Many essential metalloproteins require iron–sulfur (Fe–S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe–S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe–S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein–protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.