Tags

Type your tag names separated by a space and hit enter

Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae.
J Biol Chem. 2016 08 19; 291(34):17547-56.JB

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+Show Affiliations

From the Department of Chemistry and Biochemistry and.Chemical Analysis and Instrumentation Laboratory, New Mexico State University, Las Cruces, New Mexico 88003.Chemical Analysis and Instrumentation Laboratory, New Mexico State University, Las Cruces, New Mexico 88003.From the Department of Chemistry and Biochemistry and etyukl@nmsu.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27358409

Citation

Mukhopadyay, Roma, et al. "Heme-independent Redox Sensing By the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) From Vibrio Cholerae." The Journal of Biological Chemistry, vol. 291, no. 34, 2016, pp. 17547-56.
Mukhopadyay R, Sudasinghe N, Schaub T, et al. Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae. J Biol Chem. 2016;291(34):17547-56.
Mukhopadyay, R., Sudasinghe, N., Schaub, T., & Yukl, E. T. (2016). Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae. The Journal of Biological Chemistry, 291(34), 17547-56. https://doi.org/10.1074/jbc.M116.733337
Mukhopadyay R, et al. Heme-independent Redox Sensing By the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) From Vibrio Cholerae. J Biol Chem. 2016 08 19;291(34):17547-56. PubMed PMID: 27358409.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae. AU - Mukhopadyay,Roma, AU - Sudasinghe,Nilusha, AU - Schaub,Tanner, AU - Yukl,Erik T, Y1 - 2016/06/29/ PY - 2016/04/18/received PY - 2016/7/1/entrez PY - 2016/7/1/pubmed PY - 2017/5/13/medline KW - bacterial signal transduction KW - heme KW - histidine kinase KW - nitric oxide KW - redox signaling SP - 17547 EP - 56 JF - The Journal of biological chemistry JO - J Biol Chem VL - 291 IS - 34 N2 - 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. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/27358409/Heme_Independent_Redox_Sensing_by_the_Heme_Nitric_Oxide/Oxygen_Binding_Protein__H_NOX__from_Vibrio_cholerae_ L2 - https://linkinghub.elsevier.com/retrieve/pii/M116.733337 DB - PRIME DP - Unbound Medicine ER -