Kinetic analysis of the bisubstrate cysteine desulfurase SufS from Bacillus subtilis.

Cysteine is the major sulfur donor for thio cofactors in bacterial and eukaryotic systems. The first step in sulfur mobilization involves a PLP-dependent enzymatic mechanism. During catalysis, free cysteine is converted into alanine with the concomitant formation of a persulfide bond with the catalytic cysteine residue, thus forming a covalent enzyme intermediate. Cysteine desulfurases in their persulfurated forms serve as donors at the intersection of various cellular sulfur-requiring pathways. Most Gram-positive bacteria, including Bacillus subtilis, contain a cysteine desulfurase gene sufS located adjacent to the gene encoding the proposed Fe-S cluster scaffold SufU. In this work, we identified the participation of SufU as a substrate in the SufS catalytic mechanism. Development of a sensitive method for detection of alanine formed in the SufS reaction enabled the identification of its associated mechanistic features. Steady-state kinetic analysis of alanine formation provided evidence of a double-displacement mechanism (ping-pong) of the cysteine:SufU sulfurtransferase reaction catalyzed by SufS. Results from site-directed mutagenesis of the catalytic cysteine (SufS(C361A)) and iodoacetamide alkylation of SufU support the occurrence of persulfide sulfur transfer steps in the mechanism of SufS.