Ru(II)-Containing Wires for ET Studies in Metalloenzymes

描述(申请人提供)：本项目的目标是使用敏化连接底物(SLS)来探索一氧化氮合酶(NOS)中电子转移介导的催化的机理和机制。第一部分涉及针对一氧化氮合酶的基于蝶呤的SLS的设计和合成；既包括被酶内源性结合的天然辅因子，也包括用作蝶呤结合口袋的荧光探针的芳香类似物。第二部分将利用诱导型一氧化氮合酶的催化氧化域(INOSoxy)测定蝶呤蛋白探针的结合亲和力，利用荧光技术探索催化过程中活性部位附近的结构和电子扰动，利用X射线结晶学来阐明探针掺入蛋白质的结构效应，最后将利用电子转移研究来研究一氧化氮合酶的催化机制，特别是蝶呤蛋白辅因子的作用。在第三部分中，我们将探索这些SLS的发展，用于高通量结合分析一氧化氮合酶抑制剂。最后，我们将确定这些一氧化氮合酶特异性探针在研究其他蛋白质复杂混合物中的一氧化氮合酶的有效性。

DESCRIPTION (provided by applicant): The goal of this project is to use sensitizer-linked-substrates (SLS's) to probe the mechanism and mechanics of electron transfer-mediated catalysis in nitric oxide synthase (NOS). Part I involves the design and synthesis of pterin-based SLS's for NOS; both the natural cofactorthat is endogenously bound by the enzyme will be incorporated, as well as aromatic analogues which will serve as fluorescent probes of the pterin binding pocket. In Part II, using the catalytic oxidase domain of iNOS (iNOSoxy), the binding affinities of the pterin probes will be determined, fluorescence techniques will be used to explore structural and electronic perturbations near the active site during catalysis, x-ray crystallography will be utilized to elucidate the structural effects of incorporation of the probe into the protein, and finally, electron transfer studies will be employed to study the mechanism of NOS catalysis, especially the role of the pterin cofactor. In Part III, we will explore the development of these SLS's for use in high-throughput binding assays for NOS inhibitors. Finally, we will determine the efficacy of these NOS specific probes for studies on NOS in complex mixtures of other proteins.