STRUCTURAL STUDIES ON NITRIC OXIDE SYNTHASE

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The long range goal of this project is to study structure-function relationships in nitric oxide synthase (NOS). NOS is the enzyme responsible for the oxidation of arginine to nitric oxide (NO). In recent years nitric oxide has been recognized as a major physiological messenger molecule involved in the nervous, immune, and cardiovascular systems. Owing to the potency and importance of NO as a regulatory molecule, NOS is a complex enzyme under stringent control. The enzyme consists of a heme domain where the actual oxidation of arginine occurs and an FMN/FAD domain that serves to shuttle electrons from NADPH to the heme domain. Between the heme and flavin domains is a linker that binds another regulator molecule, calmodulin. In addition to heme, FMN, and FAD, NOS contains yet another cofactor of unknown function, tetrahydrobiopterin. Sequence alignments clearly show that the flavin domain is very similar to cytochrome P450 reductase but that the heme domain bears little resemblance to other heme proteins despite the similarity to P450 in both function and spectral properties. Our goal is to determine the crystal structure the three primary forms of NOS: macrophage inducible or iNOS, neuronal or nNOS and endothelial or eNOS. Since the most unique feature is the heme domain and since the heme domain is the primary target for rational drug design, we initially have focused on crystallizing the heme domain of all three. Crystals of the eNOS heme domain diffract to 2.5A and smaller crystals of the nNOS and iNOS heme domains have been obtained. Our initial goal will be to solve the crystal structure of the eNOS heme domain.