A labile sulfur ligand in a three-cysteine-coordinated [2Fe−2S] cluster mediates sulfide sensing in NreB

Iron-sulfur clusters are versatile protein cofactors involved in diverse biological processes, but their role in hydrogen sulfide/hydrosulfide (H2S/HS-) sensing remains largely unexplored. Here, we report that the Bacillus licheniformis sensor kinase NreB contains an unusual [2Fe-2S] cluster within its PAS domain. A 1.52-Å crystal structure reveals a distinct coordination geometry where three conserved cysteine residues and a non-cysteinyl sulfur ligand stabilize the cluster. Biochemical and native mass spectrometry support assignment of the predominant ligand state as -SH and show enrichment of -SSH/SSOH-like state upon sulfide exposure in the presence of O2, correlating with increased NreB kinase activity. Electron paramagnetic resonance spectroscopy shows that the cluster retains its oxidized [2Fe-2S]2+ state during sulfide-sensing. Molecular dynamics simulations further reveal transient solvent and HS- accessibility to the buried cluster, providing a physical basis for ligand entry. Here, we show that bacteria sense sulfide via a three-cysteine-coordinated Fe-S cluster with a labile sulfur ligand. Sulfide is an important bacterial signal, but how bacteria detect it remains unclear. Here the authors show that NreB uses an unusual iron-sulfur cluster with a labile sulfur ligand to sense sulfide and regulate kinase activity.