Heme‐Free H‐NOX from Vibrio cholerae is Activated by Oxidation via a Zinc Ligand Switch Mechanism.

The heme nitric oxide/oxygen binding proteins (H‐NOX) are bacterial homologues of the heme‐binding domain of soluble guanylate cyclase (sGC). Like sGC, they can act as sensors for nitric oxide (NO). In some gram‐negative bacteria, such as Shewanella oneidensis , H‐NOX influences the activity of downstream protein histidine kinases (HK) in an NO‐dependent manner, ultimately impacting biofilm formation and dispersal. The mechanism is generally thought to involve NO binding to the H‐NOX heme cofactor, eliciting a conformational change promoting inhibition of HK autophosphorylation activity. However, our recent work has shown that heme is not required for Vibrio cholerae H‐NOX ( Vc H‐NOX) to inhibit its associated HK ( Vc HnoK) in vitro . Rather, the heme‐free Vc H‐NOX can act as a redox sensor. Like So H‐NOX, Vc H‐NOX binds a single zinc ion, which, based on homology to the So H‐NOX crystal structure, is bound to three conserved Cys residues (C149, C174 and C182) while a fourth Cys residue (C177) is nearby. Reversible oxidation of Cys ligands at the zinc binding site leads to the formation of disulfide bonds and a conformational change allowing heme‐free H‐NOX to bind HnoK and inhibit autophosphorylation. Since zinc is not lost from the protein upon oxidation, we hypothesized that a zinc ligand switch mechanism mediates these structural changes. Since Vc H‐NOX has proven recalcitrant to crystallization, we have used a battery of solution experiments on the WT and mutant proteins to determine the structures of the zinc site in reduced and oxidized states. X‐ray absorption fine structure (EXAFS) at the zinc k‐edge for reduced and oxidized samples show clear differences consistent with sulfur ligands being replaced by oxygen/nitrogen coordination upon oxidation. Perturbation of these spectra in the C177A mutant indicates a role for this residue in zinc binding. Our previous results also showed that the C177A mutant was impaired in HnoK inhibition upon oxidation. We also employed mass spectrometry to determine the disulfide bonding pattern in the oxidized state. These data allow us to model the zinc site in both reduced and oxidized states and demonstrate how oxidation leads to a conformational change that activates Vc H‐NOX as an inhibitor of HnoK autophosphorylation. Thus, we have gained significant insight into an unusual redox sensing mechanism that may also be relevant to the large number of organisms encoding H‐NOX proteins with conserved zinc ligands. Further, this work expands our understanding of mechanisms regulating biofilm formation, a process important for bacterial pathogenicity and persistence.Mechanism of oxidative activation of Vibrio cholerae H‐NOX by a ligand switch at the zinc binding site.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .