Nitric oxide (NO) has emerged as an important signal and cytotoxic agent in the human nervous system. In the brain, NO synthesis is involved in olfaction, spatial memory, and neurotoxicity associated with stroke and ischemia, while in the periphery, neurons synthesize NO to stimulate gastric motility, penile erection, and smooth muscle relaxation. A Ca2+/calmodulin-activated NO synthase (NOS) is expressed in neurons that is dimeric and catalyzes a multistep oxidation of L-arginine, generating NO and citrulline as products. The enzyme contains four distinct prosthetic groups (FAD, FMN, tetrahydrobiopterin & heme) that are thought to participate in NO synthesis. Our model for NOS has the flavins accepting electrons from NADPH and transferring them to the heme, which then binds oxygen and catalyzes NO synthesis. Our preliminary results suggest that the flavin-to-heme electron transfer is critical for neuronal NOS function, and is somehow controlled by calmodulin, L-arginine, and enzyme dimeric structure. We propose to uncover how calmodulin. L-arginine and dimeric structure control NOS activity at the molecular level. using a variety of complementary approaches. We will locate the heme binding site within the NOS oxygenase domain by site-directed mutagenesis and alkylation-protection experiments. For both dimeric NOS and its subunits, we will probe how calmodulin and L-arginine influence overall protein conformation, heme and flavin binding domain architecture, and the relative positioning of the flavin and heme groups by a variety of techniques, including tyrosine and flavin fluorescence, visible, electron paramagnetic resonance, and Resonance Raman spectroscopies. The ability of L-arginine, calmodulin, and dimeric structure to alter the electronic characteristics of the enzyme's flavin and heme groups will be studied by reductive titration and midpoint potentiometry in conjunction with visible and electron paramagnetic spectroscopies. We will also investigate how calmodulin and L-arginine influence the kinetics of the first portion of the NO synthase reaction, using stopped-flow spectroscopy. This includes calmodulin binding to NOS, conformational changes, electron loading into the flavins, and flavin-mediated heme reduction. Together, our research will reveal exactly how the neuronal NO synthase is activated at the molecular level, and how its activity can be controlled.
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