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

## TL;DR

This paper explores how a bacterial protein called H-NOX senses nitric oxide and activates signaling pathways, revealing a new mechanism involving oxidation of a zinc ligand.

## Contribution

The study identifies a novel oxidative activation mechanism in H-NOX proteins involving zinc ligand oxidation, distinct from previously known systems.

## Key findings

- Spectroscopic analysis of Cc H-NOX reveals formation of a 5cLS Fe(II)-NO heme.
- Oxidation of the zinc ligand Cys residues activates Cc H-NOX without inhibiting HnoK autophosphorylation.
- X-ray absorption data show a change in zinc coordination upon oxidation but no zinc loss.

## Abstract

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, 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.