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Updated: Mar 7, 2026

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
Nitric Oxide-Induced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in
Minxi Rao1, Mark A Herzik1, Anthony T Iavarone1
1Department of Chemistry, ‡Department of Molecular and Cell Biology, §QB3 Institute, University of California , Berkeley, California 94720, United States.
Abstract:
Nitric oxide (NO) is implicated in biofilm regulation in several bacterial families via heme-nitric oxide/oxygen binding (H-NOX) protein signaling. Shewanella oneidensis H-NOX (So H-NOX) is associated with a histidine kinase (So HnoK) encoded on the same operon, and together they form a multicomponent signaling network whereby the NO-bound state of So H-NOX inhibits So HnoK autophosphorylation activity, affecting the phosphorylation state of three response regulators. Although the conformational changes of So H-NOX upon NO binding have been structurally characterized, the mechanism of HnoK inhibition by NO-bound So H-NOX remains unclear. In the present study, the molecular details of So H-NOX and So HnoK interaction and regulation are characterized. The N-terminal domain in So HnoK was determined to be the site of H-NOX interaction, and the binding interface on So H-NOX was identified using a combination of hydrogen-deuterium exchange mass spectrometry and surface-scanning mutagenesis. Binding kinetics measurements and analytical gel filtration revealed that NO-bound So H-NOX has a tighter affinity for So HnoK compared that of H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with binding-deficient H-NOX mutants further indicate that while formation of the H-NOX-HnoK complex is required for HnoK to be catalytically active, H-NOX conformational changes upon NO-binding are necessary for HnoK inhibition.
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