Marletta Lab

Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing hemoprotein that has been described in eukaryotes from Drosophila to humans. Genomic analysis has recently placed sGC within a larger family of proteins with Heme Nitric oxide/Oxygen binding (H-NOX) domains including prokaryotic proteins with significant homology (15-40% identity) to the heme domain of sGC. Predicted H-NOX domains were found in facultative aerobes, obligate anaerobes, and thermophiles. Genomic analysis reveals that the H-NOX domains may be linked to histidine kinases or diguanylate cyclases (obligate anaerobes) or methyl-accepting chemotaxis proteins (obligate anaerobes). Uncovering the biological function of these H-NOX domains is currently an area of intense investigation in our lab.

(A) Structural features of Tt H-NOX. (B) Heme binding pocket.

H-NOX proteins also exhibit remarkable diatomic ligand selectivity despite a similar protein fold. For example, the H-NOX domain from Vibrio cholera (a facultative aerobe) binds NO in a high spin 5-coordinate complex and excludes oxygen, while the H-NOX domain from Thermoanaerobacter tengcongensis (Tt, obligate anaerobe) has been found to bind oxygen in a low-spin 6-coordinate complex, making it the first member of the family to bind O2. Current research is focused on understanding the nature of this ligand selectivity from a molecular level and how this selectivity translates into protein function as sensors in biology.

Our lab is currently focused on investigating three main areas pertaining to H-NOXs:
1. How do H-NOX domains control ligand affinity and selectivity?
2. How do H-NOX domains regulate their associated signaling proteins?
3. What role do H-NOX domains play in vivo?

A variety of biochemical, structural, and biological methods are being used to investigate these questions. We use spectroscopic techniques, such as UV-Vis spectroscopy, stopped flow spectroscopy, laser flash photolysis, and resonance Raman spectroscopy, to probe how mutations in the heme pocket affect ligand selectivity and affinity. Both x-ray crystal and NMR solution structural studies are used to determine the effects of heme conformation and ligand binding on the conformation of H-NOX domains. Phosphorylation assays are used to probe the effect of H-NOX redox and ligation state on kinase autophosphorylation activity.

Finally, we are interested in the pathways that are controlled in vivo by H-NOX domains. In facultative aerobes, we postulate that NO produced through denitrification may serve as the signal for low O2 tension. In obligate anaerobes, we hypothesize that the fusion of the H-NOX to a methyl-accepting chemotaxis protein (MCP) may result in an O2 sensing protein that produces a chemotaxic response away from O2 or as an NO sensor. We are investigating these hypotheses through biochemical analysis of potential downstream signaling proteins and through the generation of H-NOX knockouts in vivo.