalytic cycle,
this active radical is closely controlled and cannot oxidize external peroxidase substrates. Its redox
activity is directed toward the oxidation of arachidonic acid, whose binding site is situated close to the
tyrosyl radical [57].
The recently identified members of animal heme peroxidase family are peroxidasins (peroxidasin
1 and peroxidasin 2). Peroxidasin 1 is secreted to extracellular matrix and catalyzes the oxidation of
bromide to hypobromous acid. HOBr mediates the formation of specific sulfilimine cross-links of
collagen IV in the basement membrane, which is important for the structural integrity of extracellular
matrix [58,59]. As with TPO, COX and peroxidasins generate oxidants to perform biosynthesis tasks.
These enzymes cannot produce excess of highly active oxidants and ROS and initiate oxidative stress.

2.2. Pseudo-Peroxidases
Pseudo-peroxidases are hemoproteins that perform different important functions in the body
and initially are not meant to interact with H2 O2 . However, due to changes in external conditions,
the characteristics of their active center can change, and they can display peroxidase-like activity.
The pseudo-peroxidase active sites are not specifically designed to catalyze the H2 O2 reduction to
water. The rate of reaction of heme with H2 O2 is rather low, so the first step of the pseudo-peroxidase
reaction is rate-limiting (Table 1). Therewith, hydroxyl radicals can be formed in the active center
due to homolytic splitting of H2 O2 [60,61]. Nevertheless, these proteins consume H2 O2 and oxidize
peroxidase substrates, so they may be considered as peroxidases.
Pseudo-peroxidases are different in terms of their heme and activity characteristics, but all of them
have one common feature: exposure to H2 O2 causes immediate oxidation of protein amino acids that
are close to the active center (most commonly tyrosine, tryptophan, or histidine) [62,63]. Compound
I was not detected for pseudo-peroxidases. The reaction of heme with H2 O2 results in formation of
oxoferryl heme PorFe(IV)=O, equivalent to Compound II of true peroxidases, and protein-derived
radicals (Figure 2). The protein-based tyrosyl radicals are the alternative reactive intermediates for the
oxidation of larger substrates that cannot access the heme pocket [64]. Hydroxyl radicals, which can
be formed in the pseudo-peroxidase active site due to the homolytic splitting of H2 O2 , are unlikely
responsible for tyrosyl radical formation. First, hydroxyl radicals are non-specific oxidants. Secondly,
phenoxyl radicals are the minor products of the reaction of HO• with phenolic compounds (<5%)
(Figure S1) [65–67].
Molecules 2018, 23, x FOR PEER REVIEW 6 of 27
Molecules 2018, 23, 2561 6 of 27

Figure 2. Catalytic cycle of pseudo-peroxidases [40,63,68,69]. The interaction between hydrogen

Figure 2. Catalytic
peroxide and nativecycle
ferricofpseudo-peroxidase
pseudo-peroxidasesheme [40,63,68,69].
leads the The interaction
formation between hydrogen
of Compound I which is
peroxide andoxoferryl
most likely native ferric pseudo-peroxidase
porphyrin-π-cationic heme Compound
radical. leads the formation of Compound
I immediately I whichacid
oxidizes amino is
residues (Tyr, Trp, His) which are located near the heme with formation of protein
most likely oxoferryl porphyrin-π-cationic radical. Compound I immediately oxidizes amino acid based radicals and
oxoferryl(Tyr,
residues heme. Oxoferryl
Trp, hemeare
His) which iron can oxidize
located protein
near the heme amino
withacids and peroxidase
formation of proteinsubstrates. AH is
based radicals
a peroxidase • ). The protein-based tyrosyl
and oxoferrylsubstrate that is oxidized
heme. Oxoferryl heme with
iron formation
can oxidize of a protein
radical (A amino acids and peroxidase
radicals areAH
substrates. the is
alternative reactive
a peroxidase intermediates
substrate that is ofoxidized
pseudo-peroxidases.
with formation of a radical (A•). The
protein-based tyrosyl radicals are the alternative reactive intermediates of pseudo-peroxidases.
Pseudo-peroxidases can be divided into two groups:
Pseudo-peroxidases can be divided into two groups:
(1) Proteins in which the heme iron initially does not have an amino acid ligand in a distal
(1) Proteins in which
coordination the heme iron initially
position—hemoglobin (Hb) and does not have
myoglobin an amino acid ligand in a distal
(Mb).
(2) coordination position—hemoglobin
Proteins in which the heme iron has (Hb) a sixthand myoglobinbond,
coordination (Mb).but under the influence of external
(2) Proteins
factors, inthewhich the hemeofiron
configuration has a site
the active sixthcancoordination
change, and bond, but under
this newly the influence
conferred peroxidaseof
external
activity factors,
plays anthe configuration
important role inof thethe active
cells, site can
unrelated to change, and biological
the primary this newlyfunctions
conferred of
peroxidase
the enzymes. activity plays an important role in the cells, unrelated to the primary biological
functions of the enzymes.
Cyt c and cytoglobin (Cygb) are hexacoordinated proteins with methionine/histidine residues
Cyt c and
occupying cytoglobin
the sixth (Cygb)sites
coordination are of
hexacoordinated
the heme iron (for proteins with methionine/histidine
Cygb—under reducing conditions).residues
Anionic
occupying the sixth coordination sites of the heme iron (for Cygb—under
lipids can break this bond and awaken the dormant peroxidase activity of Cyt c and Cygb [26,70–72]. reducing conditions).
AnionicLow lipids can break
temperature this bond
electron and awaken
paramagnetic the dormant
resonance (LT EPR)peroxidase activity
spectroscopy is a of cyt c and
powerful toolCygb
used
[26,70–72].
to study peroxidase activity of hemoproteins (Figure S2). EPR signal at a g-factor of about six (g ~ 6)
Low temperature
evidences the absence of electron
a ligandparamagnetic resonance (LT
at the sixth coordination EPR) of
position spectroscopy
iron. Proteinisoxidation
a powerful andtool
the
used to study peroxidase activity of hemoproteins (Figure S2). EPR signal at
formation of protein-derived (tyrosyl) radicals can be monitored by acquiring the spectra of tyrosyl a g-factor of about six
(g ~ 6) evidences
radicals the absence
at a g-factor of about of twoa ligand
(g ~ 2).atNottheonly
sixthHcoordination position of iron. Protein oxidation
2 O2 , but other small molecules including CO and
and the formation of protein-derived (tyrosyl) radicals can
NO, can interact with iron at the sixth coordination position side. NO binding be monitored by acquiring the spectraand
to both heme-Fe(III) of
tyrosyl radicals at a g-factor of about two (g ~ 2). Not only H 2O2, but other small molecules
heme-Fe(II) changes the optical absorbance spectra of hemeproteins in the Soret band region, but only
including
Fe(II)-NOCO and NO,have
complexes can characteristic
interact with LT ironEPRat the sixthofcoordination
spectra nitrosylated position side. S2).
heme (Figure NO The
binding
LT EPRto
both heme-Fe(III) and heme-Fe(II) changes the optical absorbance spectra
method has been successfully employed for the study of pseudo-peroxidases and provided unique of hemeproteins in the
Soret
useful band region, but
information only Fe(II)-NO complexes have characteristic LT EPR spectra of nitrosylated
[64,72–77].
heme (Figure S2). The LT EPR method has been successfully employed for the study of
pseudo-peroxidases and provided unique useful information [64,72–77].
Molecules 2018, 23, 2561 7 of 27

3. Myeloperoxidase
MPO is the queen of the peroxidase family. It is the only enzyme capable of generating
physiologically significant amounts of HOCl—the potent oxidizing agent that is necessary to fight
invading pathogens [78,79].
The enzyme is stored in the azurophilic granules of neutrophils, comprising approximately 5% of
cellular dry weight. During neutrophil activation, the granules release their contents, including MPO,
into the phagosomes and the extracellular space. Simultaneously, NADPH oxidase assembles on the
plasma membrane and generates superoxide radicals that dismutate to form H2 O2 [1,78]. In inflamed
tissue, steady state concentrations of H2 O2 in extracellular medium can be as high as 100 µM [6,8].
Upon phagocytizing a microbe, MPO is released into the phagosome up to a concentration of about
1 mM [35]. MPO is one of the key components of neutrophil extracellular traps (NETs). NETs are
formed during neutrophil-specific cell death, characterized by the release of DNA strands associated
with histones and decorated with about 20 different proteins [80,81].
Under physiological conditions, MPO Compound I oxidizes similar amounts of SCN− and
Cl− [82]. Nevertheless, a major function of neutrophil myeloperoxidase is the synthesis of HOCl,
which plays a cytotoxic role against bacteria and viruses at inflammatory sites and in phagosomes [79].
The concentration of MPO in plasma is normally in the range of 20–100 ng/mL, whereas under
pathology, these values increase several-fold [83–85]. Despite of the low concentration of MPO
in vivo, the enzyme has been implicated in the development of a number of widespread diseases [21],
such as cardiovascular disorders [83,86–88], Alzheimer’s disease [89,90], kidney diseases [91,92],
and rheumatoid arthritis [93].
The enzyme is a 145–160 kD homodimer comprising two identical subunits joined by a single
disulfide bridge. Each subunit is a complex of a light chain and a heavy chain; the latter contains
heme. In addition to the two ester linkages that are also present in EPO and LPO, the heme in
MPO has a unique sulfonium ion linkage that significantly distorts the prosthetic group from a
planar conformation [48,94,95]. As a result, myeloperoxidase Compound I is the strongest one- and
two-electron oxidant among all peroxidase compounds. The reduction potentials are 1.35 and 1.16 V
for the Compound I/Compound II and Compound I/native MPO couples, respectively [22,96].
The protein does not have oxidizable amino acids near the active site. In the absence of external
substrates, MPO compounds are long-lived oxidants. The stability of Compound II can be observed by
measuring the protein spectra in the Soret band region (Figure 3). The spectrum of the native enzyme
has a maximum at 430 nm. Interaction of MPO heme with H2 O2 leads to the immediate formation
of Compound I. The one-electron oxidation of Compound I by the next molecule of H2 O2 results
in formation of Compound II and superoxide [54]. Compound II has characteristic spectrum with
maximum at 454 nm. Compound II is stable and can wait for a substrate for a relatively long time [51].
Only after ~90 min, the enzyme returns to the native form. The decrease in the magnitude of the
spectrum evidences partial damage of MPO. However, protein-derived radicals cannot be detected by
LT EPR spectroscopy after the reaction of MPO with hydrogen peroxide.
Molecules 2018, 23, x FOR PEER REVIEW 8 of 27
Molecules 2018, 23, 2561 8 of 27

Figure 3. Optical spectra of myeloperoxidase and Cyt c/TOCL complexes after addition of H2 O
Figure 3. Optical spectra of myeloperoxidase and cyt c/TOCL complexes after addition of H 2 [51,97].
2O 2

[51,97]. (A) Upon addition of an excess2of2H2O2 (200 µM), MPO (1.2 µM) undergoes conversion from native
(A) Upon addition of an excess of H O (200 µM), MPO (1.2 µM) undergoes conversion from its
itsform (theform
native Soret(the
bandSoret
maximum
band at 430 nm) to
maximum at Compound
430 nm) toIICompound
having the absorbance
II having the maximum at 454 nm.
absorbance
maximum at 454 nm. In the absence of peroxidase substrates the absorbance at 454 nm slowly shifts
In the absence of peroxidase substrates the absorbance at 454 nm slowly decreases, and the peak
towards 430
decreases, andnm.the The
peakarrow
shifts indicates
towards a decrease
430 nm. The in arrow
the Soret band intensity
indicates a decreasein in
90 the
minSoret
after band
addition of
H O to MPO. (B) A progressive decrease of the absorbance of Cyt c/TOCL during
2 2 in 90 min after addition of H2O2 to MPO. (B) A progressive decrease of the absorbance of
intensity its auto-oxidation
process
cyt c/TOCLinitiated
duringbyitsthe addition of 250
auto-oxidation µM H2initiated
process O2 to thebysolution
the addition of 250c (1.5
of ferri-Cyt H2Otetraoleoyl-CL
µM µM, 2 to the

30 µM).of50ferri-cyt
solution mM sodiumc (1.5 phosphate buffer, 100
µM, tetraoleoyl-CL 30mMµM).NaCl,
50 mMpHsodium
7.4, 100phosphate
µM DTPA.buffer, 100 mM
NaCl, pH 7.4, 100 µM DTPA.
Although the major function of peroxidases in innate immunity is the production of hypohalous
acids and HOSCN,
Although these function
the major enzymes of canperoxidases
also oxidizeinperoxidase substrates
innate immunity is under plasma conditions
the production of
(neutral pHacids
hypohalous and 140
and mM NaCl;these
HOSCN, Tableenzymes
S1) [51,53]. Amino
can also acid peroxidase
oxidize residues of substrates
proteins, such
under asplasma
tyrosine and
tryptophan,
conditions are peroxidase
(neutral pH and 140 substrates
mM NaCl; [98,99].
TableDoes this mean
S1) [51,53]. Amino thatacid
MPO can directly
residues oxidize
of proteins, proteins
such
asintyrosine
plasma?and There are several
tryptophan, are mechanisms for controlling
peroxidase substrates [98,99].the
Does‘fire’ inmean
this the MPO activecan
that MPO sitedirectly
to minimize
oxidize proteins
its adverse in plasma?
impact There are several mechanisms for controlling the ‘fire’ in the MPO
on biomolecules.
active site to minimize its adverse impact on biomolecules.
3.1. Restricted Access to MPO Active Site
3.1. Restricted Access to MPO Active Site
The structure of the MPO active site prevents the enzyme from oxidizing large molecules.
TheThe structure
active site ofofMPO
the MPO active site
is located prevents
at the theaenzyme
base of deep andfrom oxidizing
narrow heme large molecules.
pocket The
inaccessible to
active site of significantly
compounds MPO is located at the
larger thanbase of a deep[100,101].
a dipeptide and narrow For heme pocket inaccessible
macromolecule oxidation, to small
compounds
substrates are significantly
needed, larger
which than a dipeptide
are the [100,101].
intermediates For macromolecule
between Compounds and oxidation, small
the biomolecules.
substrates are needed, which are the intermediates between Compounds and
At pH < 7, the major oxidant is HOCl. At neutral pH, free radicals with high reduction potentialthe biomolecules. At
pH < 7, the major oxidant is HOCl. At neutral pH, free radicals with high reduction
formed in the peroxidase cycle, mainly phenoxyl radicals and nitrogen dioxide (Tyr-O• , • NO2 ), can potential
formed
mediate in oxidation
the peroxidase cycle,constituents
of plasma mainly phenoxyl(Tableradicals
1). and nitrogen dioxide (Tyr-O●, ●NO2), can
mediate oxidation of plasma constituents (Table 1).
The accessibility of the MPO active site can be tested by substrates with different characteristics.
The accessibility of the MPO active site can be tested by substrates with different
Table 2 presents the activity of different hemoproteins toward phenolic compounds that are peroxidase
characteristics. Table 2 presents the activity of different hemoproteins toward phenolic compounds
substrates, namely guaiacol, Amplex red and etoposide. The substrates differ in molecular mass and
that are peroxidase substrates, namely guaiacol, Amplex red and etoposide. The substrates differ in
reduction potentials (Figure S3). Guaiacol oxidation with formation of tetraquaiacol can be observed
molecular mass and reduction potentials (Figure S3). Guaiacol oxidation with formation of
only by peroxidase
tetraquaiacol Compounds.
can be observed only byMPO activity
peroxidase toward thisMPO
Compounds. indicative
activitysubstrate
toward thisis about 1000-fold
indicative
higher than for other proteins. Two other tested substrates—Amplex Red and etoposide—have lower
reduction potentials compared to quaiacol. So, they are expected to be oxidized more easily. However,
MPO activity toward Amplex red is more than 10 times lower, and that for etoposide is 3 × 104 times
Molecules 2018, 23, 2561 9 of 27

lower than activity toward guaiacol. Despite its low reduction potential (Eo = ~0.56 V), etoposide is a
very poor substrate for MPO because access of this large phenolic compound (MW = 589 D) to the
MPO active site is highly constrained [102,103].

