Characterization of the catalytic disulfide bond in E. coli 4‐thiouridine synthetase to elucidate its functional quaternary structure

4-Thiouridine at position 8 in prokaryotic tRNA serves as a photosensor for near-UV light, and the posttranscriptional conversion of uridine to 4-thiouridine is catalyzed by the 4-thiouridine synthetases (s4US, also named ThiI), which fall into two classes that differ in the presence of a C-terminal rhodanese homology domain. A cysteine residue in this domain first bears a persulfide group and then forms a disulfide bond with a cysteine residue that is conserved in both classes of s4US. Recent crystal structures suggest that s4US dimerizes in the presence of RNA substrate with domains from each subunit contributing to the binding and reaction of one RNA molecule, which raises the question of whether the catalytic disulfide bond in the longer class of s4US is formed within or between subunits. The E. coli enzyme is the best-characterized member of the longer class of s4US, and it was examined after quantitative installation of the disulfide bond during a single catalytic turnover. Gel electrophoresis and proteolysis/MALDI-MS results strongly imply that the disulfide bond forms within a single subunit, which provides a vital constraint for the structural modeling of the class of s4US with an appended rhodanese homology domain and the design and interpretation of experiments to probe the dynamics of the domains during catalysis.