Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control.

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. Рeroxidases 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.

1. Introduction

pan class="Chemical">Reactive oxygen species (ical">pan class="Chemical">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 pan class="Chemical">NADPH oxidase family of enzymes is a major source of ical">pan class="Chemical">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 (H2O2). 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 [4,5,6]. A healthy physiological level of H2O2 in the plasma is 1–5 µM, but may be as high as 50 µM in inflammatory disease [6,7,8].

pan class="Chemical">Hydrogen peroxide is one of the major in vivo oxidants. It is a two-electron acceptor with reduction potential E (ical">pan class="Chemical">H2O2/H2O) of 1.32 V. However, H2O2 reacts poorly or not at all with most biological molecules due the high activation energy barrier that must be overcome to release H2O2 oxidative power [9] (see Supplementary Materials, Section S1).

The development of oxidative stress is mediated by transition n class="Chemical">metal ions, primarily with ical">pan class="Chemical">iron ions that promote H2O2 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 H2O2 and the reduction potentials of radicals and oxidants formed in the reactions. The rate constants of the reaction of H2O2 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 H2O2. Standard reduction potentials (E) are presented for oxidative couples: HOX/X-; ●NO2/NO2−; phenoxyl radical (Ph-O●)/phenolic compound (Ph-OH); HO●/H2O; HOO●/H2O2.

Form of Iron	k, M−1 s−1	Reaction Products	Eo, V pH 7	References
* free ferrous ion, Fe2+	76	hydroxyl radical, HO●	2.31	[13,14]
		lipid alkoxyl radical, LO●	≤1.06
Lig-Fe2+	102–104	HO●, LO●		[15,16]
free ferric ion, Fe3+	0.01–0.02	hydroperoxyl radical, HOO●	1.06	[14,17,18]
		lipid peroxyl radical, LOO●	≤1.00
Albumin-heme		phenoxyl radicals, Ph-O●	0.4–0.94	[19,20]
		lipid radical, L● (LO●, LOO●)	0.6	[14]
True peroxidases	(1.1–4.3) × 107	hypochlorous acid, HOCl	1.28	[21,22]
		hypobromous acid, HOBr	1.13
		hypoiodous acid, HOI	0.78
		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 + FFA-OOH	~5 × (103–105)			[27]

* Fe2+ + H2O2 = 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 pan class="Chemical">H2O2 are ical">pan class="Chemical">peroxidases. Peroxidases consume H2O2 and catalyze the one- or two-electron oxidation of a diverse array of Compounds called peroxidase substrates [1].

pan class="Chemical">Heme-ical">pan class="Chemical">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 H2O2 are several orders of magnitude higher than that of the Fenton reaction [21] (Table 1). In the presence of H2O2, 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 H2O2 [29,30,31].

The mandatory requirements for the pan class="Chemical">peroxidase activity of a ical">pan class="Chemical">heme-containing protein (i.e., its ability to form reactive intermediates) are:

The pan class="Chemical">peroxidases oical">pan class="Chemical">perate having iron in ferric form (Fe(3+) or Fe(III)).

The pan class="Chemical">heme ical">pan class="Chemical">iron of a peroxidase has five coordination bonds: four with nitrogens of tetrapyrrole ring, and the fifth heme ligand on the proximal heme side is a highly conserved imidazole ring of histidine residue linking the heme to the protein. On the distal heme side, the iron binds a water molecule. This H2O molecule is replaced by H2O2 upon activation of a peroxidase [28,32].

The pan class="Chemical">peroxidases are characterized by the sical">pan class="Chemical">pecific location of amino acids in the active center for effective coordination and use of H2O2. On the distal heme site, a conserved histidine-arginine couple is involved in a hydrogen peroxide network [28,33].

