Unconventional monooxygenation by the O₂-dependent tRNA wobble uridine hydroxylase TrhO

Modifications at the wobble position of transfer RNA (tRNA) are critical for accurate codon recognition and efficient translation. 5-Hydroxyuridine serves as a key intermediate for more complex wobble uridine derivatives commonly found in bacterial tRNAs and is synthesized by either prephenate-dependent TrhP or dioxygen-dependent TrhO. Despite its biological importance, structural and mechanistic insights into these enzymes have remained elusive. Here, we report the cryo-electron microscopy structure of Bacillus subtilis TrhO-tRNA^Ala complex. Combined with biochemical analyses, our results reveal that TrhO functions without any metal or organic cofactor, unlike most other oxygenases. We propose that the conserved C179 reacts with dioxygen to form a thiohydroperoxy intermediate, which is cleaved to produce 5-hydroxyuridine and a sulfenic acid at C179. The oxidized cysteine subsequently forms a disulfide bond with the adjacent C185, protecting the catalytic cysteine from irreversible overoxidation. These findings broaden our understanding of cofactor-independent dioxygen use in aromatic ring hydroxylation.