Abstract 1730 The E. coli cysteine desulfurase SufS uses a half-sites mechanism likely regulated by persulfide transfer to SufE

Cysteine desulfurases (CDs) are dimeric PLP-dependent enzymes that play a crucial role in mobilization of sulfur frommcysteine, generating alanine and persulfide on an active site cysteine residue. Under oxidative stress and iron starvation condition Escherichia coli utilizes a type II CD, EcSufS to initiate the biosynthesis of iron-sulfur clusters. EcSufS couples with an accessory protein, EcSufE, facilitating a protected transfer of persulfide between EcSufS and EcsufE with an 8-fold increase in EcSufS activity. Pre-steady state burst and single-turnover kinetic assays were used to investigate whether EcSufE simply acts as persulfide acceptor or if it plays a role in activating other steps in the EcSufS mechanism. EcSufS exhibits a pre-steady state burst phase of alanine formation with a kburst value of 3.7 ± 0.2 s-1 and anamplitude equivalent to 46% of total active sites. The sub-stochiometric burst amplitude indicates a half-sites mechanism, where only one active site of the EcSufS dimer is active at a time. However, in the presence of EcSufE, the pre-steady state burst is lost for alanine formation, indicating activation of a post-chemistry rate-determining step by EcSufE. Assays following persulfide formation obtain 37% equivalent active sites, confirming the half-site reactivity of EcSufS. The difference in kburst values for persulfide (24.5 ± 2.2 s-1 ) and alanine formation (3.7 ± 0.2 s-1) suggest the buildup of an intermediate in the first turnover. Single-turnover reactions followed by UV-Vis stopped-flow spectroscopy identify an intermediate with maximal absorbance at 340 nm. Formation of the 340 nm intermediate aligns with the formation of persulfide product. Decay of the intermediate matches the formation of alanine, suggesting the 340 nm intermediate is likely a PLP-alanine species. The presence of equimolar amount of EcSufE slightly decrease the rate constants for formation and decay of the intermediate, ruling out an activating role for EcSufE in these steps. Taken together, this work demonstrates i) half-site reactivity of EcSufS and ii) EcSufE is most likely activating the transfer of persulfide from EcSufS to EcSufE after alanine formation, which in turn leads to activation of the adjacent active site of EcSufS. This more detailed kinetic mechanism of EcSufS/EcSufE catalysis helps provide understanding of other type II cysteine desulfurase enzymes, such as SufS from Bacillus subtilis and Mycobacterium tuberculosis.