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是负责将精氨酸氧化为一氧化氮（NO）的酶。近年来，一氧化氮被认为是参与神经、免疫和心血管系统的主要生理信使分子。由于NO作为调控分子的效力和重要性，NOS是一种受到严格控制的复杂酶。该酶由一个血红素区域和一个FMN/FAD区域组成，前者用于精氨酸的实际氧化，后者用于将电子从NADPH传送到血红素区域。在血红素和黄素结构域之间是一个连接体，它与另一个调节分子钙调蛋白结合。除了血红素、FMN和FAD外，NOS还含有另一种辅助因子——四氢生物蝶呤。序列比对清楚地表明，黄素结构域与细胞色素P450还原酶非常相似，而血红素结构域与其他血红素蛋白几乎没有相似之处，尽管在功能和光谱特性上与P450相似。所有3种哺乳动物亚型的血红素结构域的结构已经确定。现在的目标是使用已知抑制剂以及作为该项目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，研究人员还将努力确定NOS的整体结构以及各种结构，以了解NOS的电子转移特性。此外，还计划进行各种生物化学和生物物理研究，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.