If a catalytic center of a hemoprotein meets the requirements listed above, pan class="Chemical">H2O2 is sical">pan class="Chemical">pecifically positioned in the peroxidase catalytic center and transfers its high oxidation potential to the heme. Two electrons are transferred from the enzyme to H2O2, which is reduced into water, whereby the heme is oxidized to Compound I (Figure 1). Compound I has two oxidizing equivalents: one in the oxyferryl 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 [34,35,36]. The interaction between hydrogen peroxide and native ferric peroxidase heme leads to the reduction of H2O2 to water with formation of Compound I which is oxoferryl porphyrin-π-cationic radical. Compound I can be sequentially reduced to the native enzyme via formation of Compound II by two one-electron oxidations caused by a number of simple compounds, peroxidase substrates (peroxidase cycle). AH is a peroxidase substrate that is oxidized with formation of a radical (A•). Compound I can catalyze the two-electron oxidation of (pseudo)halides, thus completing the so-called halogenation cycle. X− stands for halide ions (Cl−, Br−, I−) and thiocyanate ions (SCN−). HOX is the corresponding (pseudo)hypohalous acid.

pan class="Chemical">Coical">pan class="Gene">mpound I has very high oxidizing ability, with a reduction potential above 1.1 V, and it can oxidize many simple substances—the peroxidase substrates—through a one-electron mechanism via the intermediate Compound II—PorFe(4+)=O. At the end of the reaction, the active center returns to the native form, completing the peroxidase cycle.

Participation in the metabolism of pan class="Chemical">nitric oxide and its derivatives is an iical">pan class="Gene">mportant function of hemoproteins, including those that have peroxidase activity. This topic is discussed in a number of papers and reviews [37,38,39,40].

Tpan class="Chemical">his review summarizes the autical">pan class="Chemical">hor’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 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 pan class="Chemical">peroxidases exist, but most of them are plant, fungi, or bacteria ical">pan class="Chemical">peroxidases: horseradish peroxidase, ascorbate peroxidase, yeast cytochrome c peroxidase, lignin peroxidase etc. [41,42,43]. 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 pan class="Chemical">peroxidases are enzymes wical">pan class="Chemical">hose 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].

pan class="Chemical">Coical">pan class="Gene">mpound 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].

Altpan class="Chemical">hough plasma ical">pan class="Chemical">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 pan class="Chemical">iodide ical">pan class="Chemical">concentration in vivo (<1 µM) with the exception of the thyroid gland, iodide oxidation by peroxidases is of little importance [33,36].

pan class="Gene">MPO, EPO and ical">pan class="Gene">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:

The pan class="Chemical">heme of the ical">pan class="Chemical">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].

True pan class="Chemical">peroxidases do not have oxidizable amino acids in close proximity to ical">pan class="Chemical">heme that are able to compete with external substrates for Compound I (except for cyclooxygenase).

The activity of the enzyme toward pan class="Chemical">guaiacol, a hardly oxidizable ical">pan class="Chemical">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 n class="Chemical">peroxidases is ical">pan class="Chemical">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) [51,52,53,54,55,56]. 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 mepan class="Gene">mbers of the true ical">pan class="Chemical">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.

pan class="Chemical">COX (or ical">pan class="Chemical">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 men class="Gene">mbers of animal ical">pan class="Chemical">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-pan class="Chemical">peroxidases are hemoproteins that ical">pan class="Chemical">perform different important functions in the body and initially are not meant to interact with H2O2. 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 H2O2 reduction to water. The rate of reaction of heme with H2O2 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 H2O2 [60,61]. Nevertheless, these proteins consume H2O2 and oxidize peroxidase substrates, so they may be considered as peroxidases.

Pseudo-pan class="Chemical">peroxidases are difical">pan class="Chemical">ferent in terms of their heme and activity characteristics, but all of them have one common feature: exposure to H2O2 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 H2O2 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 H2O2, 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) [65,66,67].

Figure 2

Catalytic cycle of pseudo-peroxidases [40,63,68,69]. The interaction between hydrogen peroxide and native ferric pseudo-peroxidase heme leads the formation of Compound I which is most likely oxoferryl porphyrin-π-cationic radical. Compound I immediately oxidizes amino acid residues (Tyr, Trp, His) which are located near the heme with formation of protein based radicals and oxoferryl heme. Oxoferryl heme iron can oxidize protein amino acids and peroxidase substrates. AH is a peroxidase substrate that is oxidized with formation of a radical (A•). The protein-based tyrosyl radicals are the alternative reactive intermediates of pseudo-peroxidases.

