SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase

Abstract

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.

Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS

“…These included peptides covering active site residues, 225–236 and 356–366, and peptides covering the dimer interface, 88–100 and 243–255 (Figure 6A). Changes in the two active site regions had previously been identified by HDX-MS when SufE or an activated variant of SufE (D74R SufE) bound to SufS 14, 15 . HDX-MS results with the activated D74R SufE also indicated changes in peptide 88–100, suggesting that the dimer interface is sensitive to activated intermediates in the SufS/SufE reaction.…”

Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues

“…The addition of SufE greatly enhances SufS activity up to 200-fold over SufS alone. Previous studies have suggested that SufE also influences the SufS activesite allosterically and could impact specific steps in the cysteine desulfurase reaction as well (37,38).…”

Iron–sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding

“…Examination of the structure of the resting SufE shows a variety of interactions that hold the active site loop folded down into the interior of SufE and reveals that the Asp74 residue would play a key role for maintaining such a structure. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of the SufE D74R mutant revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS [77]. In addition, SufE D74R mutant is a better sulfur acceptor for SufS than wt SufE.…”

“…This induces a SufE conformational change by making the Cys51 active site loop more dynamic. In addition, it was shown that this mutation promotes higher interaction of SufE with SufS [77]. Therefore, this mutation enhances the ability of SufE to accept sulfur from SufS.…”