GASEOUS SIGNALING

The Smith Lab has two project areas investigating signaling cascades involving gaseous molecules: nitric oxide synthase signaling and hydrogen sulfide signaling.

NITRIC OXIDE SYNTHASE

Nitric Oxide Synthases (NOS) are multi-domain enzymes that produce nitric oxide, a signaling molecule important for vasodilation, neurotransmission, and the innate immune response. During catalysis NOS facilitates an intricate sequence of electron transfers to shuttle electrons from the reductase to the oxygenase domain. Because electron transfer is efficient only over distances of a few Ångströms, NOS function requires sequential formation and release of conformations that place electron donors (NADPH, FAD, FMN) adjacent to electron acceptors (FAD, FMN, heme).

The goal of this project is to map NOS subdomain interactions and dynamics in the presence of the calcium signaling molecule calmodulin (CaM), which activates electron transfer from FAD to FMN and from FMN to heme through incompletely understood mechanisms.

Our approach employs an innovative combination of fluorescence, mass spectrometric, enzymological, and single molecule techniques to identify conformational states in NOS and track their interchange dynamics.

This project is in collaboration with Dr. Carey Johnson (University of Kansas) and Dr. David Arnett (Northwestern College) and is supported by a multi-year National Science Foundation (NSF) grant.

REFERENCES

Hanson QM, Carley JR, Gilbreath TJ, Smith BC, Underbakke ES.Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation J Mol Biol. 2018 (7):935-947. doi: 10.1016/j.jmb.2018.02.003.

Campbell MG, Smith BC, Potter CS, Carragher B, Marletta MA. “The molecular architecture of mammalian nitric oxide synthases”. Proceedings of the National Academy of Sciences. 2014, 111, E3614-23.

Smith BC, Underbakke ES, Kulp DW, Schief WR, Marletta MA. “Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation”. Proceedings of the National Academy of Sciences. 2013, 110, E3577-86.

HYDROGEN SULFIDE (H2S)-PRODUCING ENZYMES

Smith Lab collaborator Dr. Rodney Willoughby (MCW and CHW), and colleagues proposed a novel virulence mechanism by pathogenic bacteria to explain severe complications following infections that are unresponsive to antimicrobials (Verma et al, 2013). Specifically, hydrogen sulfide (H2S) was produced by infecting bacteria, and the effects were attenuated by treatment usually deployed for reversing H2S toxicity in farm workers: nitrite and hyperbaric oxygen.

The mechanism of H2S toxicity is similar to hydrogen cyanide: H2S inhibits cytochrome c oxidase activity in mitochondria, leading to death or tissue damage characteristic of hypoxia. Being freely diffusible, H2S can intoxicate across tissue boundaries with a diffusion radius – similar to mechanical trauma, thermal burns or radiation injury – that hollows out an abscess cavity.

In this project area, we seek to identify and characterize the bacterial enzymes responsible for producing H2S at the observed levels, with a long-term goal of developing potential clinical therapeutics to target those enzymes.

REFERENCE

Verma S, Landisch R, Quirk B, Schmainda K, Prah M, Whelan H, Willoughby RE. "Presumed Hydrogen Sulfide-Mediated Neurotoxicity FollowingStreptococcus Anginosus Group Meningitis." Pediatr Infect Dis J. 2013 February ; 32(2): 189–191. doi:10.1097/INF.0b013e3182748fe9.

 

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