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 regulatory molecule, calmodulin. In addition to heme, FMN, and FAD, NOS contains yet another cofactor, 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. Structure of the heme domain for all 3 mammalian isoforms have been determined. The goal now is to determine the structure of various complexes using known inhibitors as well as new inhibitors designed and synthesized as part of this project. In addition, efforts will be made to determine the holo-NOS structure as well as various constructs geared toward understanding the electron transfer properties of NOS. A variety of biochemical and biophysical studies also are planned to complement the crystallographic work.