Table 2. Peroxidase activity of heme-containing proteins toward different phenolic compounds (25 ◦ C;
pH 7.4; H2 O2 = 100 µM) [69,72,75,103,104].

Guaiacol * Etoposide **
Amplex Red **
Substrate:Protein MW = 127 D MW = 589 D
MW = 257 D
Eo ~ 0.95 V Eo ~ 0.56 V
Hemoglobin 2.26 ± 0.35 10.5 ± 1.8 0.43 ± 0.05
Myoglobin 0.90 ± 0.05 2.2 ± 0.2 0.34 ± 0.02
Cyt c/cardiolipin (1:20) 0.15 ± 0.06 1.40 ± 0.15 0.41 ± 0.02
Cytoglobin (S-S) 0.30 ± 0.05 3.2 ± 0.20 0.28 ± 0.04
MPO 660 ± 50 54.5 ± 3.2 0.020 ± 0.005
MPO + phenol - 355 ± 45 19.5 ± 2.5
* µmol product/min × nmol protein; ** ∆magnitude (A.U.)/min × * nmol protein; Experimental conditions are
summarized in Supplementary Materials, Section S2.

The small phenolic molecules may act as co-substrates for oxidation of large and bulky
compounds, such as etoposide, because they have free access to the MPO active site and due to
the relatively high oxidizing potential of their phenoxyl radicals compared with etoposide (Table 2,
compare MPO and MPO + phenol) [103]. In this case, nonspecifically reactive phenoxyl radicals, which
oxidize different biomolecules, are converted to etoposide-O• , which reacts specifically with GSH
and protein SH groups. As the etoposide phenoxyl radical is relatively long-lived compared with the
phenol radical [102], the oxidative potential of MPO can be more readily transferred. The activity of
many proteins is sensitive to the redox state of thiols [105]. The re-oxidation of phenolic compounds
results in the decrease in MPO oxidative ability (reduction potentials: Compound I > tyrosyl-O• >
etoposide-O• ). Tyrosine is the only phenolic compound that is present in plasma in free form (Table S1).
Other phenolic compounds can enter the body as medicines or with food. These phenolic compounds
may be antioxidants (flavonoids) or may have a weak oxidative activity, thereby altering the profile of
MPO-induced damage to biomolecules [106–109].

3.2. Ceruloplasmin Is an Endogenous MPO Inhibitor

The interaction of highly cationic MPO (pI ~ 10) with anionic proteins and with the negatively
charged surfaces of different cells has been suggested to be mostly dependent on electrostatic
interactions [110]. The weak binding of MPO to albumin and binding to lipoproteins have been
demonstrated [4,84,111]. Several negatively charged proteins are present in plasma at micromolar
concentrations, like ceruloplasmin (CP), fibrinogen, and haptoglobin. Immunoprecipitation
experiments revealed that CP is the major protein in plasma that is associated with MPO [112,113].
CP is a globular anionic copper containing plasma glycoprotein (pI ~ 4.4, MW ~ 132 kD) that
has multiple functions, the main one of which is ferroxidase activity, i.e., oxidation of redox-active
Fe2+ to Fe3+ . Trapping of Fe3+ by transferrin prevents iron participation in redox chemistry [114].
CP is an abundant acute phase protein whose concentration can reach the value of 4–10 µM with
pathologies [115,116].
CP binds to MPO with stoichiometry 2:1: one molecule of CP binds to each monomer of MPO with
a dissociation constant of ~0.15 µM [117]. Binding of CP in close proximity to the MPO active center
results in inhibition of both the halogenating and peroxidase activities of MPO [118–121]. The degree
of inhibition depends on the size of the peroxidase substrate, suggesting that CP hinders access of
larger substrates to the MPO active site [121,122]. Reduction of Compound I to Compound II and
then retardation of the turnover of Compound II to native enzyme is the mechanism of CP-induced
inhibition of MPO activity [113]. Oxidation of ascorbate by MPO was observed in plasma from
Molecules 2018, 23, 2561 10 of 27

CP-knock-out mice, but no significant loss of the antioxidant in plasma from wild type animals
was detected [113]. Partially proteolized or thrombin-damaged CP loses the ability to inhibit MPO.
Therefore, thrombin can exacerbate inflammation and development of pathologies by impairing MPO
inhibitory function of CP [123].

3.3. pH-Dependent Rregulation of MPO Activity

The chlorinating activity of MPO decreases with increasing pH [124,125]. The rate constant of the
reaction of Cl- with Compound I is about 150-fold higher at pH 5.0 compared to pH 7.0, but this does
not imply a decrease in the oxidative capacity of MPO. At acidic pH, MPO catalyzes the formation of
HOCl, whereas oxidation of phenolic substrates in the peroxidase cycle can be observed at neutral
pH [104,126]. The ratio between peroxidase and chlorinating MPO activities is important for targeted
synthesis of HOCl at inflammatory sites and in phagosomes, where pH may be shifted into the acidic
range [127].
Peroxidase substrates can dose-dependently inhibit the formation of HOCl at neutral
pH [104,128,129]. The substrates of the MPO peroxidase cycle are present in plasma and the intracellular
space in low concentrations, but their diversity suggests that they may compete with halide ions during
interaction with Compound I (Table S1).

4. Eosinophil Peroxidase and Lactoperoxidase

The standard reduction potential of the redox couple Compound I/ferric enzyme of human
true peroxidases falls in the order of MPO (1.16 V) > EPO (1.10 V) > LPO (1.09 V), which is
proportional to the reduction potential of oxidants the peroxidases can produce in the halogenation
cycle (Table 1) [22,96,130]. The difference in the oxidative ability of the enzymes is due to the variations
in the active site structure and properties [28,50].
Eosinophil peroxidase is the major granule enzyme of eosinophils, which are the phagocytic
cells recruited against invading bacteria, viruses, protozoans and helminths [45]. EPO is a monomer
comprised of light and heavy chains (57 and 11 kD, respectively). Heme is bound by one ester
bond to the protein, whereas a second ester linkage is formed autocatalytically in the presence of
hydrogen peroxide [131]. At normal plasma conditions, the enzyme catalyzes oxidation of bromide
and thiocyanate to HOBr and HSCN, respectively. The enzyme has limited ability to generate HOCl
at acidic pH, so the EPO-induced chloride oxidation in vivo is meaningless [132]. EPO plays a
prominent deleterious role in pathogenesis of various common human allergic disorders, including
asthma [133–135].
More than 60% of amino acids residues are identical in EPO and MPO, and even higher homology
was demonstrated for the active-site-related residues [136]. Expectedly, the mechanisms of protection
against undesirable redox activity of EPO are similar to those described above for MPO. If the pH
of the medium changes from 7 to 5, the brominating activity increases by an order of magnitude
(from 10−7 to 10−8 M−1 s−1 ) [132]. The active center of EPO is inaccessible to plasma macromolecules,
similar to MPO [50]. EPO forms a complex with CP, but the stoichiometry of binding is 1:1. CP inhibits
the peroxidase activity of EPO but does not affect the brominating activity of the enzyme [137].
Lactoperoxidase was found in mucosal surfaces and exocrine secretions including milk, tears,
and saliva, and in nasal and lung airway fluids. LPO is a major contributor to airway defenses and to
the antimicrobial properties of exocrine gland secretions [33,36,138,139].
LPO is monomeric hemoprotein (MW ~ 78 kD). The prosthetic group is bound to the protein
autocatalytically; noncovalently bound heme becomes covalently bound through two ester linkages
after enzyme exposure to H2 O2 [140]. At physiological conditions, LPO has barely detectable activity
with bromide but oxidizes iodide and thiocyanate. SCN− is the physiological substrate of LPO because
it is much more abundant than iodide [33,141,142] Normal plasma levels of thiocyanate are 20–120 µM
(iodide < 1 µM). Thiocyanate and iodide are actively accumulated in secretory epithelial cells through
uptake from blood by the sodium–iodide symporter. The maximum concentration of thiocyanate was
Molecules 2018, 23, 2561 11 of 27
Molecules 2018, 23, x FOR PEER REVIEW 11 of 27

maximum
observed concentration
in saliva (2–4 mM), of thiocyanate
and that of was observed
iodide was inin saliva
milk (2–4 mM),
(2 µM) [33,36].and that of formed
HOSCN iodide was in
by LPO
is amilk
weak (2 µM) [33,36].
oxidizing HOSCN
agent that formed by LPO iswith
reacts primarily a weak oxidizing
thiols agent that
and inhibits reacts primarily
microbial metabolism withand
thiols and
growth [47,142]. inhibits microbial metabolism and growth [47,142].
TheThe substratechannel
substrate channelofofLPO LPOisis longer,
longer, narrower,
narrower, and and more
morehydrophobic
hydrophobiccompared compared to to
that of of
that
MPO,MPO, which
which preventsdirect
prevents directoxidation
oxidationofofmacromolecules
macromolecules by by the
the enzyme
enzyme[36,50].
[36,50].
Thyroid peroxidase is localized in membranes of thyroid
Thyroid peroxidase is localized in membranes of thyroid follicle cells. The follicle cells. The enzyme
enzyme generates
generates HOI
and is involved in the synthesis of the thyroid gland hormones: thyroxine and triiodothyronineand
HOI and is involved in the synthesis of the thyroid gland hormones: thyroxine [21].
triiodothyronine [21].
5. Hemoglobin and Myoglobin
5. Hemoglobin and Myoglobin
The main function of respiratory hemoproteins, such as Hb and Mb, is the storage and delivery
The main function of respiratory hemoproteins, such as Hb and Mb, is the storage and delivery
of oxygen into cells. Both proteins are members of the globin family, which also includes Cygb and
of oxygen into cells. Both proteins are members of the globin family, which also includes Cygb and
neuroglobin. Mammalian globins have a typical structure: a protein consists of six to eight helical
neuroglobin. Mammalian globins have a typical structure: a protein consists of six to eight helical
chains arranged in a three-on-three helix “sandwich”, forming the hydrophobic pocket of the active
chains arranged in a three-on-three helix ‘‘sandwich’’, forming the hydrophobic pocket of the active
center containing heme [25]. All members of the globin family possess NO dioxygenase activity and
center containing heme [25]. All members of the globin family possess NO dioxygenase activity and
participate in NO metabolism [143–145].
participate in NO metabolism [143–145].
HbHbconsists
consistsof of four identicalsubunits
four identical subunits (MW(MW ~ 64~kD),
64 and
kD),Mb andis aMb is a monoglobular
monoglobular protein (MW protein
~
(MW 16.7~ kD).
16.7 Proteins
kD). Proteinsperform perform their functions,
their functions, having
having iron ironactive
in the in the active
center in center in the(Fe(II))
the ferrous ferrous
(Fe(II))
state state
with with free sixth
free sixth coordination
coordination bond.bond. The characteristic
The characteristic EPR signal
EPR signal of openofhemeopen measured
heme measuredat g
at g ~ 6 can be detected from the proteins at the temperature of liquid
~6 can be detected from the proteins at the temperature of liquid nitrogen (77 K), and hemenitrogen (77 K), and heme
can becan
be easily
easilynitrosylated
nitrosylated(Figure
(Figure4)4)[69,146].
[69,146].There
Thereare
areoxidizable
oxidizable amino
amino acids
acids in in close
close proximity
proximity to heme;
to heme;
thetheaddition
addition ofofHH O
2 22 O to
2 toprotein
protein solutions
solutions causes
causes formation
formation of
of protein-derived
protein-derived radicals
radicals [64,147].
[64,147].

Figure Low-temperature
4. 4.
Figure Low-temperatureEPR EPR(77(77K)K) spectra
spectra of Hb
Hb and
and MbMb[69]:
[69]:(A)
(A)low
lowfield
fieldEPR
EPR signal
signal of of ferric
ferric
myoglobin;
myoglobin; (B)(B)
a typical
a typicalLT
LTEPR
EPRsignal
signalofofheme-nitrosylated Hb(II);(C)
heme-nitrosylated Hb(II); (C)spectrum
spectrumofof protein-derived
protein-derived
(tyrosyl)
(tyrosyl) radicals
radicals measuredinin3030s safter
measured afteraddition
additionof H22O22 to
of H toHb.
Hb.

