Mutation of Val90 to His in the pseudoperoxidase from Leishmania major enhances peroxidase activity

Pseudoperoxidase from Leishmania major (LmPP) catalyzes the breakdown of peroxynitrite though it can hardly react with H2O2. Our modeling structure predicts that a conserved His to Val switch near the distal heme pocket of LmPP may determine the profile of its H2O2 activity. To test this hypothesis, we have generated complementary mutations in the LmPP (V90H) and studied the formation of Compounds I and II. The rate of transition from high spin ferric state of V90H to Compound I by H2O2 is increased by approximately three orders relative to wild-type LmPP, which is consistent with electron donor oxidation data where the V90H mutant enzyme is ~ 30 fold more active than wild type. Thus, our data indicate that a lower rate for heterolytic cleavage of the OO bond of H2O2 in wild type LmPP is caused by the His/Val switch in heme distal site. In the catalysis of peroxynitrite scavenging, V90H LmPP has lower catalytic activity compared to the wild type enzyme. In contrast to peroxynitrite scavenging, the second order rate constant of peroxynitrite binding step in mutant enzyme does not change significantly compared to the wild-type. Spectral data suggest that the distal Val90 residue in LmPP prevents the ferryl species formation in the presence of peroxynitrite. The lower peroxynitrite scavenging activity of the mutant reflects increased peroxidase activity rather than isomerase activity.

► L. major pseudoperoxidase acts as a scavenger of peroxynitrite instead of H2O2.
► It contains an amino acid switch (His to Val) in heme distal site.
► The reactivity of V90H with H2O2 was increased ~ 2000 times.
► His/Val switch is important for altering enzymatic activity.