Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-depen-dent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is pro-tected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "11-latch" structural element that includes the alpha 6 helix, a glycine-rich loop, a 11-hairpin, and a cis-proline residue. To identify a functional role for the 11-latch, we used site -directed mutagenesis to obtain the N99D and N99A SufS var-iants. N99 is a conserved residue that connects the alpha 6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a dis-torted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high -affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demon-strate that the 11-latch does not affect the chemistry of persul-fide formation but does protect it from undesired reductants. The data also indicate the 11-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.