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-steadystate 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 C␣ 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.

SufS is a type II cysteine desulfurase and acts as the initial step in the Suf Fe-S cluster assembly pathway. In Escherichia coli this pathway is utilized under conditions of oxidative stress and is resistant to reactive oxygen species. Mechanistically this means SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE. Here, we report five x-ray crystal structures of SufS including a new structure of SufS containing an inward facing persulfide intermediate on C364. Additional structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved β-hairpin structure plays a role in mediating interactions with SufE. These new structures, along with previous HDX-MS and biochemical data identify an interaction network capable of communication between active-sites of the SufS dimer coordinating the shift between desulfurase and transpersulfurase activities.

Cysteine serves as the sulfur source for the biosynthesis of Fe–S clusters and thio‐cofactors, molecules that are required for core metabolic processes in all organisms. Therefore, cysteine desulfurases, which mobilize sulfur for its incorporation into thio‐cofactors by cleaving the Cα—S bond of cysteine, are ubiquitous in nature. SufS, a type 2 cysteine desulfurase that is present in plants and microorganisms, mobilizes sulfur from cysteine to the transpersulfurase SufE to initiate Fe–S biosynthesis. Here, a 1.5 Å resolution X‐ray crystal structure of the Escherichia coli SufS homodimer is reported which adopts a state in which the two monomers are rotated relative to their resting state, displacing a β‐hairpin from its typical position blocking transpersulfurase access to the SufS active site. A global structure and sequence analysis of SufS family members indicates that the active‐site β‐hairpin is likely to require adjacent structural elements to function as a β‐latch regulating access to the SufS active site.

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