Pseudo-pan class="Chemical">peroxidases can be divided into two groups:

Proteins in which the pan class="Chemical">heme ical">pan class="Chemical">iron initially does not have an amino acid ligand in a distal coordination position—hemoglobin (Hb) and myoglobin (Mb).

Proteins in which the pan class="Chemical">heme ical">pan class="Chemical">iron has a sixth coordination bond, but under the influence of external factors, the configuration of the active site can change, and this newly conferred peroxidase activity plays an important role in the cells, unrelated to the primary biological functions of the enzymes.

pan class="Gene">Cyt c and ical">pan class="Gene">cytoglobin (Cygb) are hexacoordinated proteins with methionine/histidine residues occupying the sixth coordination sites of the heme iron (for Cygb—under reducing conditions). Anionic lipids can break this bond and awaken the dormant peroxidase activity of cyt c and Cygb [26,70,71,72].

Low tempan class="Chemical">perature electron ical">paramagnetic resonance (LT EPR) sical">pan class="Chemical">pectroscopy is a powerful tool used to study peroxidase activity of hemoproteins (Figure S2). EPR signal at a g-factor of about six (g ~ 6) evidences the absence of a ligand at the sixth coordination position of iron. Protein oxidation and the formation of protein-derived (tyrosyl) radicals can be monitored by acquiring the spectra of tyrosyl radicals at a g-factor of about two (g ~ 2). Not only H2O2, but other small molecules including CO and NO, can interact with iron at the sixth coordination position side. NO binding to both heme-Fe(III) and heme-Fe(II) changes the optical absorbance spectra of hemeproteins in the Soret band region, but only Fe(II)-NO complexes have characteristic LT EPR spectra of nitrosylated heme (Figure S2). The LT EPR method has been successfully employed for the study of pseudo-peroxidases and provided unique useful information [64,72,73,74,75,76,77].

3. Myeloperoxidase

pan class="Gene">MPO is the queen of the ical">pan class="Chemical">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, n class="Chemical">comprising approximately 5% of cellular dry weight. During neutrophil activation, the granules release their ical">pan class="Chemical">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 H2O2 [1,78]. In inflamed tissue, steady state concentrations of H2O2 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 pan class="Chemical">conditions, ical">pan class="Gene">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 pan class="Chemical">concentration of ical">pan class="Gene">MPO in plasma is normally in the range of 20–100 ng/mL, whereas under pathology, these values increase several-fold [83,84,85]. 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 [83,86,87,88], Alzheimer’s disease [89,90], kidney diseases [91,92], and rheumatoid arthritis [93].

The enzyme is a 145–160 kD pan class="Chemical">homodimer ical">pan class="Chemical">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, n class="Gene">MPO ical">pan class="Chemical">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 H2O2 leads to the immediate formation of Compound I. The one-electron oxidation of Compound I by the next molecule of H2O2 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.

Figure 3

Optical spectra of myeloperoxidase and cyt c/TOCL complexes after addition of H2O2 [51,97]. (A) Upon addition of an excess of H2O2 (200 μM), MPO (1.2 μM) undergoes conversion from its native form (the Soret band maximum at 430 nm) to Compound II having the absorbance maximum at 454 nm. In the absence of peroxidase substrates the absorbance at 454 nm slowly decreases, and the peak shifts towards 430 nm. The arrow indicates a decrease in the Soret band intensity in 90 min after addition of H2O2 to MPO. (B) A progressive decrease of the absorbance of cyt c/TOCL during its auto-oxidation process initiated by the addition of 250 μM H2O2 to the solution of ferri-cyt c (1.5 μM, tetraoleoyl-CL 30 µM). 50 mM sodium phosphate buffer, 100 mM NaCl, pH 7.4, 100 µM DTPA.

Altpan class="Chemical">hough the major function of ical">pan class="Chemical">peroxidases in innate immunity is the production of hypohalous acids and HOSCN, these enzymes can also oxidize peroxidase substrates under plasma conditions (neutral pH and 140 mM NaCl; Table S1) [51,53]. Amino acid residues of proteins, such as tyrosine and tryptophan, are peroxidase substrates [98,99]. Does this mean that MPO can directly oxidize proteins in plasma? There are several mechanisms for controlling the ‘fire’ in the MPO active site to minimize its adverse impact on biomolecules.

