Nitric Oxide Synthase


Nitric oxide synthase (NOS) proteins are heme-based monooxygenase enzymes that convert L-arginine to L-citrulline and nitric oxide (NO). NO is a diffusible, reactive molecule that functions in control of vascular tone and blood pressure, protection tone and blood pressure, protection against pathogens and cancer, hormone regulation, nerve cell transmission, and angiogenesis. NO production in cells is a target for drug design in many different capacities as overproduction of NO has been linked to neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases, and insufficient NO production has been linked to conditions such as hypertension and cardiovascular disease. Mammalian NOS enzymes are homodimers that contain an N-terminal oxidase domain (NOSox) and C-terminal reductase domain called NOSred, and crosstalk between the two domains is regulated by a calmodulin (CaM)-binding interface. NOSox binds the L-arginine substrate, heme, and the redox-active cofactor 6R-tetrahydrobiopterin (H4B), all of which are required for an active enzyme. NOSred has binding sites for flavin cofactors as well as NADPH, and acts as a source of reducing equivalents for oxygen binding and activation at the heme in NOSox. Controlling the communication between redox-active cofactors in the NOSox and NOSred domains regulates at least two mammalian NOS isozymes. Our project goal is to provide a better understanding about the relationship between NOS structural arrangement, electron transfer and the mechanism of NO production by NOS enzymes. Bacterial NOS enzymes share many similarities to their mammalian counterparts, and because of their stripped-down domain structure and ease of purification, bacterial NOS proteins serve as useful models for investigating the mechanism of NO synthesis. We use a NOS enzyme from the thermostable Geobacillus stearothermophilus (gsNOS) as a model system for studying oxygen-activation in NO synthesis because of its unusually stable heme-oxygen complexes. Additionally, though previously identified bacterial NOS enzymes have notcontained a covalently attached reductase domain, several new cyanobacterial enzymes have been identified that possess a new domain structure that contains a fused NOSox-NOSred module. We have isolated and purified a NOS enzyme from from Synechococcus pcc7335 (spNOS) to be used as a model for NOSox-NOSred electron transfer communication. Currently there are no crystallographic structures of NOSox in complex with a NOSred, and this protein provides a new avenue for structural characterization. We are working to reveal the structural essential aspects of redox communication between spNOSox and spNOSred active sites that have been inaccessible from the mammalian systems and prior bacterial models.