RESUMO
The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base catalyst for heterolytic cleavage of an OO bond in H2O2. However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to investigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H2O2 is decreased by approximately five orders of magnitude relative to wild type, which is consistent with electron donor oxidation data where the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rate of intermediates in the mutants is not significantly lower than that for the wild type, indicating that the His68 has no major role in homolytic cleavage of an OO bond in peroxynitrite. EPR spectroscopic data suggest that the transient intermediates formed by the reaction of LmP with H2O2 exhibits an intense and stable signal similar to CCP Compound ES whereas in case of the reaction with peroxynitrite, this signal disappears, indicating that the transient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higher decay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactive species in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higher peroxynitrite degrading activity.