3.1. Restricted Access to MPO Active Site

The structure of the pan class="Gene">MPO active site prevents the enzyme from oxidizing large molecules. The active site of ical">pan class="Gene">MPO is located at the base of a deep and narrow heme pocket inaccessible to compounds significantly larger than a dipeptide [100,101]. For macromolecule oxidation, small substrates are needed, which are the intermediates between Compounds and the biomolecules. At pH < 7, the major oxidant is HOCl. At neutral pH, free radicals with high reduction potential formed in the peroxidase cycle, mainly phenoxyl radicals and nitrogen dioxide (Tyr-O●, ●NO2), can mediate oxidation of plasma constituents (Table 1).

The accessibility of the n class="Gene">MPO active site can be tested by substrates with difical">pan class="Chemical">ferent characteristics. Table 2 presents the activity of different hemoproteins toward phenolic compounds that are peroxidase substrates, namely guaiacol, Amplex red and etoposide. The substrates differ in molecular mass and reduction potentials (Figure S3). Guaiacol oxidation with formation of tetraquaiacol can be observed only by peroxidase Compounds. MPO activity toward this indicative substrate is about 1000-fold 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 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; H2O2 = 100 µM) [69,72,75,103,104].

Substrate:Protein	Guaiacol *MW = 127 DEo ~ 0.95 V	Amplex Red **MW = 257 D	Etoposide **MW = 589 DEo ~ 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; ** Δmagnitude (A.U.)/min × * nmol protein; Experimental conditions are summarized in Supplementary Materials, Section S2.

The small pan class="Chemical">phenolic molecules may act as ical">pan class="Chemical">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 [106,107,108,109].

3.2. Ceruloplasmin Is an Endogenous MPO Inhibitor

The interaction of highly cationic pan class="Gene">MPO (pI ~ 10) with anionic proteins and with the negatively charged surfaces of difical">pan class="Chemical">ferent 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].

pan class="Gene">CP is a globular anionic ical">pan class="Chemical">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].

pan class="Gene">CP binds to ical">pan class="Gene">MPO with stoichiometry 2:1: one molecule of CP binds to each monomer of MPO with a dissociation constant of ~0.15 µМ [117]. Binding of CP in close proximity to the MPO active center results in inhibition of both the halogenating and peroxidase activities of MPO [118,119,120,121]. 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 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 n class="Gene">MPO decreases with increasing pH [124,125]. The rate ical">pan class="Chemical">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].

pan class="Chemical">Peroxidase substrates can dose-deical">pan class="Chemical">pendently 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 pan class="Chemical">couple ical">pan class="Chemical">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].

pan class="Gene">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 ical">pan class="Chemical">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 [133,134,135].

More than 60% of amino acids residues are identical in EPO and pan class="Gene">MPO, and even higher ical">pan class="Chemical">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].

pan class="Gene">Lactoperoxidase was found in muical">pan class="Chemical">cosal 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].

pan class="Gene">LPO is monomeric hemoprotein (MW ~ 78 kD). The pical">pan class="Chemical">rosthetic group is bound to the protein autocatalytically; noncovalently bound heme becomes covalently bound through two ester linkages after enzyme exposure to H2O2 [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 observed in saliva (2–4 mM), and that of iodide was in milk (2 µM) [33,36]. HOSCN formed by LPO is a weak oxidizing agent that reacts primarily with thiols and inhibits microbial metabolism and growth [47,142].

The substrate channel of pan class="Gene">LPO is longer, narrower, and more hydropical">pan class="Chemical">hobic compared to that of MPO, which prevents direct oxidation of macromolecules by the enzyme [36,50].

pan class="Gene">Thyroid peroxidase is localized in memical">pan class="Chemical">branes of thyroid follicle cells. The enzyme generates HOI and is involved in the synthesis of the thyroid gland hormones: thyroxine and triiodothyronine [21].

