Lactoperoxidase is a member of the family of the mammalian heme peroxidases which have a broad spectrum of activity. Their best known effect is their antimicrobial activity that arouses much interest in
Mammalian peroxidases are distinct from plant peroxidases in size, amino acid homologies, nature of the prosthetic group, and binding of the prosthetic group to the protein. Plant peroxidases consist of approximately 300 amino acids with a noncovalently bound heme moiety, while mammalian peroxidases have 576 to 738 amino acids with a covalently bound heme moiety [
Based on amino acids homologies, peroxidases are now classified into two superfamilies. The first superfamily clusters peroxidases from plant, archea bacteria, and fungi and is classified into three classes. Class I is composed of intracellular peroxidases such as yeast cytochrome
MPO is a lysosomal constituent in neutrophils and macrophages and displays antimicrobial activity during the postinfection inflammatory process but is also involved in acute inflammatory diseases and in other pathologies such as atherosclerosis [
This review summarizes present knowledge on the mode of action of lactoperoxidase which can be extended to the mammalian peroxidases mode of action and on the specific interaction of LPO with thiocyanate and iodide, together or alone, with implications on its antimicrobial activity.
LPO is a calcium- and iron-containing glycoprotein arranged in a single polypeptide chain of about 80 kDa [
Heme peroxidases are oxidoreductase enzymes that act through different reaction mechanisms. Although some characteristics are specific to a member of the family, the same global procedure is followed by all members. The cycle begins with the transformation of the native enzyme into Compound I. Afterwards, and depending mainly on substrate concentrations, Compound I enters the halogenation cycle or the peroxidase cycle which both end by the enzyme returning to its native state (see Figure
Halogenation or peroxidase cycle of peroxidases Compound I.
The first reaction of the native enzyme starts in the presence of hydrogen peroxide, which acts as a relatively specific electron acceptor [
The native enzyme undergoes a two-electron oxidation. Two electrons are transferred from the enzyme to hydrogen peroxide which is reduced into water. Compound I is two oxidizing equivalents above the native enzyme: one is in the oxyferryl heme center and the other is present as an organic cation located on the porphyrin ring [
In the presence of a halogen (Cl−, Br−, or I−) or a pseudohalogen (SCN−), Compound I is reduced back to its native enzymatic form through a two-electron transfer while the (pseudo)halogen is oxidized into a hypo(pseudo)halide (see Figure
The oxidation rate of halogens by peroxidase-derived Compound I depends on various factors. One of these factors is the standard reduction potential of the enzyme, which differs among peroxidases and plays a role in their capacity to oxidize specific (pseudo)halides. The redox reaction can occur only if the reduction potential of the enzyme is equal or superior to the reduction potential of the substrate. The standard reduction potential at pH 7 of Compound I peroxidases and the couple of two-electron reduction HOX/X− is ranking in the following ascending rank: LPO Compound I < EPO Compound I < MPO Compound I; HOSCN/SCN− < HOI/I− ≪ HOBr/Br− < HOCl−/Cl− [
Apparent second-order rate constant at pH 7 (×104 M−1 s−1) of the reaction between myeloperoxidase Compound I or lactoperoxidase Compound I with (pseudo)halides [
The reduction potential of the Compound I/native enzyme and HOX/X− redox couples depends on reactant concentrations and pH values. At a specific reactant concentration, it decreases with increasing pH values, but slopes differ (Figure
Illustration (according to [
Concentrations of (pseudo)halogens also affect their affinity to Compound I (Figure
Illustration of the interaction between the biodisponibility of a peroxidase, the (pseudo)halogen concentration in plasma, in saliva, and in milk, and the production of oxidant molecules. MPO: myeloperoxidase; SPO: salivary peroxidase; LPO: bovine lactoperoxidase; OCl−: hypochlorite; and OSCN−: hypothiocyanite. Although chloride is the most available substrate compared to thiocyanate, bromide, and iodide, thiocyanate is the most effective substrate for the Compound I and hypothiocyanite could be produced at equal or superior levels compared to hypohalides.
