Standard reduction potentials of all couples of the peroxidase cycle of lactoperoxidase

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Abstract

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears and saliva and has physiological significance in antimicrobial defense. Its predominant physiological role is to convert hydrogen peroxide and thiocyanate in hypothiocyanite. In this study, the standard reduction potentials of all redox couples involved in the halogenation and peroxidase cycle of LPO have been determined by multi-mixing stopped-flow spectroscopy. The standard reduction potentials of the redox couples compound I/native LPO, compound I/compound II of LPO, and compound II/native LPO are (1.09 ± 0.01) V, (1.14 ± 0.02) V, and (1.04 ± 0.02) V, respectively, at pH 7 and 25 °C. Thus, for the first time, a full description of these important thermodynamic parameters of lactoperoxidase has been performed, allowing a better understanding in the substantial differences in the oxidation of two- and one-electron donors by LPO and other members of the mammalian heme peroxidase superfamily.

Introduction

The heme enzyme lactoperoxidase (LPO) is involved in host defense mechanisms being found in mucosal surfaces and exocrine secretions including milk, tears and saliva [1]. It comprises functional and structural homology with other mammalian peroxidases such as myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO) [2]. In these peroxidases the porphyrin ring is considerably distorted from planarity due to covalent bonds with the apoprotein. In the heme of LPO, two ester linkages are formed autocatalytically, namely between the heme 1- and 5-methyl groups and Glu375 and Asp225, respectively [3], [4]. Two ester bonds and a similar self-processing mechanism were also postulated for EPO [5] and TPO [6]. By contrast, in myeloperoxidase three covalent links exist between the heme and the protein [7]. In addition to the two ester linkages a thioether sulfonium bond between the β-carbon of the 2-vinyl group and Met243 is present [7]. This additional thioether bond influences the spectroscopic and catalytic properties of MPO and could have a role in modulating the redox properties as shown by site directed mutagenesis [8], [9]. Generally, the existence of covalently linked, non-planar heme and the differences in heme linkage within the mammalian peroxidase superfamily are responsible for all differences of the optical properties and all characteristics related to electron transfer reactions mediated by these proteins.

Similar to other mammalian peroxidases LPO catalyses a set of reactions that can be summarized as peroxidation and halogenation cycles. In both cycles, the native ferric enzyme is oxidized by hydrogen peroxide to compound I (reaction (1)), a ferryl porphyryl radical species where an oxygen is coupled by a double bond to the iron [10]:PorFe3++H2O2+PorFeIV=O+H2O

In the peroxidation cycle, compound I is reduced by two successive one-electron steps via compound II (PorFeIV = O) to the native enzyme (reactions (2), (3)). In these one-electron oxidation reactions, numerous substrates (AH) are oxidized to their corresponding radicals (Aradical dot) supplying one electron to compound I and compound II:+PorFeIV=O+AHPorFeIV=O+A+H+PorFeIV=O+AH+H+PorFe3++A+H2O

On the other hand, in the halogenation cycle compound I can be directly reduced to the native ferric state (reaction (4)) upon attracting two electrons from (pseudo)halides (X) whereby (pseudo)hypohalous acids (HOX) are formed.+PorFeIV=O+X-+H+PorFe3++HOX

By isomerisation the porphyryl radical of LPO compound I can be also converted into an amino acid radical species [11]. This conversion was only found in the absence of one- and two-electron donors. Apparently, this amino acid radical species has never been detected under physiological conditions [12].

The mammalian peroxidases differ in their ability to catalyze the one-electron oxidation of small molecules and the two-electron oxidation of halide ions. For example, at neutral pH, only MPO is capable to oxidize chloride at a reasonable rate [13], [14] and it is assumed that chloride and thiocyanate are competing substrates in vivo [15]. EPO can oxidize chloride only at acidic pH [16] and at normal plasma concentrations bromide and thiocyanate function as substrates [17]. For LPO it has been shown that it has barely detectable activity with bromide at neutral pH but oxidizes iodide and thiocyanate, the latter being thought to be the physiological substrate [1], [18], [19], [20], [21], [22].

The knowledge of redox properties of all conversions between enzyme intermediates during their catalytic reaction is important to understand the extraordinary reactivity and differences in reactivity between these peroxidases. The systematic study of these thermodynamic properties started only recently. Sequential-mixing stopped-flow technique was successfully used by our group to determine the standard reduction potentials of all redox couples of the peroxidase cycle of MPO at 25 °C and pH 7 [23], [24], [25]. They are 1.16, 1.35, and 0.97 V for the couples compound I/native MPO, compound I/compound II, and compound II/native MPO, respectively [23], [24]. The standard reduction potential of the couple compound I/native enzyme was determined to be 1.10 V for EPO [23].

The main objective of this paper was to determine for the first time the standard reduction potentials of the redox couples compound I/native enzyme, compound I/compound II and compound II/native enzyme of lactoperoxidase at pH 7.0 and 25 °C by using the sequential-mixing stopped-flow technique and relate these data to structural and functional properties of this enzyme.

Section snippets

Materials

Lactoperoxidase from bovine milk was purchased as a lyophilized powder (Sigma Chemical Co. type L-8257, purity index A412/A280 = 0.9). Enzyme concentration was determined by using the extinction coefficient 112,000 M−1 cm−1 at 412 nm [26]. Highly purified human myeloperoxidase was purchased from Planta Natural Products (www.peroxidase.at). Hydrogen peroxide, obtained as a 30% solution from Sigma Chemical Co. was diluted and the concentration determined by absorbance measurement at 240 nm where

Standard reduction potential of compound I/native LPO

By mixing 2 μM ferric LPO with H2O2 (1–1.5 μM) in the conventional stopped-flow mode compound I formation was induced (Fig. 1). Within milliseconds the absorbance at 412 nm decreased and, finally, reached a steady-state level at defined Soret absorbance (strongly depending on the involved concentration of the oxidant). From the differences in absorbance at 412 nm before mixing and immediately after the oxidant-mediated absorbance decrease, the equilibrium concentrations of native LPO and

Discussion

In this work, we extended successfully our trial to determine the standard reduction potentials of all redox couples of the peroxidase cycle of mammalian heme peroxidases on lactoperoxidase. By the use of stopped-flow analysis, E′° values of (1.09 ± 0.01) V for the couple compound I/native LPO, (1.04 ± 0.02) V for the couple compound II/native LPO, and (1.14 ± 0.02) V for the couple compound I/compound II were found.

With varying concentrations of hydrogen peroxide as well with peroxyacetic acid two

Abbreviations

    LPO

    lactoperoxidase

    MPO

    myeloperoxidase

    EPO

    eosinophil peroxidase

    HRP

    horseradish peroxidase

    PorFe3+

    ferric enzyme

    radical dot+PorFeIVdouble bondO

    compound I

    PorFeIVdouble bondO

    compound II

    Por

    porphyrin ring

    E′°

    standard reduction potential

    K

    equilibrium constant at pH 7

    PAA

    peroxyacetic acid

    HOX

    hypohalous acid

Acknowledgements

This work was supported by the German Research Foundation (Postgraduate Training Programme ‘Mechanisms and Applications of Non-Conventional Oxidation Reactions’), the Austrian Science Fund FWF (Grant No. P15660) and the European COST Action D21.

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