Identification and characterization of a conserved soluble guanylate cyclase-like heme domain that discriminates between nitric oxide and oxygen.

Abstract

Soluble guanylate cyclase (sGC) is a nitric oxide (NO)-sensing hemoprotein that has been identified in animals from <italic>Drosophila</italic> to humans. sGC mediates vasodilation, platelet aggregation and neurotransmission and, unlike many hemoproteins, has the unique characteristic of not binding O₂---a property which allows this enzyme to sense NO in an aerobic environment. Using the sGC heme domain as the query sequence in a BLAST search, we have identified multiple, predicted prokaryotic proteins with significant homology to this region. We have cloned and characterized the sGC-like heme domains from <italic> Vibrio cholerae</italic> and <italic>Nostoc punctiforme</italic> and find that both of them are similar to sGC and are likely prokaryotic NO-sensors. Both ORFs are found in histidine kinase-containing operons, suggesting that they regulate histidine kinases. We have also characterized the heme domain from a predicted methyl-accepting chemotaxis protein from the strict anaerobe <italic> Thermoanaerobacter tengcongensis</italic> and find that it is able to bind O₂. This is the first protein from the sGC-like heme domain-containing family that binds O₂, and we predict that this protein functions to sense and avoid O₂. Accordingly, we have named this conserved sGC-like heme domain the H-NOX (H&barbelow;eme-N&barbelow;itric oxide/ OXygen binding) domain. In order to evaluate the molecular basis for ligand discrimination in the H-NOX domain, we have obtained a crystal structure (2.1 A resolution) of the <italic>T. tengcongensis</italic> H-NOX domain, and find that a tyrosine residue, contained in the distal heme pocket, forms a hydrogen bond to the O₂ ligand. Suspecting that an O₂-binding H-NOX domain might be found in other organisms, we turned our attention to the nematode, <italic> C. elegans</italic>. The annotated <italic>C. elegans</italic> genome contains 7 predicted sGC homologues (<italic>gcy-31</italic> through <italic>gcy-37</italic>) but no ORF for nitric oxide synthase. Our work shows that the heme domain of GCY-35 binds O₂ and that <italic>gcy-35</italic> mediates attraction to low-O₂, suggesting that <italic>gcy-35</italic> is a novel O₂-sensing guanylate cyclase. We also show that low O₂ levels rapidly and reversibly disrupt social behavior and that <italic>gcy-35</italic> mediates some aspects of this behavior, suggesting that O₂ is the molecular cue for social behavior in <italic>C. elegans</italic>.