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

项目描述（由申请人提供）：本项目的目标是利用敏化剂连接底物（SLS）探索一氧化氮合酶（NOS）中电子转移介导的催化机制和力学。第一部分是基于pterin的NOS SLS的设计与合成；结合酶内源性结合的天然辅因子，以及芳香类似物作为蝶呤结合袋的荧光探针。在第二部分中，将利用iNOS的催化氧化酶结构域（iNOSoxy）来确定蝶呤探针的结合亲和，利用荧光技术探索催化过程中活性位点附近的结构和电子扰动，利用x射线晶体学来阐明探针与蛋白质结合的结构效应，最后利用电子转移研究来研究NOS催化的机理。尤其是蝶呤辅因子的作用。在第三部分中，我们将探索这些SLS用于NOS抑制剂的高通量结合分析的发展。最后，我们将确定这些NOS特异性探针在其他蛋白质复杂混合物中NOS的功效。

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.