MICROSOMAL ELECTRON TRANSPORT IN LIVER & HEART

由于NADPH-细胞色素P-450还原酶存在于每个组织中， 细胞色素P-450介导的羟基化 内源性（类固醇、脂肪酸和甘草素）， 外源性（治疗药物、环境毒物和 致癌物）发生时，了解其模式很重要 行动上 本建议旨在了解结构- 肝微粒体黄素蛋白、NADPH- 细胞色素P-450还原酶，其含有FAD和FMN作为 辅基-哺乳动物黄素酶中的一个独特的。 在 与其生理电子受体细胞色素相互作用 P-450，这种黄素蛋白行使一种机制， 2个电子顺序地插入到衬底结合中， 细胞色素P-450还原O2复合物。 这个过程需要 独特的构象，具有不同的结构域， 每种人工黄素的结合以及 产生适当的氧化还原状态， 在催化过程中细胞色素P-450的特定氧化还原状态 周转 由于没有单一的技术可以解决 这个有趣的和重要的黄素蛋白的各个方面，我们计划 在分子水平上检查其结构和功能， 各种生物物理学方法。 我们将执行：1）31 P NMR 天然猪和大鼠还原酶和酶的研究 用FMN和FAD两者的硫代磷酸酯类似物取代， 对大鼠肝还原酶的定点突变产物进行了研究， 确定对FMN和NADPH结合结构域的影响， 通过谱线加宽和/或化学位移; 2）互补和 激光共振拉曼光谱的补充研究 还原酶的NMR样品的等分试样，在具有FMN的酶上 在异咯嗪环中被13个C和15个N取代， 突变的还原酶来探测黄素的环境（氢 键合效应）; 3）研究了两种完整的结晶 和用于X射线晶体学的蛋白水解切割的还原酶 研究;和4）还原酶结合的性质的确定 磷（结合磷脂？）及其功能作用。 这 技术的组合将允许全面的， 希望，结构功能特性的结论性研究 一种独特的哺乳动物黄素蛋白。

Because NADPH-cytochrome P-450 reductase exists in every tissue in which the cytochrome P-450-mediated hydroxylations of both endogenous (steroids, fatty acids, and prostaglandins) and exogenous (therapeutic drugs, environmental toxicants and carcinogens) occur, it is important to understand its mode of action. This proposal is aimed at understanding the structure- function relationships of the liver microsomal flavoprotein, NADPH- cytochrome P-450 reductase, which contains both FAD and FMN as prosthetic groups-a unique among mammalian flavoenzymes. In interacting with its physiological electron acceptor, cytochrome(s) P-450, this flavoprotein exercises a mechanism which allows the insertion of 2 electrons sequentially into the substrate-bound- cytochrome P-450 reduced 02 complex. This process requires a unique conformation with distinct structural domains for the binding of each of the prosthetic flavins and the capability of generating the appropriate oxidation-reduction states to interact with specific redox states of cytochrome P-450 during catalytic turnover. Due to the fact that no single technique can address the various aspects of this interesting and vital flavoprotein, we plan to examine its structure and function at the molecular level by a variety of biophysical methods. We will perform: 1) 31P NMR studies on the native pig and rat reductases and on enzymes substituted with phosphorothioate analogs of both FMN and FAD and on site-directed mutagenesis products of rat liver reductase to determine effects on FMN-, and NADPH-binding domains as detected by line broadening and/or chemical shifts; 2) complementary and supplementary studies with laser resonance Raman spectroscopy on aliquots of the NMR samples of reductase, on enzyme with FMN substituted with 13C and 15N in the isoalloxazine ring, and on the mutant reductases to probe the environment of the flavins (hydrogen bonding effects); 3) studies on the crystallization of both intact and proteolytically cleaved reductase for X-ray crystallography studies; and 4) determination of the nature of reductase-bound phosphorus (bound phospholipid?) and its functional role. This combination of techniques will permit a comprehensive and, hopefully, conclusive study of the structure-function properties of this unique mammalian flavoprotein.