In this study we investigated the role of active site residues in the peroxidase activity of Orp1 (GPx3) using three different peroxide substrates. Using a structural homology model of the reduced form of Orp1, we identified Asn126 and Phe127 as evolutionarily conserved residues that line the back of the Orp1 active site and which are likely to affect the peroxidase activity of Orp1. Additionally, we identified Phe38 as a surface residue that could influence substrate specificity as it is located adjacent to Cys36, in the same position occupied by similar hydrophobic amino acids in many Orp1 homologs. We individually mutated these residues to alanine and examined the effect of each mutation in vitro and in vivo. Chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to identify Cys-SOH modification of Cys36 in response to H2O2, tert-butyl-hydroperoxide (tert-BHP), and cumene hydroperoxide (CHP) in Orp1(WT). Mutation of Asn126 and Phe127 eliminate Cys-SOH formation and peroxidase activity in response to H2O2, tert-BHP and CHP. Furthermore, the pK(a) of Cys36 is elevated closer to that of free cysteine compared to Orp1(WT). Mutation of Phe38 does not affect the peroxidase activity of Orp1 upon exposure to H2O2. The Phe38 mutation decreases Orp1 peroxidase activities in response to either tert-BHP or CHP. The in vivo sensitivity of the Phe38 mutant to both tert-BHP and CHP is increased, while the H2O2 sensitivity is unchanged. The pK(a) of Cys36 in the Phe38 mutant is 5.0, which is the same as Orp1(WT). Taken together, these results suggest that Phe38 does not play a role in the reactivity of Cys36, but does modulate the affinity of Orp1 for alkyl hydroperoxides. (C) 2010 Elsevier Inc. All rights reserved.