The
Theredox
redoxactivity
activity of
of Hb and
and Mb Mbisissuppressed
suppressed in in myocytes
myocytes andand erythrocytes,
erythrocytes, respectively,
respectively, by
bythe
thereducing
reducingenvironment
environmentofofthe thecells.
cells.The
The ferric
ferric form
form of of
HbHb comprises
comprises about
about 1%1% of of total
total HbHb in in
erythrocytes.
erythrocytes. Ferric
Ferric HbHb is effectively
is effectively converted
converted into ferrous
into ferrous protein
protein by metHb
by metHb reductasereductase [148].
[148]. Myocyte
Myocyte
injury injury orcause
or hemolysis hemolysis cause hemoprotein
hemoprotein leakage into leakage
tissuesinto
andtissues
blood and
wherebloodthe where
globinsthe doglobins
not have
do not have their normal reducing environment. Since the redox chemistry
their normal reducing environment. Since the redox chemistry is the same for Hb and Mb, is the same for Hbbutandthe
Mb, but the
aggravation of aaggravation
number of of a number isofmore
pathologies pathologies
associatedis more
with associated
hemolysis with
and the hemolysis
release ofandHbtheinto
release of Hb into the plasma, this chapter
the plasma, this chapter mainly focuses on hemoglobin.mainly focuses on hemoglobin.
AA constant
constant processofofmild
process mildintravascular
intravascular erythrocyte
erythrocyte damage
damageoccurs
occursunder
undernormal
normal conditions,
conditions,
so that free hemoglobin appears in plasma in small amounts. The inadvertently appearing ferric
so that free hemoglobin appears in plasma in small amounts. The inadvertently appearing ferric
form of Hb can be converted into ferrous Hb by ascorbate. Given the lack of oxidizing equivalents,
form of Hb can be converted into ferrous Hb by ascorbate. Given the lack of oxidizing equivalents,
the protein cannot display any peroxidase activity.
the protein cannot display any peroxidase activity.
Hemolysis occurs in many diseases such as trauma, sepsis, and sickle cell disease. Under some
Hemolysis occurs in many diseases such as trauma, sepsis, and sickle cell disease. Under some
pathological conditions, particularly as a result of microangiopathic hemolytic anemia,
pathological conditions, particularly as a result of microangiopathic hemolytic anemia, concentrations
concentrations of Hb in plasma can reach micromolar levels [149]. Under uncontrolled pathological
of conditions
Hb in plasma can reachby
accompanied micromolar
inflammation levels
and [149]. Understress,
oxidative uncontrolled
depletionpathological
of antioxidantsconditions
and
accompanied
generation of by superoxide
inflammation and oxidative
radicals by activated stress, depletioninduce
neutrophils of antioxidants
the redoxand generation
activity of the of
superoxide radicals
hemoproteins, by activated
resulting neutrophils
in damage induce
to plasma the redox
proteins andactivity
lipids of theThe
[62]. hemoproteins,
globin-based resulting
free
in damage
radicals of ferryl hemoglobin were detected in normal human blood upon H2O2 treatment [150]. were
to plasma proteins and lipids [62]. The globin-based free radicals of ferryl hemoglobin
detected in normal human blood upon H2 O2 treatment [150].
Molecules 2018, 23, 2561 12 of 27

Ferrous Hb is activated by H2 O2 to the oxoferryl form, equivalent to Compound II. The second
molecule of H2 O2 can convert the active site into the ferric form. The ferric form of the protein
is oxidized by H2 O2 to Compound I, which, in the absence of exogenous peroxidase substrates,
immediately oxidizes a tyrosine residue near the heme [77,151,152]. The peroxidase substrates can be
oxidized in two processes: by Compounds, if heme in hydrophobic pocket is accessible for a substrate;
or by a tyrosyl radical, which is exposed to the peroxidase surface (Figure 2) [64]. The relatively high
oxidation ability of Hb and Mb toward guaiacol compared to Cyt c/CL supports the participation of
Compounds in substrate oxidation (Table 2). Mediation of a substrate oxidation by tyrosyl radicals
accelerates heme reduction but decreases the oxidative potential of a peroxidase.
Self-oxidation of Hb and formation of protein-derived (tyrosyl) radicals result in protein
cross-linking and aggregation. Tyrosyl radicals recombine with the formation of very stable
carbon-carbon covalent bonds between the proteins. This process involves not only a hemoprotein
itself but other proteins in plasma. As a result, large protein hetero-oligomers (aggregates) occur [69].
Reeder et al. demonstrated the formation of a covalent link between the heme and protein
moiety for Hb or Mb treated with hydrogen peroxide [25,153]. Globins with covalently-linked
heme showed higher peroxidase activity and cellular toxicity. Oxidation of low density lipoproteins
by heme-to-protein cross-linked Mb was five times greater than that of native protein [154].
The heme-to-protein cross-linked Mb or Hb are stable forms of the proteins, and the authors identified
them in urine, kidney, and cerebrospinal fluid [25].
Contrary to Cyt c or Cygb, the peroxidase activity of Hb does not make any biological sense, so it
should be neither regulated nor limited but just abrogated. There are two special acute phase proteins
in plasma whose major function is to prevent adverse unintended consequences of Hb release [155].

5.1. Haptoglobin
Haptoglobin (Hp) is an abundant plasma protein with high binding affinity toward Hb
(Kd ~ 10−15 M, [156]), reminiscent of an antigen-antibody reaction. Human haptoglobin occurs as
three major phenotypes: Hp 1-1, Hp 2-1, and Hp 2-2. The phenotypes differ in the protein structure
and their distribution throughout different populations in different locations [157]. Nevertheless,
all Hp types function to bind free Hb and to facilitate its delivery into macrophages via the CD163
receptor-mediated pathway [158–161]. Circulating haptoglobin provides an important endogenous
defense against the toxic effects of Hb.
Hp is an acute phase protein; its normal plasma level ranges from 0.1 to 2 mg/mL, which increases
two- to five-fold during inflammation [12,156]. These amounts of Hp are sufficient to bind micromolar
levels of free Hb [69].
Trapping of free Hb by Hp results in the formation of Hb-Hp complexes. Hp protects Hb from
heme dissociation, thus preserving the peroxidase activity of hemoprotein. Hb-Hp complex formation
does not alter Hb’s ability to consume H2 O2 [24]. In complex with Hp, the oxidizing potential of Hb
is partially re-directed toward oxidation of Hp, thus delaying the oxidation of plasma constituents.
Oxidation of Hb-Hp complexes causes covalent cross-linking of the proteins and the formation of large
aggregates. The ratios of peroxidase activity were 100:50:10 for Hb, Hb-Hp complexes, and Hb-Hp
aggregates, respectively. The aggregates still displayed significant peroxidase activity toward external
molecules [69].
Hb-Hp aggregates were uptaken by macrophages at rates exceeding those for Hb-Hp complexes.
The engulfed Hb-Hp aggregates induced intracellular oxidative stress and a dose-dependent
cytotoxicity. Neither Hb nor Hb-Hp complexes showed significant toxic effects under the same
incubation conditions [69]. Long-living protein-based (tyrosyl) radicals in Hb-Hp aggregates have
high oxidizing potential and may be responsible for the cytotoxicity of Hb-Hp aggregates. Notably,
the lifespan of protein-based radicals in aggregates (10–20 min) is comparable to the lifespan of Hb-Hp
complexes in blood (<10 min) [12,69].
Molecules 2018, 23, 2561 13 of 27

Hb-Hp aggregates were found in septic plasma [69]. In the hemolysis setting, Hb may intensify the
potentially fatal effects of sepsis syndrome in patients with trauma, infection, or hypotension [162,163].
Mb, released from damaged muscle (rhabdomyolysis), cannot be bound by Hp, leading to kidney
damage [62].

5.2. Hemopexin
Hx is a plasma protein that functions to sequestrate free heme or extract heme from other proteins
and transport it into hepatocytes by receptor-mediated endocytosis [12]. The enzymatic degradation of
heme by the heme oxygenase enzyme system leads to the formation of biliverdin, with the concomitant
liberation of CO and reduced heme iron [164]. The human body generates more than 12 mL of CO per
day from heme catabolism [165].
In adults, serum Hx concentration ranges between 0.4 and 1.5 g/L. In the acute phase,
Hx concentration increases two- to three-fold [12,155]. When Hp is exhausted, free ferrous Hb can be
oxidized in plasma to its ferric form. Non-covalently bound heme of Hb(III) can be easily uptaken by
hemopexin (but not from ferrous Hb or Hb bound to Hp) [155,166].
When free Hb(III) is exposed to H2 O2 , self-oxidation of Hb causes conformational changes in
the heme pocket environment, facilitating the release of heme from the protein. Free heme is a
highly hydrophobic compound that cannot exist as a soluble molecule under neutral pH, heme is
trapped immediately by albumin or lipoproteins, which have middle affinity for heme but can compete
with Hx due to their high concentrations in plasma. Albumin has special sites for heme binding
characterized by Kd ~ 2 × 10−8 M [167], whereas incorporation of heme into lipoproteins can initiate
their oxidation and aggregation. Albumin-heme complexes have peroxidase activity and oxidize
phenolic compounds [19,167], but this activity is unlikely relative to the in vivo situation. Having
high binding capacity toward heme (Kd ~ 10−13 M), Hx sequesters heme from other proteins in a ratio
of 1 mol heme per 1 mol Hx and inhibits peroxidase-like activity of heme by 80–90% [19,155]. Thus,
despite the relatively long life time of the complex Hx-Hb in plasma (7–8 h, [12]), bound heme does
not have significant adverse effects on cells and macromolecules.

6. Intracellular Pseudo-Peroxidases

6.1. Cytochrome c/Cardiolipin Complexes

Cyt c is a heme protein located in the intermembrane space of mitochondria where it shuttles
electrons between complex III (ubiquinol:cytochrome c reductase) and complex IV (cytochrome c
oxidase), participating in the life-supporting synthesis of ATP [168]. Another important function of
cyt c is participation in programmed cell death through apoptosis. Cyt c is released from mitochondria
into the cytosol and amplifies signals that are generated by other apoptotic pathways. Both electron
transport function and propagation of apoptosis are well-accommodated by six-coordinated heme
iron in the active center of the protein [169].
The heme of Cyt c (MW ~ 12.5 kD) is linked by a covalent bond to the only SH group of the
protein. The heme iron is coordinated by His18 at the proximal position and by Met80 on the distal
side. Significant conformational changes accompanied by elimination of the distal heme ligand may
be induced by strong denaturating agent such as guanidine chloride or by protein oxidation by
hypochlorous acid [170,171]. The peroxidase activity of Cyt c increases substantially after protein
unfolding and oxidation.
Similar structural changes in Cyt c, accompanied by the loss or exchange of Met80 and an increase
in peroxidase activity, were observed when Cyt c interacted with negatively charged phospholipid
membranes [26,172–175]. The strength of phospholipids in inducing activation of Cyt c into peroxidase
ranks as follows: CL ≈ phosphatidic acid (PA) > phosphoinositols (PI) > phosphatidylserine (PS) >>
phosphatidylcholine (PC) [70]. The interaction of Cyt c with CL is of great importance because both
 mitochondrial membrane markedly increases [177]. As a consequence, the concentration of cyt
c/TLCL complexes increases. CL causes partial unfolding of cyt c and weakening of the
coordination bond between heme-iron and Met80. Contrary to Mb (Figure 4A), characteristic LT
EPR signal at g~6 could be hardy resolved under special experimental conditions in cyt c/CL EPR
spectra at 77 K [26]. Less than 7% of the protein loses the distal heme ligand upon interaction with
Molecules 2018, 23, 2561 14 of 27
CL. Lys 79, His26, or His33 are the likely candidates for the substitution of the position of Met80
[178,179]. Because these amino acids are weak ligands of heme iron in cyt c/CL complexes, small
molecules
molecules,are components
like H2O2, NO, of theCO,
and mitochondria
gain accessinterior. Millimolar
to the heme site oflevels of (ferri)Cyt
the protein (Figurec are5) contained
[180,181].
in
Thethedependence
intermembrane space
of the [176]. and shape of cyt c-CO and cyt c-NO complex spectra on CL
magnitude
Cardiolipin
concentration is a mitochondria-specific
shows that the increase in phospholipid
CL potentiates thatconformational
is located predominantly
changes ofincyt the cinner
and
leaflet
enhancesof theheme
inner mitochondria
accessibility membrane
for small inmolecules
normal cells. Tetralinoleoyl
(Figure S4) [75].cardiolipin (TLCL) is
At relatively specific
high CL
for cardiac and skeletal muscles; in other cells, highly diversified forms of CL
concentrations (cyt c:CL > 1:15), a three-line signal with a splitting of 17 G emerges at g = 2.009 in are present. In apoptotic
cells,
the LT theEPR
CL spectra
content of both in the outer
nitrosylated cytleaflet
c/CL of the inner
(Figure 5B). membrane
This three-lineand signal
in the outer
is duemitochondrial
to the shift in
membrane markedly
electron density increases
in Fe-N bond to[177]. As a consequence,
nitrogen (nuclear spin the concentration
I = 1) of Cyt c/TLCL
due to the cleavage complexes
of the His18-Fe(II)
bond [40]. CL causes partial unfolding of Cyt c and weakening of the coordination bond between
increases.
heme-iron and Met80.
Exposure of cyt Contrary to Mb (Figure
c/CL complexes 4A), characteristic
to hydrogen peroxide LT EPR signal
results at g ~ 6 could
in immediate be hardy
formation of
resolved under special
protein-derived experimental
(tyrosyl) conditions
radicals (Figure 5C). inForCytcytc/CL
c/CL,EPRno spectra
evidence at of
77 Compound
K [26]. Less than I or II7%wasof
the protein
found; loses amino
protein the distal heme
acids ligand upondonate
immediately interaction with CL.
electrons Lys 79, His26,
to porphyrin or His33
radical and are the likely
probably to
candidates for the substitution
oxoferryl intermediate (Figureof2).theInposition
the absenceof Met80 [178,179]. Because
of antioxidants these amino
or peroxidase acids the
substrates, are weak
heme
of cyt c/CL
ligands complexes
of heme Cyt c/CLwithin
iron indegrades several
complexes, minutes
small after addition
molecules, like H2 Oof 2, H 2O2 and
NO, (Figure
CO, 3B).gainAmong
access
thethe
to four tyrosines
heme site ofof thecyt c, Tyr67
protein is a likely
(Figure electron The
5) [180,181]. donor for Compounds
dependence of the formed
magnitudein theand cytshape
c active
of
site c-CO
Cyt withandfurther
Cyt c-NOtransfer of the
complex radical
spectra on CLreaction to othershows
concentration oxidizable
that theamino
increaseacids
in CL[63]. As the
potentiates
oxidative ability
conformational of tyrosyl
changes of Cyt radicals is lower
c and enhances heme than that of peroxidase
accessibility Compounds,
for small molecules (Figurecyt S4) c/CL
[75].
complexes
At relativelydisplay
high CLrelatively
concentrationslower(Cyt peroxidase activity
c:CL > 1:15), toward signal
a three-line quaiacolwithcompared
a splittingtoof other17 G
hemoproteins,
emerges but their
at g = 2.009 capacity
in the LT EPRtospectra
oxidize of anitrosylated
lipid-soluble Cytphenolic compound
c/CL (Figure etoposide
5B). This three-line (Eo signal
= 0.56
V,due
is [102]) is the
to the shifthighest (Table
in electron 2). The
density active
in Fe-N sitetoofnitrogen
bond cyt c/CL(nuclear
is occupied
spin Iby = 1)andue
acylto chain of CL.
the cleavage
Lipids
of and lipid soluble
the His18-Fe(II) compounds are better substrates for cyt c/CL peroxidase.
bond [40].

