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.