UC Berkeley

Nitric oxide (NO) signaling in mammals occurs through the activation of soluble guanylate cyclase (sGC), which results in the stimulation of cGMP mediated signaling pathways important for blood vessel homeostasis and neurotransmission. sGC is a heterodimeric hemoprotein comprised, typically, of alpha1 and beta1 subunits, with the heme cofactor responsible for selective NO binding located at the N-terminus of the beta1 subunit. The heme domain of sGC belongs to a larger class of proteins termed Heme-Nitric oxide/OXygen binding (H-NOX) domains, named as such to encompass their ability to serve as either NO or oxygen sensors. Functional characterization of H-NOX proteins from prokaryotes has provided important insight into the roles these proteins serve in biological contexts. However, fundamental questions remain about the mechanisms of NO activation and signal transduction within H-NOX signaling pathways.

Extensive studies on several members of the H-NOX family have established the functional importance of topological features within the protein scaffold that tune the electronic properties of the heme. However, there is little knowledge regarding the functional role of H-NOX protein structure in modulating gas diffusion as a means for tuning the ligand-binding properties. Using X-ray crystallography with xenon as a probe for gas diffusion pathways, a bifurcated tunnel network between the solvent and interior heme site was mapped in a prokaryotic H-NOX protein. Site-directed mutagenesis and kinetic measurements demonstrate that blocking the tunnels to hinder gas diffusion has important consequences on gas-binding affinity. These data suggest that this tunnel network in H-NOX proteins may serve functional roles in controlling the flux of ligands important for tuning gas-mediated signaling.

A key molecular event during NO-induced activation of H-NOX proteins is loss of the heme-histidine bond and formation of a five-coordinate nitrosyl complex. Although this has been known for quite some time, molecular details into this process have remained elusive. Using X-ray crystallography, structures of a prokaryotic H-NOX protein in the ferrous-unliganded as well as both six-coordinate and five-coordinate nitrosyl complexes were reported. These structures show that several features in the unliganded state maintain the heme in a distorted conformation that relaxes towards planarity following NO binding and loss of the heme-histidine bond. Because the heme and protein conformations are intimately coupled, relaxation of the heme towards planarity results in a pronounced conformational change in the H-NOX protein involving a rotational displacement of the distal subdomain about the proximal subdomain. It is hypothesized that this conformational change is the method by which H-NOX proteins communicate NO binding to downstream signaling partners.

The observation that H-NOX proteins can bind NO as five-coordinate complexes with NO located in either the distal or proximal heme pockets presented the possibility that these two nitrosyl complexes would yield different NO signaling lifetimes as a result of their intrinsically different dissociation rates. To test this hypothesis, two different chemical based NO traps were employed to measure the dissociation rate constants from distal and proximal-bound, five-coordinate NO complexes in a prokaryotic H-NOX protein. Unexpectedly, under all conditions tested, similar rate constants for NO dissociation are observed between these two nitrosyl species. As such, it is hypothesized that the rate-determining step for dissociation of NO from both five-coordinate NO species is loss of the NO-iron bond.

Prokaryotic H-NOX proteins are often found in predicted operons with putative signaling proteins, predominantly histidine kinases. Importantly, histidine kinases are commonly part of two-component signaling systems, which serve as a basic stimulus-response pathway critical for prokaryotes to sense and respond to extracellular stimuli. Furthermore, it has been established that the H-NOX: histidine kinase signaling couple from Shewanella oneidensis regulates the motility of the organism in response to NO. To obtain insight into the mechanism by which H-NOX proteins regulate histidine kinase autophosphorylation upon binding NO, various biophysical techniques were employed to determine the architecture of the H-NOX: histidine kinase complex from S. oneidensis. From these data, it is hypothesized that the NO-induced conformational change in the H-NOX protein allosterically inhibits histidine kinase activity by eliciting a series of conformational changes in the histidine kinase that places the phospho-accepting histidine in an inaccessible conformation.