ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA.Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese. Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis. We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide. Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo. We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis. To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S 2؊ rather than S 0 .

The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283-8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.The metabolism of many sulfur-containing biomolecules remains incompletely understood. Among the metabolic pathways requiring further elucidation are those leading to ironsulfur clusters (1-5), biotin (6 -8), molybdopterin (9), lipoic acid (10), thiamin (8, 11), and sulfur-containing bases in RNA (12). The sulfur-containing nucleosides include 4-thiouridine (s 4 U), 1 which is found at position 8 of some bacterial tRNA ( Fig. 1) and serves as a photosensor for near-UV light (12). The s 4 U undergoes a photoactivated 2 ϩ 2 cycloaddition with cytidine 13 when the tRNA is exposed to light of a wavelength similar to the 334 nm absorbance maximum of s 4 U (13-15). The resulting cross-linked tRNA are poor aminoacylation substrates (16), and the accumulation of uncharged tRNA arrests growth by triggering the stringent response (17, 18). Lipsett and co-workers (19,20) investigated the enzymology of s 4 U biosynthesis in Escherichia coli and reported that the overall reaction utilized cysteine as the sulfur donor and required ATP as a substrate. Lipsett and co-workers (20) concluded that two enzymes were required and that one of them also plays a role in thiamin biosynthesis and requires the cofactor PLP for activity (21,22). By using a genetic screen based on the role of s 4 U as a photosensor (18,(22)(23)(24), the genetic loci of two ...

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