The role of compound III in reversible and irreversible inactivation of lactoperoxidase

Abstract

In the presence of iodide (I−, 10 mM) and hydrogen peroxide in a large excess (H2O2, 0.1–10 mM) catalytic amounts of lactoperoxidase (2 nM) are very rapidly irreversibly inactivated without forming compound III (cpd III). In contrast, in the absence of I− cpd III is formed and inactivation proceeds very slowly. Increasing the enzyme concentration up to the micromolar range significantly accelerates the rate of inactivation. The present data reveal that irreversible inactivation of the enzyme involves cleavage of the prosthetic group and liberation of heme iron. The rate of enzyme destruction is well correlated with the production of molecular oxygen (O2), which originates from the oxidation of excess H2O2. Since H2O2 and O2per se do not affect the heme moiety of the peroxidase, we suggest that the damaging species may be a primary intermediate of the H2O2 oxidation, such as oxygen in its excited singlet state (1ΔgO2), superoxide radicals (O−2), or consequently formed hydroxyl radicals (OH⋅).

Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione

“…The phenol instead stimulated the formation of an intermediate (Fig. 8B, curve c) whose Soret band at 423 nm and two distinct peaks at 550 and 589 nm in the visible region are the same as those of compound III reported previously (43,44). With time, compound III returned to the native ferric enzyme (Fig.…”

“…Compound II of LPO is predominant whenever LPO is exposed to a small excess of H 2 O 2 (21,(43)(44)(45). In this work, we prepared compound II by adding H 2 O 2 to the ferric enzyme at a molar ratio of 2:1.…”

High Dissociation Rate Constant of Ferrous-Dioxy Complex Linked to the Catalase-like Activity in Lactoperoxidase

“…Kohler et al (66) have also shown that the stoichiometry of O 2 release to the consumption of H 2 O 2 was 1:2, typical of catalase activity. It is important to note that this ratio is applied when high or low H 2 O 2 concentrations have been used (41). Several kinetic mechanisms have been proposed to provide a mechanistic explanation for these experimental findings and to explain the buildup of the LPO ferrous-dioxy complex during steady state catalysis (43,66).…”

Enantioselective Epoxidation and Carbon–Carbon Bond Cleavage Catalyzed by Coprinus cinereus Peroxidase and Myeloperoxidase

“…Originally it was believed that only steric restrictions accounted for lack of oxygen transfer by most peroxidases, since it was shown that enlarging the accessibility to the oxoferryl species in the active site of HRP for aromatic substrates by site-directed mutagenesis (Phe 41 3 Leu and Phe 41 3 Thr), led to a mutant enzyme that was capable of enantioselective epoxidation of styrene and cis-␤-methylstyrene (22). Cytochrome c peroxidase, which has a more accessible oxoferryl than HRP, could also epoxidize styrene, but no e.e.…”

“…Compound II can, in its turn, react with H 2 O 2 to give compound III, which is believed to be a dead end species and catalytically inactive (41). We recently demonstrated that the presence of compound III and an excess of H 2 O 2 should be avoided in the enantioselective sulfoxidation of thioanisole catalyzed by CiP and lactoperoxidase (10).…”