Although plasma Cl− concentrations are 1,000-fold higher than Br− and SCN−, MPO Compound I oxidizes similar amounts of SCN− and Cl− [
Alternatively, Compound I can shift to the peroxidase cycle, which consists of two sequential one-electron transfers back to the enzyme that yield (i) Compound II and (ii) the native enzyme, while the substrate is oxidized into a radical (Figure
Compound I is not specific regarding the one-electron donor; it can be exogenous or endogenous, and a lot of candidates have been described [
During the first step of the peroxidase cycle, the cation located in the porphyrin ring undergoes a one-electron reduction with formation of Compound II and concomitant oxidizing of one one-electron substrate [
The standard reduction potential of the couple Compound I/Compound II is high and allowed the one-electron oxidation by Compound I of a wide range of substrates [
The peroxidase cycle has been described as a possible catalytic sink for nitric oxide (NO) [
However, thiocyanate can act as a one-electron donor and be part of the peroxidase cycle, with the sequential formation of two thiocyanate radicals [
In the presence of both one- and two-electron donors, competition for oxidation can occur and favor the halogenation or the peroxidase cycle. The presence of EDTA inhibits the oxidation of iodide due to competition for binding to Compound I [
The function of heme peroxidases can be inhibited in several ways that could be classified into three categories. The first one could represent an inhibition of the enzyme by (i) molecules or proteins and (ii) external conditions such as pH and temperature. For example, cyanide, azide, nitrite, mercaptomethylimidazole, thiourea, superoxide, high levels of nitric oxide, and high levels of thiocyanate bind to the native enzyme and alter Compound I formation [
The second group of inhibitors could concern substances or proteins which are able to interfere with the catalytic mechanism. For example, catalase consumes H2O2 and will stop the formation of Compound I [
The third class could be related to substances or proteins which are buffering active molecules produced during the catalytic reaction. For example, presence of thiosulfate, thioglycolate, glutathione, dithiothreitol, cysteine, NAD(P)H, and tyrosine will reduce the antimicrobial activity through reacting with OCl−, OBr−, OI−, or OSCN− [
LPO concentrations in cow’s milk are around 30 mg L−1 depending on season, diet, and calving and breeding season [
Thiocyanate is oxidized in a two-electron reaction that yields hypothiocyanite. Hypothiocyanite has a p
The acid form has a higher oxidation potential and is more soluble in nonpolar media so that it passes through hydrophobic barriers such as cell membranes more easily but it is less stable than the basic form (OX−) [
SCN− is the two-electron donor with the lowest reduction potential and therefore forms the hypothiocyanite acid with the lowest oxidative power compared to hypohalous acids. Hypohalous acids rank as follows, with increasing oxidative strength: OSCN− < OI− < OBr− < OCl− [
Target group of hypothiocyanite, hypoiodite, and iodine. Due to its low oxidation power, hypothiocyanite is relatively specific and is not reactive against all thiols.
Sulfhydryl oxidation by OSCN− generates sulfenyl thiocyanate, in equilibrium with sulfenic acid [
The cycle of reactions shows that thiocyanate acts like a cofactor for LPO (Figure
Illustration of the cofactor role of SCN− or I−. When the necessary conditions are fulfilled, that is, (i) no substrate competitor for SCN− or I− for binding to lactoperoxidase, (ii) enough peroxidase, H2O2 and SCN− or I−, (iii) enough R-SH, and (iv) no incorporation of SCN− or I− in stable byproducts, the quantity of OSCN− or OI− produced depends only on the amount of H2O2. SCN−: thiocyanate; I−: iodide; H2O2: hydrogen peroxide; LPO: lactoperoxidase; R-SH: peptide or protein with a thiol moiety; R-S-SCN or R-S-I sulfenyl thiocyanate or iodide; R-SOH: sulfenic acid; OSCN−: hypothiocyanite; and OI−: hypoiodite.
Although the target of OSCN− is a thiol moiety, not all sulfhydryls are equally sensitive to OSCN−; albumin, cysteine, mercaptoethanol, dithiothreitol, glutathione, and 5-thio-2-nitrobenzoic acid are all oxidized but
Some authors suggest that (SCN)2 is formed during the enzymatic reaction and then chemically hydrolyzed into hypothiocyanite [
Hypothiocyanite is less stable in acid conditions, with high concentrations of SCN− and in the presence of (SCN)2, and it is thought to break down
A recent study, based notably on spectroscopic and chromatographic methods, proposes the following net equation within the 4–7 pH range:
The proportions of end anions were different at pH 4 and pH 7; at pH 7, the proportion of CNO− was higher, SCN− formation was slower, and no CN− was detected [
It might seem easier to produce hypothiocyanite chemically in
Hypothiocyanite inhibitors have been described. For example CN−, a weak acid buffer, dissolved carbonate, excess hydrogen peroxide, hydrofluoric acid, metallic ions, glycerol, or ammonium sulfate accelerates the decomposition of OSCN−, whereas sulfonamide stabilizes it [
Appropriate concentrations of substrates induce enhanced activity [
The biological activity of hypothiocyanite is summarized in Figure
Biological activity of hypothiocyanite on bacteria and possible defense mechanism of the bacteria. Reversible inhibition is observed in that (i) hypothiocyanite is not reactive against all thiols and (ii) if hypothiocyanite is removed or diluted, the pathogen recovers. Irreversible inhibition is linked to (i) long period of incubation, (ii) the bacterial species, and (iii) hypothiocyanite concentration. HOSCN/OSCN−: acidic or basic form of hypothiocyanite and GSH: glutathione.
