Activation of the H‐NOX redox sensor in Vibrio cholerae by a zinc ligand switch mechanism

Abstract

Heme Nitric Oxide/Oxygen (H-NOX) binding proteins are bacterial heme binding proteins that interact with nitric oxide (NO) or oxygen (O2). In some gram-negative bacteria, such as Shewanella oneidensis, H-NOX influences the activity of downstream protein histidine kinases in an NO-dependent manner, ultimately impacting biofilm formation and dispersal. Vibrio cholerae has a homologous H-NOX to Shewanella oneidensis, and the histidine kinase (HnoK) and downstream proteins mediating the signaling cascade responsible for biofilm formation are conserved in this organism. We have expressed and purified the H-NOX and HnoK from Vibrio cholera and demonstrated that HnoK kinase activity is inhibited by both the holo and apo protein. Interestingly, the inhibitory function of H-NOX on HnoK is lost when the protein is reduced with DTT. Further investigation showed that cysteine residues in H-NOX are reversibly oxidized to form disulfide bonds, mediating HnoK binding and inhibitory activity and implicating this protein as a redox sensor. Currently our focus is on the structural details of reduced and oxidized H-NOX. There is a ligand switch mechanism at a conserved zinc site whereby cysteine ligands to the zinc are replaced by other protein side chains in the oxidized state. Detailed structural information on the interaction of HNOX with HnoK may be important for V. cholerae pathogenicity and environmental persistence. Support or Funding InformationNational Science Foundation (IIA-1301346) Apo Vc H-NOX signaling mediated by zinc ligand switch