Figure5.5. Cardiolipin
Figure Cardiolipin induces
induces protein
proteinunfolding
unfoldingof cyt cc accompanied by the loss or
ofCyt or exchange
exchange ofof the
the
distal
distal heme
heme iron
iron ligand
ligand resulting
resulting in
in an
an increased
increased accessibility
accessibility of heme for small molecules [26,75].
(A)
(A)absorbance
absorbancespectra
spectraofof
native
native c(II)/CL
Cytcyt c(II)/CLcomplexes
complexesand complexes
and incubated
complexes withwith
incubated CO; CO;
(B) LT
(B)EPR
LT
spectrum of nitrosylated Cyt c(II)/CL complexes, (C) LT EPR spectrum of Cyt c(III)/CL
EPR spectrum of nitrosylated cyt c(II)/CL complexes, (C) LT EPR spectrum of cyt c(III)/CL complexes
exposed
complexesto H 2 O2 forto
exposed 30Hs.2O2 for 30 s;

Exposure of Cyt c/CL complexes to hydrogen peroxide results in immediate formation of

protein-derived (tyrosyl) radicals (Figure 5C). For Cyt c/CL, no evidence of Compound I or II was
found; protein amino acids immediately donate electrons to porphyrin radical and probably to
oxoferryl intermediate (Figure 2). In the absence of antioxidants or peroxidase substrates, the heme of
Cyt c/CL complexes degrades within several minutes after addition of H2 O2 (Figure 3B). Among the
four tyrosines of Cyt c, Tyr67 is a likely electron donor for Compounds formed in the Cyt c active site
with further transfer of the radical reaction to other oxidizable amino acids [63]. As the oxidative ability
of tyrosyl radicals is lower than that of peroxidase Compounds, Cyt c/CL complexes display relatively
lower peroxidase activity toward quaiacol compared to other hemoproteins, but their capacity to
Molecules 2018, 23, 2561 15 of 27

oxidize a lipid-soluble phenolic compound etoposide (Eo = 0.56 V, [102]) is the highest (Table 2).
The active site of Cyt c/CL is occupied by an acyl chain of CL. Lipids and lipid soluble compounds are
better substrates for Cyt c/CL peroxidase.
The peroxidase activity of Cyt c/CL toward external peroxidase substrates is low; the major
biological function of these complexes is oxidation of the polyunsaturated fatty acids (PUFA) of
CL. Cyt c/CL peroxidase activity is implicated in the propagation of apoptosis. In mitochondria,
the peroxidase activity of the Cyt c/CL complex is specific toward oxidation of PUFA-CL, yielding
FA-hydroperoxides. More abundant phospholipids, PC and PE, do not undergo peroxidation [26].
Cyt c dissociates from peroxidized CL; peroxidized CL participates in mitochondrial membrane
permeabilization, resulting in the release of Cyt c and other pro-apoptotic factors from mitochondria
to cytosol [177]. The second important function of Cyt c/CL peroxidase is the generation of a high
diversity of polyunsaturated molecular species for lipid mediator production. Cyt c-oxydized CL
undergoes phospholipase A2 -catalysed hydrolysis thus generating multiple oxygenated fatty acids,
including well-known lipid mediators [182].
To activate Cyt c into peroxidase, H2 O2 , which is formed due to a leaking electron transport
chain (from O2 •− dismutation), reacts with the protein active site. After several catalytic cycles, lipid
hydroperoxides accumulated. Fatty acid hydroperoxides more effectively react with the Cyt c active
site compared to H2 O2 , so they can accelerate the CL peroxidation (Table 1) [27]. Accumulation of
LOOH could heavily potentiate Cyt c/CL peroxidase activity, promoting cell death. Implication
of Cyt c peroxidase activity in neurodegenerative disorders may be due to the Cyt c/CL complex
oxidation and formation of protein-lipid aggregates [183,184].
The mechanisms preventing elevation of Cyt c/CL peroxidase activity in mitochondria are based
on the restriction of H2 O2 and substrate access to Cyt c active site [40,75,177]:

(1) In resting-state mitochondria, most CLs and Cyt c are spatially separated; therefore, the peroxidase
activity of Cyt c is not significant.
(2) The active center of Cyt c is occupied by an acyl chain of CL, which prevents access of peroxidase
substrates to Tyr67 in the active site.
(3) Tyrosyl radicals exposed on the Cyt c surface can recombine with the formation of very stable
dityrosine bonds. As a result, Cyt c/CL complexes can form aggregates, and the effective
concentration of Cyt c that can react with H2 O2 decreases.
(4) Mitochondrial NO, from external sources and generated by mitochondrial nitric-oxide synthase,
plays a regulatory role in the control of peroxidase activity of Cyt c/CL complexes.

Design and synthesis of mitochondria-targeted inhibitors of Cyt c/CL complex peroxidase activity
is a promising field of investigation for the development of new anti-apoptotic drugs [185–188].

6.2. Cytoglobin
Cygb is a recently discovered protein that is a member of the vertebrate globin family. All globins,
namely Hb, Mb, Cygb (MW ~ 21 kD), and neuroglobin (Ngb, MW ~ 17 kD), have a high α–helice
globular structure, heme group in the active site, and similar functions: oxygen transport and
participation in NO metabolism [62,143–145]. Cygb and Ngb have been reported to protect against
cell dysfunctions under hypoxia and oxidative stress, which is not the case for Hb and Mb. Ngb is
predominantly expressed in the brain. Cygb was discovered in 2001 in hepatic stellate cells [189].
Further studies revealed that the protein is expressed ubiquitously in all tissues and has a number
of specific functions, e.g., collagen synthesis, antifibrotic activity, and suppression of cell oncogenic
transformation [190]. The low amount of Cygb in cells suggests the probable signaling activity of the
protein [71,72].
Unlike Hb and Mb, the heme iron of Cygb and Ngb has a sixth coordination bond [73,143].
The ferrous forms of the proteins reversibly bind oxygen in competition with the distal His81 ligand.
Cygb, but not Ngb, was demonstrated to have substantial peroxidase activity [62,189].
Molecules 2018, 23, 2561 16 of 27

The protein heme group of Cygb is sensitive to oxidation of Cys38 and Cys83 which are exposed
to the protein surface. The diatomic ligand binding to the protein heme and its nitrite reductase activity
is controlled by formation of the disulfide bridge [144,191,192]. Under an oxidative environment,
the heme group is transformed into ferric state and stabilized in penta-coordinated state upon
oxidation of Cys residues to an internal disulfide. This form of enzyme possesses peroxidase activity
(Table 2) [143].
The protein with disulfide bridge has a low temperature EPR signal at g ~ 6 [73,76]. The peroxidase
mechanism of Cygb is similar to that of the pseudo-peroxidases discussed above. Interaction of the
Cygb heme with H2 O2 results in formation of Fe(IV) oxoferryl π cation, which is converted to Fe(IV)
oxoferryl and protein-derived (tyrosyl) radical detected by LT EPR and by spin trapping [76].
Reeder et al. demonstrated that the ferric form of Cygb lost its distal His81 ligand after binding of
oleic acid or CL [71]. Cygb is potent lipid oxidant. It oxidizes lipids five-fold faster than Mb. However,
due to the low concentration of Cygb in cells and the protein degradation under strong oxidative
conditions [76], Cygb-induced lipid oxidation is unlikely to cause extensive cellular damage [71,143].
The authors proposed that the physiological function of Cygb is to generate cell signaling lipid
molecules under an oxidative environment.
Fatty acid binding is dependent on the redox status of Cys38/Cys83 and occurs only in the Cygb
with intramolecularly cross-linked disulfide and already pre-existing significant peroxidase activity of
protein [71]. Fatty acids may act only as a co-activator of the peroxidase function of Cygb.
Searching for physiologically relevant lipid regulators of Cygb, Tejero et al. reported that highly
negatively charged anionic phospholipids—particularly phosphatidylinositolphosphates (PIP3 and
PIP2)—affect the structural organization of the protein and modulate its iron state [72]. Binding of
anionic lipids to ferric Cygb resulted in displacement of His81 by a water molecule. Thus, redox
catalytic activity can be conferred on Cygb both conjointly and/or independently of cysteine oxidation.
The increase in Cygb peroxidase activity correlated with the negative charges of the phospholipids:
PIP3(−4) > PIP2(−3) > TOCL(−2) > DOPA(−1). Essentially, the anionic phospholipids induce the
peroxidase activity of Cygb at physiologically relevant concentrations. Cygb’s peroxidase activity
can be utilized for the peroxidation of anionic phospholipids yielding mono-oxygenated molecular
species [72].
The large increase in Cygb peroxidase activity by PIP2 and PIP3 poses new alternatives for the
physiological roles of Cygb [72]. Although PIP2 and PIP3 constitute less than 1% of the total membrane
lipids, they are components of signaling pathways regulating cell proliferation and survival [193].
Upon oxidative stress, the increase in the amount of PIP3 and the PIP3/PIP2 ratio correlates with Cygb
up-regulation. Thus, possible signaling through a Cygb/PIP3 interaction would be amplified under
the stress conditions pathway through lipid peroxidation.
Double regulation of Cygb peroxidase activity by the redox state of SH groups and lipid binding
can provide fine turning of the synthesis of signaling lipid molecules.

7. Conclusions
Peroxidases form a network in the body that may exert both beneficial and adverse effects.
Peroxidases have physiological functions that substantially contribute to immunity and to a number
of metabolic and signaling processes. However, under certain circumstances, they exert deleterious
effects, such as damage to macromolecues in plasma, the activation of cell death, etc. There are special
mechanisms to minimize adverse effects of peroxidases (Table 3). The major of them are specific active
site structures and/or different types of regulating molecules. Apart from small molecules like NO or
CO that interact with the heme iron, special proteins or lipids can bind to hemoproteins and regulate
their peroxidase activity. These mechanisms may be a basis for pharmacological strategies aiming to
increase beneficial and to minimize deleterious effects of peroxidases.
Molecules 2018, 23, 2561 17 of 27

Table 3. The major mechanisms for regulation of peroxidase activity of hemoproteins.

The Major In Vivo Mechanisms for Activity Regulation

Hemoproteins Peroxidase
Products Other
Protein Structure
Macromolecules
Myeloperoxidase HOCl, HOSCN Ceruloplasmin
heme is located at the
True peroxidases Eosinophil base of a deep and
HOBr, HOSCN Ceruloplasmin
peroxidase narrow heme pocket
Lactoperoxidase HOSCN
Haptoglobin,
Hemoglobin L• ; • NO2 ; Ph-O• Hb(III) loses heme
Hemopexin
Myoglobin L• ; • NO2 ; Ph-O• Hemopexin
Pseudo- a CL fatty acid occupies
peroxidases Cyt c/CL complexes L• ; Ph-O•
Cyt c active site
oxidation of Cys38 and Fatty acids and
Cytoglobin L• ; Ph-O• Cys83 residues to an negatively charged
internal disulfide lipids

Supplementary Materials: The following are available online, Section S1: Oxidation-reduction potential,
Section S2: Experimental conditions for peroxidase activity measurements, Table S1: Plasma concentrations
and second order rate constants for the reactions of selected MPO substrates with Compound I and Compound II,
Figure S1: Dityrosine formation induced by Fenton chemistry or catalytically active myoglobin, Figure S2:
Low-temperature EPR spectra of high spin ferric heme and protein-based radicals of pseudo-peroxidases,
Figure S3: Phenolic compounds—peroxidase substrates, Figure S4: CO binding to Cyt c depends on
cardiolipin concentration.
Funding: The study of true peroxidases was funded by Russian Foundation for Basic Research according to the
research project No. 16-04-00873.
Acknowledgments: The author is grateful to Valerian E. Kagan (Center for Free Radical and Antioxidant Health,
University of Pittsburgh) for the opportunity to work in his laboratory where the creative environment stimulated
my research and, to some degree, the work on this review. The sections about pseudo-peroxidases were written
with the support by an internal grant from Sechenov University (Moscow, Russian Federation) within the
framework of Russian academic excellence project “5-100”.
Conflicts of Interest: The author declares no conflict of interest.