5. Hemoglobin and Myoglobin

The main function of respiratory hemoproteins, such as pan class="Chemical">Hb and ical">pan class="Gene">Mb, is the storage and delivery 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 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 participate in NO metabolism [143,144,145].

pan class="Chemical">Hb ical">pan class="Chemical">consists of four identical subunits (MW ~ 64 kD), and Mb is a monoglobular protein (MW ~ 16.7 kD). Proteins perform their functions, having iron in the active center in the ferrous (Fe(II)) state with free sixth coordination bond. The characteristic EPR signal of open heme measured at g ~6 can be detected from the proteins at the temperature of liquid nitrogen (77 K), and heme can be easily nitrosylated (Figure 4) [69,146]. There are oxidizable amino acids in close proximity to heme; the addition of H2O2 to protein solutions causes formation of protein-derived radicals [64,147].

Figure 4

Low-temperature EPR (77 K) spectra of Hb and Mb [69]: (A) low field EPR signal of ferric myoglobin; (B) a typical LT EPR signal of heme-nitrosylated Hb(II); (C) spectrum of protein-derived (tyrosyl) radicals measured in 30 s after addition of H2O2 to Hb.

The redox activity of pan class="Chemical">Hb and ical">pan class="Gene">Mb is suppressed in myocytes and erythrocytes, respectively, by the reducing environment of the cells. The ferric form of Hb comprises about 1% of total Hb in erythrocytes. Ferric Hb is effectively converted into ferrous protein by metHb reductase [148]. Myocyte injury or hemolysis cause hemoprotein leakage into tissues and blood where the globins do not have their normal reducing environment. Since the redox chemistry is the same for Hb and Mb, but the aggravation of a number of pathologies is more associated with hemolysis and the release of Hb into the plasma, this chapter mainly focuses on hemoglobin.

A pan class="Chemical">constant process of mild intravascular erythrocyte damage occurs under normal ical">pan class="Chemical">conditions, 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, the protein cannot display any peroxidase activity.

pan class="Disease">Hemolysis occurs in many diseases such as ical">pan class="Disease">trauma, sepsis, and sickle cell disease. Under some pathological conditions, particularly as a result of microangiopathic hemolytic anemia, concentrations of Hb in plasma can reach micromolar levels [149]. Under uncontrolled pathological conditions accompanied by inflammation and oxidative stress, depletion of antioxidants and generation of superoxide radicals by activated neutrophils induce the redox activity of the hemoproteins, resulting in damage to plasma proteins and lipids [62]. The globin-based free radicals of ferryl hemoglobin were detected in normal human blood upon H2O2 treatment [150].

pan class="Chemical">Ferrous Hb is activated by ical">pan class="Chemical">H2O2 to the oxoferryl form, equivalent to Compound II. The second molecule of H2O2 can convert the active site into the ferric form. The ferric form of the protein is oxidized by H2O2 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 pan class="Chemical">Hb and formation of protein-derived ical">pan class="Chemical">(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 pan class="Chemical">covalent link between the ical">pan class="Chemical">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].

pan class="Chemical">Contrary to ical">pan class="Gene">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

pan class="Gene">Haptoglobin (Hp) is an abundant plasma protein with high binding affinity toward ical">pan class="Chemical">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 [158,159,160,161]. 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 pan class="Disease">inflammation [12,156]. These amounts of Hp are sufficient to bind micromolar levels of free ical">pan class="Chemical">Hb [69].

Trapping of free pan class="Chemical">Hb by Hp results in the formation of ical">pan class="Gene">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 H2O2 [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].

pan class="Gene">Hb-Hp aggregates were uptaken by macrophages at rates exceeding tical">pan class="Chemical">hose 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].

pan class="Gene">Hb-Hp aggregates were found in septic plasma [69]. In the ical">pan class="Disease">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

pan class="Gene">Hx is a plasma protein that functions to sequestrate free ical">pan class="Chemical">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 pan class="Gene">Hx ical">pan class="Chemical">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 n class="Chemical">Hb(III) is exposed to ical">pan class="Chemical">H2O2, 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

n class="Gene">Cyt c is a ical">pan class="Chemical">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 pan class="Chemical">heme of ical">pan class="Gene">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 n class="Gene">cyt c, acical">pan class="Chemical">companied by the loss or exchange of Met80 and an increase in peroxidase activity, were observed when cyt c interacted with negatively charged phospholipid membranes [26,172,173,174,175]. 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 molecules are components of the mitochondria interior. Millimolar levels of (ferri)cyt c are contained in the intermembrane space [176].

