Unveiling the Significance of Arginine, a second shell conserved residue on structure, stability, and activity of Non- Seleno human Peroxiredoxin 6

1-Cys Peroxiredoxin 6 (Prdx6) is a unique bifunctional antioxidant enzyme highly expressed in mammalian lungs. It plays a dual role in cancer, acting as both a suppressor and promoter during different stages of tumor development. Prdx6 exhibits two distinct enzymatic activities: phospholipase A2 (PLA2) activity for phospholipid cleavage and peroxidase activity that reduces reactive oxygen species. The peroxidase catalytic site comprises Pro40, Thr44, Cys47, and Arg132, while second-shell residues—Glu50, Leu71, Ser72, His79, and Arg155—surround the active site and are conserved across peroxiredoxin families. Despite growing interest in Prdx6 as a therapeutic target, the structural determinants that stabilize its active conformation remain poorly understood. Particularly, the α5-helix region of Prdx6 can shift between fully folded and locally unfolded conformations to facilitate substrate binding and catalysis. This study focused on Arg155, a conserved second-shell residue, to assess its role in enzyme structure and function. Using site-directed mutagenesis, we generated Arg155 mutants and compared them with wild-type Prdx6 through biochemical, biophysical, and in silico analyses. The mutants displayed reduced thermal and urea stability, diminished peroxidase activity, and conformational alterations. These losses were attributed to the absence of stabilizing interactions normally provided by the Arg155 side chain. Our findings highlight Arg155 as a critical determinant of Prdx6 structural stability and catalytic efficiency. Understanding its role may aid in developing selective inhibitors or modulators of Prdx6 for oxidative stress-related diseases, including cancer.