Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron para-magnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp(233)(center dot+)] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate -binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state ki-netic experiments, molecular dynamic simulations, and bio-informatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Automated summary

This study solved the structure and examined the reactivity of the Trypanosoma cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP), a hybrid heme peroxidase in the protozoan parasite Trypanosoma cruzi. The results showed that TcAPx-CcP has overall structural similarity to other peroxidases, with differences in substrate-binding regions. Additionally, TcAPx-CcP preserves the electron transfer pathway from cytochrome c to heme, preferentially oxidizing cytochrome c but still capable of oxidizing ascorbate.