Cardiolipin is a mitocn class="Chemical">hondria-sical">pan class="Chemical">pecific phospholipid that is located predominantly in the inner leaflet of the inner mitochondria membrane in normal cells. Tetralinoleoyl cardiolipin (TLCL) is specific for cardiac and skeletal muscles; in other cells, highly diversified forms of CL are present. In apoptotic cells, the CL content both in the outer leaflet of the inner membrane and in the outer 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 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, like H2O2, NO, and CO, gain access to the heme site of the protein (Figure 5) [180,181]. The dependence of the magnitude and shape of cyt c-CO and cyt c-NO complex spectra on CL concentration shows that the increase in CL potentiates conformational changes of cyt c and enhances heme accessibility for small molecules (Figure S4) [75]. At relatively high CL concentrations (cyt c:CL > 1:15), a three-line signal with a splitting of 17 G emerges at g = 2.009 in the LT EPR spectra of nitrosylated cyt c/CL (Figure 5B). This three-line signal is due to the shift in electron density in Fe-N bond to nitrogen (nuclear spin I = 1) due to the cleavage of the His18-Fe(II) bond [40].

Figure 5

Cardiolipin induces protein unfolding of cyt c accompanied by the loss or exchange of the distal heme iron ligand resulting in an increased accessibility of heme for small molecules [26,75]. (A) absorbance spectra of native cyt c(II)/CL complexes and complexes incubated with CO; (B) LT EPR spectrum of nitrosylated cyt c(II)/CL complexes, (C) LT EPR spectrum of cyt c(III)/CL complexes exposed to H2O2 for 30 s.

Exposure of pan class="Gene">cyt c/CL ical">pan class="Chemical">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 H2O2 (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 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 n class="Chemical">peroxidase activity of ical">pan class="Gene">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 pan class="Gene">cyt c into ical">pan class="Chemical">peroxidase, H2O2, 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 H2O2, 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 pan class="Gene">cyt c/CL ical">pan class="Chemical">peroxidase activity in mitochondria are based on the restriction of H2O2 and substrate access to cyt c active site [40,75,177]:

In resting-state mitocpan class="Chemical">hondria, most ical">pan class="Chemical">CLs and cyt c are spatially separated; therefore, the peroxidase activity of cyt c is not significant.

The active center of pan class="Gene">cyt c is occupied by an acyl chain of CL, which prevents access of ical">pan class="Chemical">peroxidase substrates to Tyr67 in the active site.

pan class="Chemical">Tyrosyl radicals exposed on the ical">pan class="Gene">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 H2O2 decreases.

Mitocpan class="Chemical">hondrial NO, from external sources and generated by mitocical">pan class="Chemical">hondrial nitric-oxide synthase, plays a regulatory role in the control of peroxidase activity of cyt c/CL complexes.

Design and synthesis of mitocpan class="Chemical">hondria-targeted inhibitors of ical">pan class="Gene">cyt c/CL complex peroxidase activity is a promising field of investigation for the development of new anti-apoptotic drugs [185,186,187,188].

6.2. Cytoglobin

n class="Gene">Cygb is a recently disical">pan class="Chemical">covered 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 [62,143,144,145]. 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 pan class="Chemical">Hb and ical">pan class="Gene">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].

The protein pan class="Chemical">heme group of ical">pan class="Gene">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 pan class="Chemical">disulfide ical">pan class="Chemical">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 H2O2 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].

The protein with pan class="Chemical">disulfide ical">pan class="Chemical">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 H2O2 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 pan class="Chemical">ferric form of ical">pan class="Gene">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.

pan class="Chemical">Fatty acid binding is deical">pan class="Chemical">pendent 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 n class="Chemical">lipid regulators of ical">pan class="Gene">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 pan class="Gene">Cygb ical">pan class="Chemical">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 pan class="Gene">Cygb ical">pan class="Chemical">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

pan class="Chemical">Peroxidases form a network in the body that may exert both beneficial and adverse efical">pan class="Chemical">fects. 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.

Table 3

The major mechanisms for regulation of peroxidase activity of hemoproteins.

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