The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 °C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 °C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 °C, the second-order rate constants for O2 binding are 2.3 × 105 and 8.9 × 105 M−1 s−1 for isozyme H1 and 6.2 × 104 and 3.5 × 105 M−1 s−1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate contants for the oxycomplex of isozyme H1 (3.8 × 10−3 s−1) and isozyme H8 (1.0 × 10−3 s−1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 × 107 M−1) and H8 (6.2 × 107 M−1) are higher than that of myoglobin (1.9 × 106 M−1). O2 binding to ferroperoxidase of isozyme H1 was found to increase upon ionization of a group with pKa = 6.63 and ΔHion = 7.2 kcal/mol, indicative of an imidazole group of a His residue. The characteristics of the oxycomplex of lignin peroxidases suggest that the electronic structure and geometric configuration in their heme environment are different from that in myoglobin or horseradish peroxidase.
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