Crystal structure of Leishmania major peroxidase and characterization of the compound I tryptophan radical

Abstract

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp 208 in LmP and Trp 191 in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp 191 forms a stable cationic radical after reaction with H 2O 2 to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp 208 in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp 208 using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp 191 and Trp 208, respectively, which is absent in APX. This region provides stability to the Trp 191 radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp 208 forms a stable cationic radical. In LmP conversion of the Cys 197 to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.