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 thatVibrio 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.

Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio cholerae

“…A possible explanation for why V. cholerae encodes both H-NOX and NosP could be drawn from consideration of a recent proposal suggesting that V. cholerae H-NOX may be involved in heme-independent redox sensing. 12 This finding suggests that VcH-NOX may act as a dual NO/redox sensor, depending on the environment. Therefore, under certain circumstances NosP may act as a primary NO sensor.…”

“…14 In addition, the inhibition of the H-NOX-associated histidine kinase was shown to be independent of NO binding to H-NOX, as the ferric unligated form of H-NOX inhibited the kinase comparably. 12 …”

Bacterial Haemoprotein Sensors of NO

“…Many bacterial H-NOX domains have now been cloned, expressed, purified, and spectroscopically characterized (Arora & Boon, 2012; Boon et al, 2006; Dai, Farquhar, Arora, & Boon, 2012; Henares et al, 2012; Karow et al, 2004; Liu et al, 2012; Ma, Sayed, Beuve, & van den Akker, 2007; Mukhopadyay, Sudasinghe, Schaub, & Yukl, 2016; Price, Chao, & Marletta, 2007; Tsai, Berka, Martin, & Olson, 2012; Tsai et al, 2010; Wang et al, 2010; Wu, Liu, Berka, & Tsai, 2015). UV/vis spectroscopy, paired with resonance Raman (rR) spectroscopy, has demonstrated that ferrous H-NOX domains from facultative aerobes form high-spin 5-coordinate Fe(II)-unligated complexes (Boon et al, 2006; Karow et al, 2004).…”

“…To date, these simple H-NOX/HaHK signalling systems have been characterized in S. oneidensis (Plate & Marletta, 2012), V. cholerae (Mukhopadyay et al, 2016; Plate & Marletta, 2012), and P. atlantica (Arora & Boon, 2012) (Fig. 6).…”

Nitric Oxide Regulation of H-NOX Signaling Pathways in Bacteria

“…58 The effect of heme-free (apo Vc H-NOX) and heme-bound Vc H-NOX on Vc HaHK autophosphorylation activity was assessed using purified proteins. It was found that Vc HaHK activity is inhibited by heme-bound Fe(III), Fe(II)-NO and apo Vc H-NOX, with the heme-bound forms being the most effective inhibitors in vitro 58 Moreover, the authors investigated the mechanism of heme-independent (apo Vc H-NOX) inhibition of Vc HaHK autophosphorylation activity and observed that only the cysteine oxidized form of apo Vc H-NOX could inhibit Vc HaHK activity, and that this inhibition was due to a stable interaction with Vc HaHK. 58 Since Vc H-NOX may have dual functionality, it would be interesting to determine if both functions can regulate biofilm formation and pathogenicity in Vibrio cholerae .…”