LAUSR

The 5‐substituted 2‐thiouridines (R5S2Us) present in the first (wobble) position of the anticodon of transfer RNAs (tRNAs) contribute to accuracy in reading mRNA codons and tuning protein synthesis. Previously, we showed that, under oxidative stress conditions in vitro, R5S2Us were sensitive to hydrogen peroxide (H2O2) and that their oxidative desulfuration produced 5‐substituted uridines (R5Us) and 4‐pyrimidinone nucleosides (R5H2Us) at a ratio that depended on the pH and an R5 substituent. Here, we demonstrate that the desulfuration of 2‐thiouridines, either alone or within an RNA/tRNA chain, is catalyzed by cytochrome c (cyt c). Its kinetics are similar to those of Fenton‐type catalytic 2‐thiouridine (S2U) desulfuration. Cyt c/H2O2‐ and FeII‐mediated reactions deliver predominantly 4‐pyrimidinone nucleoside (H2U)‐type products. The pathway of the cyt c/H2O2‐peroxidase‐mediated S2U→H2U transformation through uridine sulfenic (U‐SOH), sulfinic (U‐SO2H), and sulfonic (U‐SO3H) intermediates is confirmed by LC–MS. The cyt c/H2O2‐mediated oxidative damage of S2U‐tRNA may have biological relevance through alteration of the cellular functions of transfer RNA.