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

Soluble guanylate cyclase (sGC) is a nitric oxide (NO)-sensing hemoprotein that has been identified in animals from Drosophila 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 Vibrio cholerae and Nostoc punctiforme 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 Thermoanaerobacter tengcongensis 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 T. tengcongensis 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, C. elegans. The annotated C. elegans genome contains 7 predicted sGC homologues (gcy-31 through gcy-37) but no ORF for nitric oxide synthase. Our work shows that the heme domain of GCY-35 binds O2 and that gcy-35 mediates attraction to low-O2, suggesting that gcy-35 is a novel O 2-sensing guanylate cyclase. We also show that low O2 levels rapidly and reversibly disrupt social behavior and that gcy-35 mediates some aspects of this behavior, suggesting that O2 is the molecular cue for social behavior in C. elegans.PhDBiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124831/2/3163837.pd