The sulfhydryl moiety is essential for the activity of numerous enzymes and proteins. Inhibition of bacterial glycolysis through the oxidation of hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, and glucose-6-phosphate dehydrogenase has been observed [
Furthermore, the activity of the entire system (enzyme + substrates) is known to be more effective than hypothiocyanite alone, whether enzymatically or chemically produced. This has been explained by the production of short-lived, highly reactive intermediates such as O2SCN− and O3SCN− by the enzyme or by the oxidation of OSCN− in conditions of excess H2O2 [
Iodide is oxidized by Compound I through a single two-electron transfer that yields oxidized I− in the form of I2 or HOI [
Based on the inorganic chemistry of iodine in water and literature on enzymatic oxidation of iodide, the active molecules have been described as follows (Figure Under pH 6 and in the presence of iodide, only I2, I−, and In solution within a 6–9 pH range and with a maximum 1 mM iodide, a mixture of HOI/I2OH/I2/ In iodine solution, without iodide or when available iodide has been oxidized, the number of I2-derived molecules decreases with decreasing I2 concentrations. At 1,000 At high I− and 1% I2 concentrations as in Lugol solution,
Illustration of the molecules that can be present after oxidation of iodide by lactoperoxidase in presence of H2O2. The active species depend mainly on the concentration of iodide (upper part) and the pH (lower part). The species with an oxidant power are represented in bold.
The stability of HOI and I2 is linked to their disproportionation in iodate, which has no oxidative activity in neutral and basic pH conditions [
I2 stability increases at higher pH values and higher iodide concentrations [
The oxidative strength of I2 is between that of the corresponding hypohalous acid HOI and the hypoiodite ion OI− and ranks as follows: 0.485 V (OI−) < 0.536 V (I2) < 0.987 V (HOI) [
HOI reacts through very rapid oxidation of thiol groups, oxidation of NAD(P)H, oxidation of
In some conditions, that is, (i) enough iodide, H2O2, and peroxidase, (ii) no accumulation of oxidized iodide, and (iii) no incorporation of iodide into stable byproducts such as tyrosine residues, iodide acts as a cofactor (Figure
In the case of high concentrations of I− and/or H2O2, inhibition of tyrosine iodation has been observed [
Both reactions deplete the amount of the active oxidizing agent I2. In the absence of tyrosine, oxidized iodide reacts with nucleophilic molecules such as I−, Cl−, or OH− to form I2,
HOI can be produced chemically through oxidation of I− by Cl2 or O3, with a short half-life due to overoxidation of HOI by Cl2 and O3 [
The biological action of oxidized iodide (Figure
Biological activity of hypoiodite or iodine on bacteria. Irreversible inhibition is observed and could be linked to (i) oxidation of thiol groups, NAD(P)H, and thioether groups, (ii) high reactivity of HOI/I2 against thiol and reduced nicotinamide nucleotides, and (iii) the incorporation of iodide in tyrosine residue of protein (iodination of protein). HOI/OI−: acid or basic form of hypoiodite and I2: iodine.
Due to the cofactor role of I−, inhibition of respiration in
The activity of the I− peroxidase system is more effective against
CN−, azide, EDTA, and SCN− inhibit the formation of oxidized iodide [
LPO-H2O2-I− in presence of
The combination of SCN− with I− in the lactoperoxidase system has been poorly studied. Tackling the enzymatic mechanism is tricky, and contradictory results have been found about microbial activity in the concomitant presence of SCN− and I−.
In the presence of SCN− and I−, there is competition between the two substrates for oxidation by lactoperoxidase [
The molecular evolution of heme peroxidases and the preservation of their catalytic domain [
The enzymatic reactions involving mammalian peroxidases are complex and various molecules can promote or reduce dramatically the antibacterial activity of the peroxidase system. In order to favor the halogenation cycle required in
The authors declare that there is no conflict of interests regarding the publication of this paper.