Abbreviations
CL cardiolipin
COX cyclooxygenase
CP ceruloplasmin
Cytg cytoglobin
Cyt c cytochrome c
EPO eosinophil peroxidase
EPR electron paramagnetic resonance
FFA free fatty acids
Hb hemoglobin
Hp haptoglobin
Hx hemopexin
L lipid
LPO lactoperoxidase
LT low temperature
Mb myoglobin
MPO myeloperoxidase
Ngb neuroglobin
Ph phenolic compound
Por heme porphyrin ring
TLCL tetralinoleoyl cardiolipin
TOCL tetraoleoyl cardiolipin
Molecules 2018, 23, 2561 18 of 27

References
1. Al Ghouleh, I.; Khoo, N.K.H.; Knaus, U.G.; Griendling, K.K.; Touyz, R.M.; Thannickal, V.J.; Barchowsky, A.;
Nauseef, W.M.; Kelley, E.E.; Bauer, P.M.; et al. Oxidases and peroxidases in cardiovascular and lung disease:
New concepts in reactive oxygen species signaling. Free Radic. Biol. Med. 2011, 51, 1271–1288. [CrossRef]
[PubMed]
2. Sies, H. Oxidative stress: A concept in redox biology and medicine. Redox Biol. 2015, 4, 180–183. [CrossRef]
[PubMed]
3. Fischer, H. Mechanisms and Function of DUOX in Epithelia of the Lung. Antioxid. Redox Signal. 2009, 11,
2453–2465. [CrossRef] [PubMed]
4. Carr, A.C.; McCall, M.R.; Frei, B. Oxidation of LDL by myeloperoxidase and reactive nitrogen species:
Reaction pathways and antioxidant protection. Arterioscler. Thromb. Vasc. Biol. 2000, 20, 1716–1723.
[CrossRef] [PubMed]
5. Dröge, W. Free Radicals in the Physiological Control of Cell Function. Physiol. Rev. 2002, 82, 47–95. [CrossRef]
[PubMed]
6. Forman, H.J.; Bernardo, A.; Davies, K.J.A. What is the concentration of hydrogen peroxide in blood and
plasma? Arch. Biochem. Biophys. 2016, 603, 48–53. [CrossRef] [PubMed]
7. Lacy, F.; O’Connor, D.T.; Schmid-Schönbein, G.W. Plasma hydrogen peroxide production in hypertensives
and normotensive subjects at genetic risk of hypertension. J. Hypertens. 1998, 16, 291–303. [CrossRef]
[PubMed]
8. Schröder, E.; Eaton, P. Hydrogen peroxide as an endogenous mediator and exogenous tool in cardiovascular
research: Issues and considerations. Curr. Opin. Pharmacol. 2008, 8, 153–159. [CrossRef] [PubMed]
9. Winterbourn, C.C. The biological chemistry of hydrogen peroxide. Methods Enzymol 2013, 528, 3–25.
[CrossRef] [PubMed]
10. Frazer, D.M.; Anderson, G.J. The regulation of iron transport. Biofactors 2014, 40, 206–214. [CrossRef]
[PubMed]
11. Stoyanovsky, D.A.; Tyurina, Y.Y.; Shrivastava, I.; Bahar, I.; Tyurin, V.A.; Protchenko, O.; Jadhav, S.;
Bolevich, S.B.; Kozlov, A.V.; Vladimirov, Y.A.; et al. Iron Catalysis of Lipid Peroxidation in Ferroptosis:
Regulated Enzymatic or Random Free Radical Reaction? Free Radic. Biol. Med. 2018. [CrossRef] [PubMed]
12. Delanghe, J.R.; Langlois, M.R. Hemopexin: A review of biological aspects and the role in laboratory medicine.
Clin. Chim. Acta 2001, 312, 13–23. [CrossRef]
13. Walling, C. Fenton’s reagent revisited. Acc. Chem Res. 1975, 8, 125–131. [CrossRef]
14. Buettner, G.R. The pecking order of free radicals and antioxidants: Lipid peroxidation, alpha-tocopherol,
and ascorbate. Arch. Biochem. Biophys. 1993, 300, 535–543. [CrossRef] [PubMed]
15. Imlay, J.A. Pathways of Oxidative Damage. Annu. Rev. Microbiol. 2003, 57, 395–418. [CrossRef] [PubMed]
16. Rush, J.D.; Maskos, Z.; Koppenol, W.H. Reactions of iron (II) nucleotide complexes with hydrogen peroxide.
FEBS Lett. 1990, 261, 121–123. [CrossRef]
17. Chen, R.; Pignatello, J.J. Role of quinone intermediates as electron shuttles in Fenton and photoassisted
Fenton oxidations of aromatic compounds. Environ. Sci. Technol. 1997, 31, 2399–2406. [CrossRef]
18. Augusto, O.; Miyamoto, S. Oxygen Radicals and Related Species. Princ. Free Radic. Biomed. 2012, 1, 1–23.
19. Grinberg, L.N.; O’Brien, P.J.; Hrkal, Z. The effects of heme-binding proteins on the peroxidative and catalatic
activities of hemin. Free Radic. Biol. Med. 1999, 27, 214–219. [CrossRef]
20. DeFelippis, M.R.; Murthy, C.P.; Faraggi, M.; Klapper, M.H. Pulse radiolytic measurement of redox potentials:
The tyrosine and tryptophan radicals. Biochemistry 1989, 28, 4847–4853. [CrossRef] [PubMed]
21. Davies, M.J.; Hawkins, C.L.; Pattison, D.I.; Rees, M.D. Mammalian Heme Peroxidases: From Molecular
Mechanisms to Health Implications. Antioxid. Redox Signal. 2008, 10, 1199–1234. [CrossRef] [PubMed]
22. Arnhold, J.; Monzani, E.; Furtmüller, P.G.; Zederbauer, M.; Casella, L.; Obinger, C. Kinetics and
thermodynamics of halide and nitrite oxidation by mammalian heme peroxidases. Eur. J. Inorg. Chem. 2006,
3801–3811. [CrossRef]
23. Stanbury, D.M. Reduction Potentials Involving Inorganic Free Radicals in Aqueous Solution. Adv. Inorg.
Chem. 1989, 33, 69–138.
Molecules 2018, 23, 2561 19 of 27

24. Buehler, P.W.; Abraham, B.; Vallelian, F.; Linnemayr, C.; Pereira, C.P.; Cipollo, J.F.; Jia, Y.; Mikolajczyk, M.;
Boretti, F.S.; Schoedon, G.; et al. Haptoglobin preserves the CD163 hemoglobin scavenger pathway by
shielding hemoglobin from peroxidative modification. Blood 2009, 113, 2578–2586. [CrossRef] [PubMed]
25. Reeder, B.J. The redox activity of hemoglobins: From physiologic functions to pathologic mechanisms.
Antioxid. Redox Signal. 2010, 13, 1087–1123. [CrossRef] [PubMed]
26. Kagan, V.E.; Tyurin, V.A.; Jiang, J.; Tyurina, Y.Y.; Ritov, V.B.; Amoscato, A.A.; Osipov, A.N.; Belikova, N.A.;
Kapralov, A.A.; Kini, V.; et al. Cytochrome c Acts as a Cardiolipin Oxygenase Required for Release of
Proapoptotic Factors. Nat. Chem. Biol. 2005, 1, 223–232. [CrossRef] [PubMed]
27. Belikova, N.A.; Tyurina, Y.Y.; Borisenko, G.; Tyurin, V.; Samhan Arias, A.K.; Yanamala, N.; Furtmüller, P.G.;
Klein-Seetharaman, J.; Obinger, C.; Kagan, V.E. Heterolytic reduction of fatty acid hydroperoxides by
cytochrome c/cardiolipin complexes: Antioxidant function in mitochondria. J. Am. Chem. Soc. 2009, 131,
11288–11289. [CrossRef] [PubMed]
28. Furtmüller, P.G.; Zederbauer, M.; Jantschko, W.; Helm, J.; Bogner, M.; Jakopitsch, C.; Obinger, C. Active
site structure and catalytic mechanisms of human peroxidases. Arch. Biochem. Biophys. 2006, 445, 199–213.
[CrossRef] [PubMed]
29. Furtmüller, P.G.; Burner, U.; Jantschko, W.; Regelsberger, G.; Obinger, C. Two-electron reduction and
one-electron oxidation of organic hydroperoxides by human myeloperoxidase. FEBS Lett. 2000, 484, 139–143.
[CrossRef]
30. Bolscher, B.G.; Wever, R. A kinetic study of the reaction between human myeloperoxidase, hydroperoxides
and cyanide. Inhibition by chloride and thiocyanate. Biochim. Biophys. Acta 1984, 788, 1–10. [CrossRef]
31. Aoshima, H.; Yoshida, Y.; Taniguchi, H. Reaction between Lipid Hydroperoxide and Hemoglobin Studied by
a Spectrophotometric and a Spin Trapping Method. Agric. Biol. Chem. 1986, 50, 1777–1783. [CrossRef]
32. Du, J.F.; Li, W.; Li, L.; Wen, G.B.; Lin, Y.W.; Tan, X. Regulating the coordination state of a heme protein by a
designed distal hydrogen-bonding network. ChemistryOpen 2015, 4, 97–101. [CrossRef] [PubMed]
33. Bafort, F.; Parisi, O.; Perraudin, J.P.; Jijakli, M.H. Mode of Action of Lactoperoxidase as Related to Its
Antimicrobial Activity: A. Review. Enzyme Res. 2014, 2014, 517164. [CrossRef] [PubMed]
34. Arnhold, J.; Furtmüller, P.G.; Obinger, C. Redox properties of myeloperoxidase. Redox Rep. 2003, 8, 179–186.
[CrossRef] [PubMed]
35. Nussbaum, C.; Klinke, A.; Adam, M.; Baldus, S.; Sperandio, M. Myeloperoxidase: A Leukocyte-Derived
Protagonist of Inflammation and Cardiovascular Disease. Antioxid. Redox Signal. 2013, 18, 692–713.
[CrossRef] [PubMed]
36. Flemmig, J.; Gau, J.; Schlorke, D.; Arnhold, J. Lactoperoxidase as a potential drug target. Expert Opin.
Ther. Targets 2016, 20, 447–461. [CrossRef] [PubMed]
37. Osipov, A.N.; Borisenko, G.G.; Vladimirov, Y.A. Biological activity of hemoprotein nitrosyl complexes.
Biochemistry 2007, 72, 1491–1504. [CrossRef]
38. Abu-Soud, H.M.; Hazen, S.L. Nitric oxide is a physiological substrate for mammalian peroxidases. J. Biol.
Chem. 2000, 275, 37524–37532. [CrossRef] [PubMed]
39. Eiserich, J.P.; Baldus, S.; Brennan, M.L.; Ma, W.; Zhang, C.; Tousson, A.; Castro, L.; Lusis, A.J.; Nauseef, W.M.;
White, C.R.; et al. Myeloperoxidase, a leukocyte-derived vascular NO oxidase. Science 2002, 296, 2391–2394.
[CrossRef] [PubMed]
40. Ascenzi, P.; Coletta, M.; Wilson, M.T.; Fiorucci, L.; Marino, M.; Polticelli, F.; Sinibaldi, F.; Santucci, R.
Cardiolipin-cytochrome c complex: Switching cytochrome c from an electron-transfer shuttle to a myoglobin-
and a peroxidase-like heme-protein. IUBMB Life 2015, 67, 98–109. [CrossRef] [PubMed]
41. Shigeto, J.; Tsutsumi, Y. Diverse functions and reactions of class III peroxidases. New Phytol. 2016, 209,
1395–1402. [CrossRef] [PubMed]
42. Pettigrew, G.W.; Echalier, A.; Pauleta, S.R. Structure and mechanism in the bacterial dihaem cytochrome c
peroxidases. J. Inorg. Biochem. 2006, 100, 551–567. [CrossRef] [PubMed]
43. Pandey, V.P.; Awasthi, M.; Singh, S.; Tiwari, S.; Dwivedi, U.N. A Comprehensive Review on Function and
Application of Plant Peroxidases. Biochem. Anal. Biochem. 2017, 6, 1–16. [CrossRef]
44. Klebanoff, S.J. Myeloperoxidase: Friend and foe. J. Leukocyte Biol. 2005, 77, 598–625. [CrossRef] [PubMed]
45. Malik, A.; Batra, J.K. Antimicrobial activity of human eosinophil granule proteins: Involvement in host
defence against pathogens. Crit. Rev. Microbiol. 2012, 38, 168–181. [CrossRef] [PubMed]
Molecules 2018, 23, 2561 20 of 27

