title: Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae.
abstract: Heme nitric oxide/oxygen (H-NOX)-binding proteins act as nitric oxide (NO) sensors among various bacterial species. In several cases, they act to mediate communal behavior such as biofilm formation, quorum sensing, and motility by influencing the activity of downstream signaling proteins such as histidine kinases (HisKa) in a NO-dependent manner. An H-NOX/HisKa regulatory circuit was recently identified in Vibrio cholerae, and the H-NOX protein has been spectroscopically characterized. However, the influence of the H-NOX protein on HisKa autophosphorylation has not been evaluated. This process may be important for persistence and pathogenicity in this organism. Here, we have expressed and purified the V. cholerae HisKa (HnoK) and H-NOX in its heme-bound (holo) and heme-free (apo) forms. Autophosphorylation assays of HnoK in the presence of H-NOX show that the holoprotein in the Fe(II)-NO and Fe(III) forms is a potent inhibitor of HnoK. Activity of the Fe(III) form and aerobic instability of the Fe(II) form suggested that Vibrio cholerae H-NOX may act as a sensor of the redox state as well as NO. Remarkably, the apoprotein also showed robust HnoK inhibition that was dependent on the oxidation of cysteine residues to form disulfide bonds at a highly conserved zinc site. The importance of cysteine in this process was confirmed by mutagenesis, which also showed that holo Fe(III), but not Fe(II)-NO, H-NOX relied heavily upon cysteine for activation. These results highlight a heme-independent mechanism for activation of V. cholerae H-NOX that implicates this protein as a dual redox/NO sensor.
authors.affiliation: From the Department of Chemistry and Biochemistry and.
authors.forename: Roma
authors.initials: R
authors.lastname: Mukhopadyay
authors.affiliation: Chemical Analysis and Instrumentation Laboratory, New Mexico State University, Las Cruces, New Mexico 88003.
authors.forename: Nilusha
authors.initials: N
authors.lastname: Sudasinghe
authors.affiliation: Chemical Analysis and Instrumentation Laboratory, New Mexico State University, Las Cruces, New Mexico 88003.
authors.forename: Tanner
authors.initials: T
authors.lastname: Schaub
authors.affiliation: From the Department of Chemistry and Biochemistry and etyukl@nmsu.edu.
authors.forename: Erik T
authors.initials: ET
authors.lastname: Yukl
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D055550:Protein Stability; D014734:Vibrio cholerae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006418:Heme
keywords: bacterial signal transduction; heme; histidine kinase; nitric oxide; redox signaling