A Cys201Ser mutation enhances PHD2-dependent hydroxylation reaction rate and protects from protein oxidation. (A) Conservation of cysteine residues (Cys201 and Cys208 in human PHD2) in all 3 human (Hs) and mouse (Mm) PHD isoforms. (B) Increased reaction rate of Cys201Ser mutant PHD2 as measured by the hydroxylation-dependent VBC binding assay. Shown are mean values ± SEM of 3 independent experiments. Linear regression analyses were performed, revealing highly different slopes (P < .0001). (C) The Cys201Ser mutation confers resistance of PHD2 to H₂O₂-mediated inhibition of hydroxylation activity. Shown are mean values ± SEM of a representative experiment performed in triplicates. (D) GSH can rescue PHD2 wild type and Cys201Ser hydroxylation activities after H₂O₂-mediated enzyme damage. In brief, enzyme preparations were preincubated with 1mM H₂O₂ for 30 minutes (H₂O₂) or left untreated for a similar period (control). For rescue experiments, enzymes after H₂O₂ treatment were incubated with 5mM GSH (H₂O₂ + GSH) for 15 minutes. Hydroxylation reactions were carried out at standard assay conditions for 60 minutes. Note that all reactions contained 2mM ascorbate freshly added when hydroxylation reactions were started. Data are given as mean values ± SEM of 3 independent experiments normalized to hydroxylation activities of control reactions obtained after 60 minutes.