46. Pattison, D.I.; Davies, M.J.; Hawkins, C.L. Reactions and reactivity of myeloperoxidase-derived oxidants:
Differential biological effects of hypochlorous and hypothiocyanous acids. Free Radic. Res. 2012, 46, 975–995.
[CrossRef] [PubMed]
47. Barrett, T.J.; Hawkins, C.L. Hypothiocyanous acid: Benign or deadly? Chem. Res. Toxicol. 2012, 25, 263–273.
[CrossRef] [PubMed]
48. Singh, P.K.; Iqbal, N.; Sirohi, H.V.; Bairagya, H.R.; Kaur, P.; Sharma, S.; Singh, T.P. Structural basis of activation
of mammalian heme peroxidases. Prog. Biophys. Mol. Biol. 2018, 133, 49–55. [CrossRef] [PubMed]
49. Capeillere-Blandin, C. Oxidation of guaiacol by myeloperoxidase: A two-electron-oxidized guaiacol transient
species as a mediator of NADPH oxidation. Biochem. J. 1998, 336, 395–404. [CrossRef] [PubMed]
50. Abu-Soud, H.M.; Hazen, S.L. Interrogation of heme pocket environment of mammalian peroxidases with
diatomic ligands. Biochemistry 2001, 40, 10747–10755. [CrossRef] [PubMed]
51. Vlasova, I.I.; Sokolov, A.V.; Arnhold, J. The free amino acid tyrosine enhances the chlorinating activity of
human myeloperoxidase. J. Inorg. Biochem. 2012, 106, 76–83. [CrossRef] [PubMed]
52. Eiserich, J.P.; Hristova, M.; Cross, C.E.; Jones, A.D.; Freeman, B.A.; Halliwell, B.; van der Vliet, A. Formation
of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998, 391, 393–397.
[CrossRef] [PubMed]
53. Meotti, F.C.; Jameson, G.N.L.; Turner, R.; Harwood, D.T.; Stockwell, S.; Rees, M.D.; Thomas, S.R.; Kettle, A.J.
Urate as a physiological substrate for myeloperoxidase: Implications for hyperuricemia and inflammation.
J. Biol. Chem. 2011, 286, 12901–12911. [CrossRef] [PubMed]
54. Furtmüller, P.G.; Jantschko, W.; Regelsberger, G.; Jakopitsch, C.; Moguilevsky, N.; Obinger, C. A transient
kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase. FEBS Lett. 2001,
503, 147–150. [CrossRef]
55. Marquez, L.A.; Dunford, H.B. Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase
compounds I. and II. Implications for lipoprotein peroxidation studies. J. Biol. Chem. 1995, 270, 30434–30440.
[CrossRef] [PubMed]
56. Burner, U.; Furtmüller, P.G.; Kettle, A.J.; Koppenol, W.H.; Obinger, C. Mechanism of reaction of
myeloperoxidase with nitrite. J. Biol. Chem. 2000, 275, 20597–20601. [CrossRef] [PubMed]
57. Tsai, A.L.; Kulmacz, R.J. Tyrosyl radicals in prostaglandin H synthase-1 and -2. Prostaglandins Other
Lipid Mediat. 2000, 62, 231–254. [CrossRef]
58. Soudi, M.; Paumann-Page, M.; Delporte, C.; Pirker, K.F.; Bellei, M.; Edenhofer, E.; Stadlmayr, G.;
Battistuzzi, G.; Boudjeltia, K.Z.; Furtmüller, P.G.; et al. Multidomain human peroxidasin 1 is a highly
glycosylated and stable homotrimeric high spin ferric peroxidase. J. Biol. Chem. 2015, 290, 10876–10890.
[CrossRef] [PubMed]
59. Lázár, E.; Péterfi, Z.; Sirokmány, G.; Kovács, H.A.; Klement, E.; Medzihradszky, K.F.; Geiszt, M.
Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking.
Free Radic. Biol. Med. 2015, 83, 273–282. [CrossRef] [PubMed]
60. Cadenas, E.; Alicia, I.V.; Alberto, B.; Britton, C. Low level chemiluminescence of the cytochrome c-catalyzed
decomposition of hydrogen peroxide. FEBS Lett. 1980, 113, 141–144. [CrossRef]
61. Metelitsa, D.I.; Karaseva, E.I. Initiation and inhibition of free-radical processes in biochemical peroxidase
systems: A review. Prikl. Biokhim. Mikrobiol. 2007, 43, 537–564. [CrossRef] [PubMed]
62. Reeder, B.J. Redox and Peroxidase Activities of the Hemoglobin Superfamily: Relevance to Health and
Disease. Antioxid. Redox Signal. 2017, 26, 763–776. [CrossRef] [PubMed]
63. Kapralov, A.A.; Yanamala, N.; Tyurina, Y.Y.; Castro, L.; Samhan-Arias, A.; Vladimirov, Y.A.; Maeda, A.;
Weitz, A.A.; Peterson, J.; Mylnikov, D.; et al. Topography of tyrosine residues and their involvement
in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes.
Biochim. Biophys. Acta-Biomembr. 2011, 1808, 2147–2155. [CrossRef] [PubMed]
64. Reeder, B.J.; Grey, M.; Silaghi-Dumitrescu, R.L.; Svistunenko, D.A.; Bülow, L.; Cooper, C.E.; Wilson, M.T.
Tyrosine residues as redox cofactors in human hemoglobin: Implications for engineering nontoxic blood
substitutes. J. Biol. Chem. 2008, 283, 30780–30787. [CrossRef] [PubMed]
65. Prasse, C.; Ford, B.; Nomura, D.K.; Sedlak, D.L. Unexpected transformation of dissolved phenols to toxic
dicarbonyls by hydroxyl radicals and UV light. Proc. Natl. Acad. Sci. USA 2018, 115, 2311–2316. [CrossRef]
[PubMed]
Molecules 2018, 23, 2561 21 of 27

66. Lundqvist, M.J.; Eriksson, L.A. Hydroxyl Radical Reactions with Phenol as a Model for Generation of
Biologically Reactive Tyrosyl Radicals. J. Phys. Chem. B 2000, 104, 848–855. [CrossRef]
67. Jayathilaka, P.B.; Pathiraja, G.C.; Bandara, A.; Subasinghe, N.D.; Nanayakkara, N. Theoretical study of phenol
and hydroxyl radical reaction mechanism in aqueous medium by the DFT/B3LYP/6-31+G(d,p)/CPCM
model. Can. J. Chem. 2014, 92, 809–813. [CrossRef]
68. Svistunenko, D.A.; Reeder, B.J.; Wankasi, M.M.; Silaghi-Dumitrescu, R.L.; Cooper, C.E.; Rinaldo, S.;
Cutruzzolà, F.; Wilson, M.T. Reaction of Aplysia limacina metmyoglobin with hydrogen peroxide. Dalt. Trans.
2007, 66, 840–850. [CrossRef] [PubMed]
69. Kapralov, A.; Vlasova, I.I.; Feng, W.; Maeda, A.; Walson, K.; Tyurin, V.A.; Huang, Z.; Aneja, R.K.; Carcillo, J.;
Bayir, H.; et al. Peroxidase activity of hemoglobin·haptoglobin complexes. Covalent aggreation and oxidative
stress in plasma and macrophages. J. Biol. Chem. 2009, 284, 30395–30407. [CrossRef] [PubMed]
70. Kapralov, A.A.; Kurnikov, I.V.; Vlasova, I.I.; Belikova, N.A.; Tyurin, V.A.; Basova, L.V.; Zhao, Q.; Tyurina, Y.Y.;
Jiang, J.; Bayir, H.; et al. The hierarchy of structural transitions induced in cytochrome c by anionic
phospholipids determines its peroxidase activation and selective peroxidation during apoptosis in cells.
Biochemistry 2007, 46, 14232–14244. [CrossRef] [PubMed]
71. Reeder, B.J.; Svistunenko, D.A.; Wilson, M.T. Lipid binding to cytoglobin leads to a change in haem
co-ordination: A role for cytoglobin in lipid signalling of oxidative stress. Biochem. J. 2011, 434, 483–492.
[CrossRef] [PubMed]
72. Tejero, J.; Kapralov, A.A.; Baumgartner, M.P.; Sparacino-Watkins, C.E.; Anthonymutu, T.S.; Vlasova, I.I.;
Camacho, C.J.; Gladwin, M.T.; Bayir, H.; Kagan, V.E. Peroxidase activation of cytoglobin by anionic
phospholipids: Mechanisms and consequences. Biochim. Biophys. Acta-Mol. Cell Biol. Lipids 2016, 391–401.
[CrossRef] [PubMed]
73. Vinck, E.; Van Doorslaer, S.; Dewilde, S.; Moens, L. Structural Change of the Heme Pocket Due to Disulfide
Bridge Formation Is Significantly Larger for Neuroglobin than for Cytoglobin. J. Am. Chem. Soc. 2004, 126,
4516–4517. [CrossRef] [PubMed]
74. Beckerson, P.; Svistunenko, D.; Reeder, B. Effect of the distal histidine on the peroxidatic activity of monomeric
cytoglobin. F1000Research 2015, 4, 87. [CrossRef] [PubMed]
75. Vlasova, I.I.; Tyurin, V.A.; Kapralov, A.A.; Kurnikov, I.V.; Osipov, A.N.; Potapovich, M.V.; Stoyanovsky, D.A.;
Kagan, V.E. Nitric oxide inhibits peroxidase activity of cytochrome c• cardiolipin complex and blocks
cardiolipin oxidation. J. Biol. Chem. 2006, 281, 14554–14562. [CrossRef] [PubMed]
76. Ferreira, J.C.; Marcondes, M.F.; Icimoto, M.Y.; Cardoso, T.H.S.; Tofanello, A.; Pessoto, F.S.; Miranda, E.G.A.;
Prieto, T.; Nascimento, O.R.; Oliveira, V.; et al. Intermediate tyrosyl radical and amyloid structure in
peroxide-activated cytoglobin. PLoS ONE 2015, 10, E0136554. [CrossRef] [PubMed]
77. Reeder, B.J.; Svistunenko, D.A.; Cooper, C.E.; Wilson, M.T. The Radical and Redox Chemistry of Myoglobin
and Hemoglobin: From In Vitro Studies to Human Pathology. Antioxid. Redox Signal. 2004, 6, 954–967.
[PubMed]
78. Klebanoff, S.J.; Kettle, A.J.; Rosen, H.; Winterbourn, C.C.; Nauseef, W.M. Myeloperoxidase: A front-line
defender against phagocytosed microorganisms. J. Leukocyte Biol. 2013, 93, 185–198. [CrossRef] [PubMed]
79. Arnhold, J.; Flemmig, J. Human myeloperoxidase in innate and acquired immunity. Arch. Biochem. Biophys.
2010, 500, 92–106. [CrossRef] [PubMed]
80. Yang, H.; Biermann, M.H.; Brauner, J.M.; Liu, Y.; Zhao, Y.; Herrmann, M. New insights into neutrophil
extracellular traps: Mechanisms of formation and role in inflammation. Front. Immunol. 2016, 7, 302.
[CrossRef] [PubMed]
81. Khan, M.A.; Philip, L.M.; Cheung, G.; Vadakepeedika, S.; Grasemann, H.; Sweezey, N.; Palaniyar, N.
Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis. Front. Med. 2018, 5, 19.
[CrossRef] [PubMed]
82. Van Dalen, C.J.; Whitehouse, M.W.; Winterbourn, C.C.; Kettle, A.J. Thiocyanate and chloride as competing
substrates for myeloperoxidase. Biochem. J. 1997, 327, 487–492. [CrossRef] [PubMed]
83. Baldus, S.; Heeschen, C.; Meinertz, T.; Zeiher, A.M.; Eiserich, J.P.; Münzel, T.; Simoons, M.L.; Hamm, C.W.
Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation 2003, 108,
1440–1445. [CrossRef] [PubMed]
Molecules 2018, 23, 2561 22 of 27

84. Sokolov, A.V.; Ageeva, K.V.; Cherkalina, O.S.; Pulina, M.O.; Zakharova, E.T.; Prozorovskii, V.N.;
Aksenov, D.V.; Vasilyev, V.B.; Panasenko, O.M. Identification and properties of complexes formed by
myeloperoxidase with lipoproteins and ceruloplasmin. Chem. Phys. Lipids 2010, 163, 347–355. [CrossRef]
[PubMed]
85. Teng, N.; Maghzal, G.J.; Talib, J.; Rashid, I.; Lau, A.K.; Stocker, R. The roles of myeloperoxidase in coronary
artery disease and its potential implication in plaque rupture. Redox Rep. 2017, 22, 51–73. [CrossRef]
[PubMed]
86. Zhang, Z.L.; Brennan, M.L.; Fu, X.M.; Aviles, R.J.; Pearce, G.L.; Penn, M.S.; Topol, E.J.; Sprecher, D.L.;
Hazen, S.L. Association between myeloperoxidase levels and risk of coronary artery disease. JAMA 2001,
286, 2136–2142. [CrossRef] [PubMed]
87. Mocatta, T.J.; Pilbrow, A.P.; Cameron, V.A.; Senthilmohan, R.; Frampton, C.M.; Richards, A.M.;
Winterbourn, C.C. Plasma Concentrations of Myeloperoxidase Predict Mortality After Myocardial Infarction.
J. Am. Coll. Cardiol. 2007, 49, 1993–2000. [CrossRef] [PubMed]
88. Schindhelm, R.K.; Van Der Zwan, L.P.; Teerlink, T.; Scheffer, P.G. Myeloperoxidase: A useful biomarker for
cardiovascular disease risk stratification? Clin. Chem. 2009, 55, 1462–1470. [CrossRef] [PubMed]
89. Green, P.S.; Mendez, A.J.; Jacob, J.S.; Crowley, J.R.; Growdon, W.; Hyman, B.T.; Heinecke, J.W. Neuronal
expression of myeloperoxidase is increased in Alzheimer’s disease. J. Neurochem. 2004, 90, 724–733.
[CrossRef] [PubMed]
90. Tzikas, S.; Schlak, D.; Sopova, K.; Gatsiou, A.; Stakos, D.; Stamatelopoulos, K.; Stellos, K.; Laske, C. Increased
Myeloperoxidase Plasma Levels in Patients with Alzheimer’s Disease. J. Alzheimer’s Dis. 2014, 39, 557–564.
[CrossRef] [PubMed]
91. Malle, E.; Buch, T.; Grone, H.-J. Myeloperoxidase in kidney disease. Kidney Int. 2003, 64, 1956–1967.
[CrossRef] [PubMed]
92. Thomson, E.; Brennan, S.; Senthilmohan, R.; Gangell, C.L.; Chapman, A.L.; Sly, P.D.; Kettle, A.J. Identifying
peroxidases and their oxidants in the early pathology of cystic fibrosis. Free Radic. Biol. Med. 2010, 49,
1354–1360. [CrossRef] [PubMed]
93. Stamp, L.K.; Khalilova, I.; Tarr, J.M.; Senthilmohan, R.; Turner, R.; Haigh, R.C.; Winyard, P.G.; Kettle, A.J.
Myeloperoxidase and oxidative stress in rheumatoid arthritis. Rheumatology 2012, 51, 1796–1803. [CrossRef]
[PubMed]
94. Kooter, I.M.; Moguilevsky, N.; Bollen, A.; Van Der Veen, L.A.; Otto, C.; Dekker, H.L.; Wever, R. The sulfonium
ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.
J. Biol. Chem. 1999, 274, 26794–26802. [CrossRef] [PubMed]
95. Malle, E.; Furtmüller, P.G.; Sattler, W.; Obinger, C. Myeloperoxidase: A target for new drug development?
Br. J. Pharmacol. 2007, 152, 838–854. [CrossRef] [PubMed]
96. Arnhold, J.; Furtmüller, P.G.; Regelsberger, G.; Obinger, C. Redox properties of the couple compound I/native
enzyme of myeloperoxidase and eosinophil peroxidase. Eur. J. Biochem. 2001, 268, 5142–5148. [CrossRef]
[PubMed]
97. Vlasova, I.I.; Vakhrusheva, T.V.; Sokolov, A.V.; Kostevich, V.A.; Gusev, A.A.; Gusev, S.A.; Melnikova, V.I.;
Lobach, A.S. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of
hypochlorous acid, the oxidant capable of degrading nanotubes. Toxicol. Appl. Pharmacol. 2012, 264, 131–142.
[CrossRef] [PubMed]
98. Pattison, D.I.; Davies, M.J. Reactions of myeloperoxidase-derived oxidants with biological substrates:
Gaining chemical insight into human inflammatory diseases. Curr. Med. Chem. 2006, 13, 32713290. [CrossRef]
99. Davies, M.J. The oxidative environment and protein damage. Biochim. Biophys. Acta-Proteins Proteomics 2005,
1703, 93–109. [CrossRef] [PubMed]
100. Zhang, R.; Shen, Z.; Nauseef, W.M.; Hazen, S.L. Defects in leukocyte-mediated initiation of lipid peroxidation
in plasma as studied in myeloperoxidase-deficient subjects: Systematic identification of multiple endogenous
diffusible substrates for myeloperoxidase in plasma. Blood 2002, 99, 1802–1810. [CrossRef] [PubMed]
101. Ramos, D.R.; García, M.V.; Canle, L.M.; Santaballa, J.A.; Furtmüller, P.G.; Obinger, C. Myeloperoxidase-
catalyzed chlorination: The quest for the active species. J. Inorg. Biochem. 2008, 102, 1300–1311. [CrossRef]
[PubMed]
Molecules 2018, 23, 2561 23 of 27

