Structural Studies on Nitric Oxide Synthase

本项目的长期目标是研究一氧化氮合酶（NOS）的结构-功能关系。NOS是负责精氨酸氧化成一氧化氮（NO）的酶。近年来，一氧化氮已被认为是参与神经、免疫和心血管系统的主要生理信使分子。由于NO作为调节分子的效力和重要性，NOS是严格控制下的复杂酶。该酶由一个血红素结构域和一个FMN/FAD结构域组成，血红素结构域发生精氨酸的实际氧化，FMN/FAD结构域用于将电子从NADPH穿梭到血红素结构域。在血红素和黄素结构域之间是一个连接体，它与另一种调节分子钙调素结合。除了血红素、FMN和FAD之外，NOS还含有另一种辅因子四氢生物蝶呤。序列比对清楚地表明，黄素结构域是非常相似的细胞色素P450还原酶，但血红素结构域几乎没有相似之处，其他血红素蛋白质，尽管相似的P450在功能和光谱特性。已确定所有3种哺乳动物同种型的血红素结构域的结构。现在的目标是使用已知的抑制剂以及作为该项目的一部分设计和合成的新抑制剂来确定各种复合物的结构。此外，还将努力确定全息NOS结构以及各种结构，以了解NOS的电子转移特性。还计划进行各种生物化学和生物物理学研究，以补充晶体学工作。

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.