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.
Highlights
The toxic gas nitric oxide (NO) is known to play multiple roles in human physiology [1]
Purification of holo heme-nitric oxide/oxygen binding (H-NOX) required the addition of hemin to the culture medium, whereas apoprotein was isolated from cultures grown in LB medium without supplementation (Fig. 1B)
We have assayed the ability of H-NOX from V. cholerae to inhibit autophosphorylation of the signaling kinase HnoK
Summary
The toxic gas nitric oxide (NO) is known to play multiple roles in human physiology [1]. The apoprotein showed robust HnoK inhibition that was dependent on the oxidation of cysteine residues to form disulfide bonds at a highly conserved zinc site. The results indicate that Cys-177 is important for H-NOX activity in both the holo Fe(III) and apo states.
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