Abstract
Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5'-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for Cα deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.
Highlights
Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins
SufS belongs to a family of cysteine desulfurases responsible for liberating sulfur from free cysteine resulting in an enzymebound S-sulfanylcysteine species
The final pyridoxal 5-phosphate (PLP) absorbance after purification of SufS H123A shows a slight decrease in extinction coefficient along with a subtle blue shift to around 418 nm, compared with 422 nm observed in WT SufS
Summary
1.10 Ϯ 0.06 0.09 Ϯ 0.01 0.07 Ϯ 0.02 a 1 M SufS with 4 M SufE. 16.1 Ϯ 1.2 0 0 two active-site mutants assisted in trapping Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction. 1.10 Ϯ 0.06 0.09 Ϯ 0.01 0.07 Ϯ 0.02 a 1 M SufS with 4 M SufE. 16.1 Ϯ 1.2 0 0 two active-site mutants assisted in trapping Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction. The mutants facilitated the direct structural observation of these two reaction intermediates and associated conformational changes to the SufS active site. Emerging from the analysis is a rich picture of the functional roles of several amino acids conserved across the SufS family including the unanticipated, essential role of Cys-364 in positioning the Cysaldimine for C␣ deprotonation, and the essential role of His-123 in protonating the Ala-enamine intermediate
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