Trypanosoma cruzi trypanothione reductase is inactivated by peroxidase-generated phenothiazine cationic radicals

Trypanosoma cruzi trypanothione reductase (TR) was irreversibly inhibited b y peroxidase/H2O2/phenothiazine (PTZ) systems. TR inactivation depended on (a) time of incubation with the phenothiazine system; (b) the peroxidase na ture and (c) the PTZ structure and concentration. With the most effective s ystems, TR inactivation kinetics were biphasic, with a relatively fast init ial phase during which about 75% of the enzyme activity was lost, followed by a slower phase leading to total enzyme inactivation. GSH prevented TR in activation by the peroxidase/H2O2/PTZ(+.) systems. Production of PTZ(+.) ca tion radicals by PTZ peroxidation was essential for TR inactivation. Horser adish peroxidase, leukocyte myeloperoxidase (MPO) and the pseudo-peroxidase myoglobin (Mb) were effective catalysts of PTZ(+.) production. Promazine, thioridazine, chlorpromazine, propionylpromazine prochlorperazine, perphena zine and trimeprazine were effective constituents of the HRP/H2O2/PTZ syste m. The presence of substituents at the PTZ nucleus position 2 exerted signi ficant influence on PTZ activity, as shown by the different effects of 2-tr ifluoromethyl and 2-H or 2-chlorophenothiazines. The PTZ(+.) cation radical s disproportionation regenerated the non-radical PTZ molecule and produced the PTZ sulfoxide that was inactive on TR. Thiol compounds including GSH in teracted with PTZ(+.) cation radicals transferring an electron from the sul fide anion to the PTZ(+.), thus nullifying the PTZ(+.) biological and chemi cal activities.