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

Trypanosoma cruzi trypanothione reductase (TR) was irreversibly inhibited by peroxidase/H₂O₂/phenothiazine (PTZ) systems. TR inactivation depended on (a) time of incubation with the phenothiazine system; (b) the peroxidase nature and (c) the PTZ structure and concentration. With the most effective systems, TR inactivation kinetics were biphasic, with a relatively fast initial phase during which about 75% of the enzyme activity was lost, followed by a slower phase leading to total enzyme inactivation. GSH prevented TR inactivation by the peroxidase/H₂O₂/PTZ⁺ systems. Production of PTZ⁺ cation radicals by PTZ peroxidation was essential for TR inactivation. Horseradish peroxidase, leukocyte myeloperoxidase (MPO) and the pseudo-peroxidase myoglobin (Mb) were effective catalysts of PTZ⁺ production. Promazine, thioridazine, chlorpromazine, propionylpromazine prochlorperazine, perpenazine and trimeprazine were effective constituents of the HRP/H₂O₂/PTZ system. The presence of substituents at the PTZ nucleus position 2 exerted significant influence on PTZ activity, as shown by the different effects of 2-trifluoromethyl and 2-H or 2-chlorophenothiazines. The PTZ⁺ cation radicals disproportionation regenerated the non-radical PTZ molecule and produced the PTZ sulfoxide that was inactive on TR. Thiol compounds including GSH interacted with PTZ⁺ cation radicals transferring an electron from the sulfide anion to the PTZ⁺, thus nullifying the PTZ⁺ biological and chemical activities.