Oxidative Activation of the Heme Nitric Oxide/Oxygen-Binding Protein (H-NOX) from Caulobacter crescentus.

The heme nitric oxide/oxygen-binding proteins (H-NOX) are bacterial homologues of the sensor domain of mammalian soluble guanylate cyclase (sGC), a multidomain enzyme that catalyzes the production of cyclic guanosine monophosphate (cGMP) in response to NO. In facultative anaerobes, H-NOX proteins sense nitric oxide (NO) and regulate various communal behaviors including biofilm formation, motility, virulence, and quorum sensing. Rupture of the proximal heme iron-histidine bond during the formation of a five-coordinate low-spin ferrous nitrosyl (5cLS Fe(II)-NO) heme is thought to be required for H-NOX activation, allowing them to interact with downstream signaling partners such as diguanylate cyclases (DGC), phosphodiesterases (PDE), or histidine kinases (HK). Some H-NOX homologues also contain a conserved Cys-ligated zinc-binding site, which can respond to oxidative stress, at least in vitro. Although classified as an obligate aerobe, Caulobacter crescentus encodes an apparent NO-sensing hnox gene adjacent to that of the HK gene hnok. Spectroscopic analysis of the Cc H-NOX protein reveals characteristics similar to those of other NO-sensing H-NOX homologues, including the formation of a 5cLS Fe(II)-NO heme. Surprisingly, this form is completely noninhibitory to HnoK autophosphorylation, in contrast to what has been observed for every other related system to date. Rather, oxidation of the zinc ligand Cys residues activates Cc H-NOX. X-ray absorption fine structure (EXAFS) data reveal a change in zinc coordination upon oxidation but no loss of zinc. This work illustrates the breadth of H-NOX-signaling mechanisms and expands our understanding of signaling pathways in which this widespread protein participates.