Inactivation of Coprinus cinereus peroxidase by 4-chloroaniline during turnover: comparison with horseradish peroxidase and bovine lactoperoxidase

The peroxidase from Coprinus cinereus (CPX) catalyzed oxidative oligomeriza tion of 4-chloroaniline (4-CA) forming several products: N-(4-chlorophenyl) -benzoquinone monoamine (dimer D), 4,4'-dichloroazobenzene (dimer E); 2-(4- chloroanilino)-N-(4-chlorophenyl)-benzoquinone (trimer F); 2-amino-5-chloro benzoquinone-di-4-chloroanil (trimer G); 2-(4-chloroanilino)-5-hydroxybenzo quinone-di-4-chloroanil (tetramer H) and 2-amino-5-(-4-chlroanilino)-benzoq uinone (tetramer I). In the presence of 4-CA and H2O2, CPX was irreversibly inactivated within 10 min. Inactivation of CPX in the presence of H2O2 was a time-dependent, first-order process when the concentration of 4-CA was v aried between 0 and 2.5 mM. The apparent dissociation constant (K-j) for CP X and 4-CA was 0.71 mM. The pseudo-first order rate constant for inactivati on (k(inact)), was 1.15 x 10 (- 2) s(-1). Covalent incorporation of 20 mole C-14-4-CA per mole of inactivated CPX was observed. The partition ratio wa s about 2200 when either 4-CA or H2O2 was used as the limiting substrate. T hese results shrew that 4-CA is a metabolically activated inactivator (i.e. a suicide substrate). Unmodified heme and hydroxymethyl heme were isolated from native, 4-CA-inactivated and H2O2-incubated CPX. Inactivation resulte d in significant losses in both heme contents. Analysis of tryptic peptides from 4-CA-inactivated CPX by. MALDI-TOF/ MS and UV-VIS spectrophotometry s uggested that trimer G and tetramer H were the major 4-CA derivatives that were covalently bound, including to apeptide (MCDAGFSPDEVVDLLAAHSLASQEGLNSA IFR) containing the heme binding site. These studies show that heme destruc tion and covalent modification of the polypeptide chain art: both important for the inactivation of CPX. These results were compared with similar stud ies on 4-CA-inactivated horseradish peroxidase (HRP) and bovine lactoperoxi dase (LPO) during the oxidation of 4-CA. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.