102. Tyurina, Y.Y.; Kini, V.; Tyurin, V.A.; Vlasova, I.I.; Jiang, J.; Kapralov, A.A.; Belikova, N.A.; Yalowich, J.C.;
Kurnikov, I.V.; Kagan, V.E. Mechanisms of cardiolipin oxidation by cytochrome c: Relevance to pro- and
antiapoptotic functions of etoposide. Mol. Pharmacol. 2006, 70, 706–717. [CrossRef] [PubMed]
103. Vlasova, I.I.; Feng, W.-H.; Goff, J.P.; Giorgianni, A.; Do, D.; Gollin, S.M.; Lewis, D.W.; Kagan, V.E.;
Yalowich, J.C. Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+
cells. Mol. Pharmacol. 2011, 79, 479–487. [CrossRef] [PubMed]
104. Vlasova, I.I.; Arnhold, J.; Osipov, A.N.; Panasenko, O.M. pH-dependent regulation of myeloperoxidase
activity. Biochemistry 2006, 71, 667–677. [CrossRef] [PubMed]
105. Summers, F.A.; Morgan, P.E.; Davies, M.J.; Hawkins, C.L. Identification of plasma proteins that are susceptible
to thiol oxidation by hypochlorous acid and N-chloramines. Chem. Res. Toxicol. 2008, 21, 1832–1840.
[CrossRef] [PubMed]
106. Flemmig, J.; Remmler, J.; Röhring, F.; Arnhold, J. (-)-Epicatechin regenerates the chlorinating activity of
myeloperoxidase in vitro and in neutrophil granulocytes. J. Inorg. Biochem. 2014, 130, 84–91. [CrossRef]
[PubMed]
107. Kirchner, T.; Flemmig, J.; Furtmüller, P.G.; Obinger, C.; Arnhold, J. (-)-Epicatechin enhances the chlorinating
activity of human myeloperoxidase. Arch. Biochem. Biophys. 2010, 495, 21–27. [CrossRef] [PubMed]
108. Tafazoli, S.; O’Brien, P.J. Peroxidases: A role in the metabolism and side effects of drugs. Drug Discov Today
2005, 10, 617–625. [CrossRef]
109. Papież, M.A. The influence of curcumin and (–)-epicatechin on the genotoxicity and myelosuppression
induced by etoposide in bone marrow cells of male rats. Drug Chem. Toxicol. 2013, 36, 93–101. [CrossRef]
[PubMed]
110. Kubala, L.; Kolářová, H.; Víteček, J.; Kremserová, S.; Klinke, A.; Lau, D.; Chapman, A.L.P.; Baldus, S.;
Eiserich, J.P. The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding
of myeloperoxidase to proteins of the extracellular matrix. Biochim. Biophys. Acta-Gen. Subj. 2013, 1830,
4524–4536. [CrossRef] [PubMed]
111. Tiruppathi, C.; Naqvi, T.; Wu, Y.; Vogel, S.M.; Minshall, R.D.; Malik, A.B. Albumin mediates the transcytosis
of myeloperoxidase by means of caveolae in endothelial cells. Proc. Natl. Acad. Sci. USA 2004, 101, 7699–7704.
[CrossRef] [PubMed]
112. Sokolov, A.V.; Pulina, M.O.; Ageeva, K.V.; Ayrapetov, M.I.; Berlov, M.N.; Volgin, G.N.; Markov, A.G.;
Yablonsky, P.K.; Kolodkin, N.I.; Zakharova, E.T.; et al. Interaction of ceruloplasmin, lactoferrin, and
myeloperoxidase. Biochem. 2007, 72, 409–415. [CrossRef]
113. Chapman, A.L.P.; Mocatta, T.J.; Shiva, S.; Seidel, A.; Chen, B.; Khalilova, I.; Paumann-Page, M.E.;
Jameson, G.N.L.; Winterbourn, C.C.; Kettle, A.J. Ceruloplasmin is an endogenous inhibitor of
myeloperoxidase. J. Biol. Chem. 2013, 288, 6465–6477. [CrossRef] [PubMed]
114. Bielli, P.; Calabrese, L. Structure to function relationships in ceruloplasmin: A “moonlighting” protein.
Cell. Mol. Life Sci. 2002, 59, 1413–1427. [CrossRef] [PubMed]
115. Lopez-Avila, V. Ceruloplasmin levels in human sera from various diseases and their correlation with patient’s
age and gender. Health 2009, 1, 104–110. [CrossRef]
116. Varfolomeeva, E.Y.; Semenova, E.V.; Sokolov, A.V.; Aplin, K.D.; Timofeeva, K.E.; Vasilyev, V.B.; Filatov, M.V.
Ceruloplasmin decreases respiratory burst reaction during pregnancy. Free Radic. Res. 2016, 50, 909–919.
[CrossRef] [PubMed]
117. Griffin, S.V.; Chapman, P.T.; Lianos, E.A.; Lockwood, C.M. The inhibition of myeloperoxidase by
ceruloplasmin can be reversed by anti-myeloperoxidase antibodies. Kidney Int. 1999, 55, 917–925. [CrossRef]
[PubMed]
118. Samygina, V.R.; Sokolov, A.V.; Bourenkov, G.; Petoukhov, M.V.; Pulina, M.O.; Zakharova, E.T.; Vasilyev, V.B.;
Bartunik, H.; Svergun, D.I. Ceruloplasmin: Macromolecular Assemblies with Iron-Containing Acute Phase
Proteins. PLoS ONE 2013, 8, e67145. [CrossRef] [PubMed]
119. Park, Y.S.; Suzuki, K.; Mumby, S.; Taniguchi, N.; Gutteridge, J.M. Antioxidant binding of caeruloplasmin to
myeloperoxidase: Myeloperoxidase is inhibited, but oxidase, peroxidase and immunoreactive properties of
caeruloplasmin remain intact. Free Radic. Res. 2000, 33, 261–265. [CrossRef] [PubMed]
120. Segelmark, M.; Persson, B.; Hellmark, T.; Wieslander, J. Binding and inhibition of myeloperoxidase (MPO):
A major function of ceruloplasmin? Clin. Exp. Immunol. 1997, 108, 167–174. [CrossRef] [PubMed]
Molecules 2018, 23, 2561 24 of 27

121. Sokolov, A.; Ageeva, K.; Pulina, M.; Cherkalina, O.; Samygina, V.; Vlasova, I.I.; Panasenko, O.; Zakharova, E.;
Vasilyev, V. Ceruloplasmin and myeloperoxidase in complex affect the enzymatic properties of each other.
Free Radic. Res. 2008, 42, 989–998. [CrossRef] [PubMed]
122. Panasenko, O.M.; Chekanov, A.V.; Vlasova, I.I.; Sokolov, A.V.; Ageeva, K.V.; Pulina, M.O.; Cherkalina, O.S.;
Vasil’ev, V.B. Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte
myeloperoxidase assayed by chemiluminescence. Biophysics 2008, 53, 268–272. [CrossRef]
123. Sokolov, A.V.; Acquasaliente, L.; Kostevich, V.A.; Frasson, R.; Zakharova, E.T.; Pontarollo, G.; Vasilyev, V.B.;
De Filippis, V. Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin:
Relevance in rheumatoid arthritis. Free Radic. Biol. Med. 2015, 86, 279–294. [CrossRef] [PubMed]
124. Zgliczynski, J.M.; Selvaraj, R.J.; Paul, B.B.; Stelmaszynska, T.; Poskitt, P.K.; Sbarra, A.J. Chlorination by the
myeloperoxidase-H2O2-Cl- antimicrobial system at acid and neutral pH. Proc. Soc. Exp. Biol. Med. 1977, 154,
418–422. [CrossRef] [PubMed]
125. Bakkenist, A.R.J.; De Boer, J.E.G.; Plat, H.; Wever, R. The halide complexes of myeloperoxidase and the
mechanism of the halogenation reactions. Biochim. Biophys. Acta-Enzymol. 1980, 613, 337–348. [CrossRef]
126. Heinecke, J.W.; Li, W.; Daehnke, H.L.; Goldstein, J.A. Dityrosine, a Specific Marker of Oxidation, is
synthesized by the myeloperoxidase-hydrogen peroxide system of human neutrophils and macrophages.
J. Biol. Chem. 1993, 268, 4069–4077. [PubMed]
127. Simmen, H.P.; Battaglia, H.; Giovanoli, P.; Blaser, J. Analysis of pH, pO2 and pCO2 in drainage fluid allows
for rapid detection of infectious complications during the follow-up period after abdominal surgery. Infection
1994, 22, 386–389. [CrossRef] [PubMed]
128. Jantschko, W.; Furtmüller, P.G.; Zederbauer, M.; Neugschwandtner, K.; Lehner, I.; Jakopitsch, C.; Arnhold, J.;
Obinger, C. Exploitation of the unusual thermodynamic properties of human myeloperoxidase in inhibitor
design. Biochem. Pharmacol. 2005, 69, 1149–1157. [CrossRef] [PubMed]
129. Dunford, H.B.; Hsuanyu, Y. Kinetics of oxidation of serotonin by myeloperoxidase compounds I. and II.
Biochem. Cell Biol. 1999, 77, 449–457. [CrossRef] [PubMed]
130. Furtmüller, P.G.; Arnhold, J.; Jantschko, W.; Zederbauer, M.; Jakopitsch, C.; Obinger, C. Standard reduction
potentials of all couples of the peroxidase cycle of lactoperoxidase. J. Inorg. Biochem. 2005, 99, 1220–1229.
[CrossRef] [PubMed]
131. Oxvig, C.; Thomsen, A.R.; Overgaard, M.T.; Sørensen, E.S.; Højrup, P.; Bjerrum, M.J.; Gleich, G.J.;
Sottrup-Jensen, L. Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in
human eosinophil peroxidase. The ester with Asp-93 is only partially formed in vivo. J. Biol. Chem. 1999,
274, 16953–16958. [CrossRef] [PubMed]
132. Furtmüller, P.G.; Burner, U.; Regelsberger, G.; Obinger, C. Spectral and kinetic studies on the formation
of eosinophil peroxidase compound I and its reaction with halides and thiocyanate. Biochemistry 2000, 39,
15578–15584. [CrossRef] [PubMed]
133. Wang, J.; Slungaard, A. Role of eosinophil peroxidase in host defense and disease pathology. Arch. Biochem.
Biophys. 2006, 445, 256–260. [CrossRef] [PubMed]
134. Martin, L.B.; Kita, H.; Leiferman, K.M.; Gleich, G.J. Eosinophils in Allergy: Role in Disease, Degranulation,
and Cytokines. Int. Arch. Allergy Immunol. 1996, 109, 207–215. [CrossRef] [PubMed]
135. Erzurum, S.C. New insights in oxidant biology in asthma. Ann. Am. Thorac. Soc. 2016, 13, S35–S39.
[CrossRef] [PubMed]
136. Sakamaki, K.; Ueda, T.; Nagata, S. The evolutionary conservation of the mammalian peroxidase genes.
Cytogenet. Genome Res. 2002, 98, 93–95. [CrossRef] [PubMed]
137. Sokolov, A.V.; Kostevich, V.A.; Zakharova, E.T.; Samygina, V.R.; Panasenko, O.M.; Vasilyev, V.B. Interaction
of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal
effect on enzymatic properties. Free Radic. Res. 2015, 49, 800–811. [CrossRef] [PubMed]
138. Sarr, D.; Tóth, E.; Gingerich, A.; Rada, B. Antimicrobial actions of dual oxidases and lactoperoxidase.
J. Microbiol. 2018, 56, 373–386. [CrossRef] [PubMed]
139. Conner, G.E.; Salathe, M.; Forteza, R. Lactoperoxidase and hydrogen peroxide metabolism in the airway.
Am. J. Respir. Crit. Care Med. 2002, 166, S57–S61. [CrossRef] [PubMed]
140. Lardinois, O.M.; Medzihradszky, K.F.; Ortiz De Montellano, P.R. Spin trapping and protein cross-linking of
the lactoperoxidase protein radical. J. Biol. Chem. 1999, 274, 35441–35448. [CrossRef] [PubMed]
Molecules 2018, 23, 2561 25 of 27

