"Structural/Functional Modularity in Nitric Oxide Synthase"; Principal Investigator: Bettie Sue Masters, Ph.D.; Agency: National Institute of General Medical Sciences; Type: R01 (GM52419, Years 9-12) Period: 4/1/2004-3/31/2008.

     The nitric oxide synthases are a family of enzymes that synthesize nitric oxide from L-arginine through a series of monooxygenation reactions.  They are bidomain enzymes, consisting of a heme-containing oxygenase domain and a flavin-containing reductase domain connected by a calmodulin binding site. These enzymes are involved in neurotransmission, immune responses (cytotoxicity) and vasodilation.  The hypothesis of this grant is that the NOS isoforms, sharing common prosthetic groups, cofactors and mechanisms, are structurally unique in order to perform specific functions in the cell types in which they occur.

Original aims of this grant activity featured a modular approach to the structural aspects of neuronal nitric oxide synthase (nNOS) but were extended to include inducible and endothelial NOS isoforms and have permitted the expression of isolated modules and domains of all three isoforms in E. coli. NOS isoforms have been purified for study using a variety of biophysical approaches, including x-ray crystallography, electroparamagnetic resonance, UV/vis spectroscopy, and circular dichroism.  We were among the first to identify the existence of heme in these enzymes and were the first to report the presence of a structural zinc atom (both shown in the figure above).  We have also identified and characterized several intrinsic regulatory elements within the reductase domain that modulate the flow of electrons, derived from NADPH, through the enzyme.