Elucidation of sulfur transfer mediated by ThiI

In some bacterial tRNAs, uridine-8 is modified to 4-thiouridine (s 4U), which acts as a near-UV photosensor. The biosynthesis of s 4U involves two enzymes, IscS and ThiI that were originally named for their involvement in the biosynthesis of iron-sulfur clusters and thiamin, respectively. Escherichia coli ThiI has two conserved cysteine residues, Cys-456 and Cys-344, and site-directed mutagenesis has shown that both are critical for ThiI function. Mueller has proposed two mechanisms that differ primarily in whether an enzymic persulfide group or bisulfide attacks uridine. The results presented in this dissertation provide direct evidence for two key intermediates shared by these mechanisms.; Single turnover experiments provided indirect evidence that both a persulfide group at Cys-456 and a disulfide bond between Cys-344 and Cys-456 form during the catalytic cycle. These intermediates were then definitively identified by mass spectrometry. The direct observation of the trapped persulfide group rules out the formation of alternative higher order polysulfide species at Cys-456.; The nucleophilic persulfide mechanism postulates the formation of a disulfide-linked adduct between ThiI and tRNA. Gel shift experiments run under non-reducing conditions revealed a retarded band that contained both RNA and protein and that disappeared upon reduction. These results are consistent with a disulfide linkage between ThiI and tRNA. Preliminary MALDI-MS data showed that Cys-456 of ThiI is linked to uridine-8 through a disulfide bond. These results strongly favor the nucleophilic persulfide mechanism, but they must be reproduced before one can confidently conclude that it is the true mechanism of s4U biosynthesis. These preliminary observations would be the first case in which an enzymic persulfide group acts as a nucleophile during a catalytic cycle in a biological system and would therefore have implications for sulfur metabolism generally.