141. Furtmüller, P.G.; Jantschko, W.; Regelsberger, G.; Jakopitsch, C.; Arnhold, J.; Obinger, C. Reaction of
lactoperoxidase compound I with halides and thiocyanate. Biochemistry 2002, 41, 11895–11900. [CrossRef]
[PubMed]
142. Skaff, O.; Pattison, D.I.; Davies, M.J. Hypothiocyanous acid reactivity with low-molecular-mass and protein
thiols: Absolute rate constants and assessment of biological relevance. Biochem. J. 2009, 422, 111–117.
[CrossRef] [PubMed]
143. Ascenzi, P.; Marino, M.; Polticelli, F.; Coletta, M.; Gioia, M.; Marini, S.; Pesce, A.; Nardini, M.; Bolognesi, M.;
Reeder, B.J.; et al. Non-covalent and covalent modifications modulate the reactivity of monomeric
mammalian globins. Biochim. Biophys. Acta-Proteins Proteomics 2013, 1834, 1750–1756. [CrossRef] [PubMed]
144. Reeder, B.J.; Ukeri, J. Strong modulation of nitrite reductase activity of cytoglobin by disulfide bond oxidation:
Implications for nitric oxide homeostasis. Nitric Oxide-Biol. Chem. 2018, 72, 16–23. [CrossRef] [PubMed]
145. Liu, X.; El-Mahdy, M.A.; Boslett, J.; Varadharaj, S.; Hemann, C.; Abdelghany, T.M.; Ismail, R.S.; Little, S.C.;
Zhou, D.; Thuy, L.T.T.; et al. Cytoglobin regulates blood pressure and vascular tone through nitric oxide
metabolism in the vascular wall. Nat. Commun. 2017, 8, 1–14. [CrossRef] [PubMed]
146. Kagan, V.E.; Day, B.W.; Elsayed, N.M.; Gorbunov, N.V. Dynamics of haemoglobin. Nature 1996, 383, 30–31.
[CrossRef] [PubMed]
147. Svistunenko, D.A. Reaction of haem containing proteins and enzymes with hydroperoxides: The radical
view. Biochim. Biophys. Acta-Bioenerg. 2005, 1707, 127–155. [CrossRef] [PubMed]
148. Hultquist, D.E.; Passon, P.G. Catalysis of methaemoglobin reduction by erythrocyte cytochrome B5 and
cytochrome B5 reductase. Nat. New Biol. 1971, 229, 252–254. [CrossRef] [PubMed]
149. Jeffers, A.; Gladwin, M.T.; Kim-Shapiro, D.B. Computation of plasma hemoglobin nitric oxide scavenging in
hemolytic anemias. Free Radic. Biol. Med. 2006, 41, 1557–1565. [CrossRef] [PubMed]
150. Svistunenko, D.A.; Patel, R.P.; Voloshchenko, S.V.; Wilson, M.T. The globin-based free radical of ferryl
hemoglobin is detected in normal human blood. J. Biol. Chem. 1997, 272, 7114–7121. [CrossRef] [PubMed]
151. Svistunenko, D.A.; Dunne, J.; Fryer, M.; Nicholls, P.; Reeder, B.J.; Wilson, M.T.; Bigotti, M.G.; Cutruzzolà, F.;
Cooper, C.E. Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen
peroxide. Biophys. J. 2002, 83, 2845–2855. [CrossRef]
152. Lu, N.; He, Y.; Chen, C.; Tian, R.; Xiao, Q.; Peng, Y.Y. Tyrosine can protect against oxidative stress through
ferryl hemoglobin reduction. Toxicol. Vitr. 2014, 28, 847–855. [CrossRef] [PubMed]
153. Reeder, B.J.; Svistunenko, D.A.; Sharpe, M.A.; Wilson, M.T. Characteristics and mechanism of formation of
peroxide-induced heme to protein cross-linking in myoglobin. Biochemistry 2002, 41, 367–375. [CrossRef]
[PubMed]
154. Vuletich, J.L.; Osawa, Y.; Aviram, M. Enhanced Lipid Oxidation by Oxidatively Modified Myoglobin: Role of
Protein-Bound Heme. Biochem. Biophys. Res. Commun. 2000, 269, 647–651. [CrossRef] [PubMed]
155. Schaer, D.J.; Vinchi, F.; Ingoglia, G.; Tolosano, E.; Buehler, P.W. Haptoglobin, hemopexin and related defense
pathways-basic science, clinical perspectives and drug development. Front. Physiol. 2014, 5, 1–13. [CrossRef]
[PubMed]
156. Levy, A.; Asleh, R.; Blum, S.; Levy, N.; Miller Lotan, R.; Kalet Litman, S.; Anbinder, Y.; Lache, O.; Nakhoul, F.;
Asaf, R.; et al. Haptoglobin: Basic and clinical aspects. Antioxid. Redox Signal. 2010, 12, 293–304. [CrossRef]
[PubMed]
157. Langlois, M.R.; Delanghe, J.R. Biological and clinical significance of haptoglobin polymorphism in humans.
Clin. Chem. 1996, 42, 1589–1600. [PubMed]
158. Moestrup, S.K.; Moller, H.J. CD163: A regulated hemoglobin scavenger receptor with a role in the
anti-inflammatory response. Ann. Med. 2004, 36, 347–354. [CrossRef] [PubMed]
159. Schaer, D.J. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in
the absence of haptoglobin. Blood 2006, 107, 373–380. [CrossRef] [PubMed]
160. Kristiansen, M.; Graversen, J.H.; Jacobsen, C.; Sonne, O.; Hoffman, H.J.; Law, S.K.A.; Moestrup, S.K.
Identification of the haemoglobin scavenger receptor. Nature 2001, 409, 198–201. [CrossRef] [PubMed]
161. Vallelian, F.; Pimenova, T.; Pereira, C.P.; Abraham, B.; Mikolajczyk, M.G.; Schoedon, G.; Zenobi, R.;
Alayash, A.I.; Buehler, P.W.; Schaer, D.J. The reaction of hydrogen peroxide with hemoglobin induces
extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger
pathways. Free Radic. Biol. Med. 2008, 45, 1150–1158. [CrossRef] [PubMed]
Molecules 2018, 23, 2561 26 of 27

162. Su, D.; Roth, R.I.; Yoshida, M.; Levin, J. Hemoglobin increases mortality from bacterial endotoxin.
Infect. Immun. 1997, 65, 1258–1266. [PubMed]
163. Effenberger-Neidnicht, K.; Hartmann, M. Mechanisms of Hemolysis During Sepsis. Inflammation 2018, 41,
1569–1581. [CrossRef] [PubMed]
164. Kim, H.P.; Ryter, S.W.; Choi, A.M.K. CO As a Cellular Signaling Molecule. Annu. Rev. Pharmacol. Toxicol.
2006, 46, 411–449. [CrossRef] [PubMed]
165. Piantadosi, C.A. Biological Chemistry of Carbon Monoxide. Antioxid. Redox Signal. 2002, 4, 259–270.
[CrossRef] [PubMed]
166. Miller, Y.I.; Smith, A.; Morgan, W.T.; Shaklai, N. Role of hemopexin in protection of low-density lipoprotein
against hemoglobin-induced oxidation. Biochemistry 1996, 35, 13112–13117. [CrossRef] [PubMed]
167. Monzani, E.; Bonafè, B.; Fallarini, A.; Redaelli, C.; Casella, L.; Minchiotti, L.; Galliano, M. Enzymatic
Properties of Human Hemalbumin. Biochim. Biophys. Acta 2001, 1547, 302–312. [CrossRef]
168. Turrens, J.F. Mitochondrial formation of reactive oxygen species. J. Physiol. 2003, 552, 335–344. [CrossRef]
[PubMed]
169. Ow, Y.L.P.; Green, D.R.; Hao, Z.; Mak, T.W. Cytochrome c: Functions beyond respiration. Nat. Rev. Mol.
Cell Biol. 2008, 9, 532–542. [CrossRef] [PubMed]
170. Diederix, R.E.M.; Ubbink, M.; Canters, G.W. Peroxidase Activity as a Tool for Studying the Folding of c-Type
Cytochromes. Biochemistry 2002, 41, 13067–13077. [CrossRef] [PubMed]
171. Chen, Y.R.; Deterding, L.J.; Sturgeon, B.E.; Tomer, K.B.; Mason, R.P. Protein oxidation of cytochrome c by
reactive halogen species enhances its peroxidase activity. J. Biol. Chem. 2002, 277, 29781–29791. [CrossRef]
[PubMed]
172. Radi, R.; Turrens, J.F.; Freeman, B.A. Cytochrome c-catalyzed membrane lipid peroxidation by hydrogen
peroxide. Arch. Biochem. Biophys. 1991, 288, 118–125. [CrossRef]
173. Nantes, I.L.; Zucchi, M.R.; Nascimento, O.R.; Faljoni-Alario, A. Effect of heme iron valence state on the
conformation of cytochrome c and its association with membrane interfaces: A CD and EPR investigation.
J. Biol. Chem. 2001, 276, 153–158. [CrossRef] [PubMed]
174. Mandal, A.; Hoop, C.L.; Delucia, M.; Kodali, R.; Kagan, V.E.; Ahn, J.; Van Der Wel, P.C.A. Structural Changes
and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c. Biophys. J. 2015,
109, 1873–1884. [CrossRef] [PubMed]
175. Vladimirov, G.K.; Vikulina, A.S.; Volodkin, D.; Vladimirov, Y.A. Structure of the complex of cytochrome c
with cardiolipin in non-polar environment. Chem. Phys. Lipids 2018, 214, 35–45. [CrossRef] [PubMed]
176. Boveris, A.; Cadenas, E. Mitochondrial Production of Hydrogen Peroxide Regulation by Nitric Oxide and
the Role of Ubisemiquinone. IUBMB Life 2000, 50, 245–250. [CrossRef] [PubMed]
177. Kagan, V.E.; Tyurina, Y.Y.; Bayir, H.; Chu, C.T.; Kapralov, A.A.; Vlasova, I.I.; Belikova, N.A.; Tyurin, V.A.;
Amoscato, A.; Epperly, M.; et al. The “pro-apoptotic genies” get out of mitochondria: Oxidative lipidomics
and redox activity of cytochrome c/cardiolipin complexes. Chem. Biol. Interact. 2006, 163, 15–28. [CrossRef]
[PubMed]
178. Sinibaldi, F.; Howes, B.D.; Droghetti, E.; Polticelli, F.; Piro, M.C.; Di Pierro, D.; Fiorucci, L.; Coletta, M.;
Smulevich, G.; Santucci, R. Role of lysines in cytochrome c -cardiolipin interaction. Biochemistry 2013, 52,
4578–4588. [CrossRef] [PubMed]
179. Oellerich, S.; Wackerbarth, H.; Hildebrandt, P. Conformational equilibria and dynamics of cytochrome c
induced by binding of sodium dodecyl sulfate monomers and micelles. Eur. Biophys. J. 2003, 32, 599–613.
[CrossRef] [PubMed]
180. Silkstone, G.; Kapetanaki, S.M.; Husu, I.; Vos, M.H.; Wilson, M.T. Nitric Oxide Binding to the Cardiolipin
Complex of Ferric Cytochrome c. Biochemistry 2012, 51, 6760–6766. [CrossRef] [PubMed]
181. Kapetanaki, S.M.; Silkstone, G.; Husu, I.; Liebl, U.; Wilson, M.T.; Vos, M.H. Interaction of Carbon Monoxide
with the Apoptosis-Inducing Cytochrome c—Cardiolipin Complex. Biochemistry 2009, 48, 1613–1619.
[CrossRef] [PubMed]
182. Tyurina, Y.Y.; Poloyac, S.M.; Tyurin, V.A.; Kapralov, A.A.; Jiang, J.; Anthonymuthu, T.S.; Kapralova, V.I.;
Vikulina, A.S.; Jung, M.Y.; Epperly, M.W.; et al. A mitochondrial pathway for biosynthesis of lipid mediators.
Nat. Chem. 2014, 6, 542–552. [CrossRef] [PubMed]
Molecules 2018, 23, 2561 27 of 27

183. Bayir, H.; Kapralov, A.A.; Jiang, J.; Huang, Z.; Tyurina, Y.Y.; Tyurin, V.A.; Zhao, Q.; Belikova, N.A.;
Vlasova, I.I.; Maeda, A.; et al. Peroxidase mechanism of lipid-dependent cross-linking of synuclein with
cytochrome c. Protection against apoptosis versus delayed oxidative stress in Parkinson disease. J. Biol.
Chem. 2009, 284, 15951–15969. [CrossRef] [PubMed]
184. Patriarca, A.; Polticelli, F.; Piro, M.C.; Sinibaldi, F.; Mei, G.; Bari, M.; Santucci, R.; Fiorucci, L. Conversion of
cytochrome c into a peroxidase: Inhibitory mechanisms and implication for neurodegenerative diseases.
Arch. Biochem. Biophys. 2012, 522, 62–69. [CrossRef] [PubMed]
185. Atkinson, J.; Kapralov, A.A.; Yanamala, N.; Tyurina, Y.Y.; Amoscato, A.A.; Pearce, L.; Peterson, J.; Huang, Z.;
Jiang, J.; Samhan-Arias, A.K.; et al. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates
radiation-induced death. Nat. Commun. 2011, 2, 497. [CrossRef] [PubMed]
186. Stoyanovsky, D.A.; Vlasova, I.I.; Belikova, N.A.; Kapralov, A.; Tyurin, V.; Kagan, V.E. Activation of NO
donors in mitochondria: Peroxidase metabolism of (2-hydroxyamino-vinyl)-triphenyl-phosphonium by
cytochrome c releases NO and protects cells against apoptosis. FEBS Lett. 2008, 582, 725–728. [CrossRef]
[PubMed]
187. Bakan, A.; Kapralov, A.A.; Bayir, H.; Hu, F.; Kagan, V.E.; Bahar, I. Inhibition of Peroxidase Activity of
Cytochrome c: De Novo Compound Discovery and Validation. Mol. Pharmacol. 2015, 88, 421–427. [CrossRef]
[PubMed]
188. Lamade, A.M.; Kenny, E.M.; Anthonymuthu, T.S.; Soysal, E.; Clark, R.S.B.; Kagan, V.E.; Bayır, H. Aiming
for the target: Mitochondrial drug delivery in traumatic brain injury. Neuropharmacology 2018. [CrossRef]
[PubMed]
189. Kawada, N.; Kristensen, D.B.; Asahina, K.; Nakatani, K.; Minamiyama, Y.; Seki, S.; Yoshizato, K.
Characterization of a Stellate Cell Activation-associated Protein (STAP) with Peroxidase Activity Found in
Rat Hepatic Stellate Cells. J. Biol. Chem. 2001, 276, 25318–25323. [CrossRef] [PubMed]
190. Bholah, T.C.; Neergheen-Bhujun, V.S.; Hodges, N.J.; Dyall, S.D.; Bahorun, T. Cytoglobin as a Biomarker
in Cancer: Potential Perspective for Diagnosis and Management. Biomed. Res. Int. 2015, 2015, 824514.
[CrossRef] [PubMed]
191. Beckerson, P.; Reeder, B.J.; Wilson, M.T. Coupling of disulfide bond and distal histidine dissociation in
human ferrous cytoglobin regulates ligand binding. FEBS Lett. 2015, 589, 507–512. [CrossRef] [PubMed]
192. Tsujino, H.; Yamashita, T.; Nose, A.; Kukino, K.; Sawai, H.; Shiro, Y.; Uno, T. Disulfide bonds regulate binding
of exogenous ligand to human cytoglobin. J. Inorg. Biochem. 2014, 135, 20–27. [CrossRef] [PubMed]
193. Cantley, L.C. The phosphoinositide 3-kinase pathway. Science 2002, 296, 1655–1657. [CrossRef] [PubMed]

© 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).