MICROSOMAL ELECTRON TRANSPORT IN LIVER & HEART

因为NADPH-细胞色素P-450还原酶存在于 其中细胞色素P-450介导的羟化反应 内源性(类固醇、脂肪酸和前列腺素)和 外源(治疗药物、环境毒物和 致癌物)发生时，了解其致癌机制是很重要的 行动。这项建议旨在了解结构- 肝微粒体黄素蛋白NADPH-1的功能关系 同时含有FAD和FMN AS的细胞色素P-450还原酶 假体基团--哺乳动物黄素酶中独一无二的一种。在……里面 与其生理电子受体细胞色素的相互作用(S) P-450，这种黄素蛋白行使一种机制，使 将2个电子顺序插入到衬底束缚中- 细胞色素P-450还原02复合体。此过程需要一个 具有不同结构域的独特构象 每种假体黄素的结合和能力 产生适当的氧化-还原状态以相互作用 与催化过程中细胞色素P-450的特定氧化还原状态有关 营业额。由于没有一种单一的技术可以解决 这种有趣而又重要的黄素蛋白的各个方面，我们计划 在分子水平上研究其结构和功能 各种生物物理方法。我们将进行：1)31P核磁共振 天然猪、鼠还原酶及其酶的研究 用FMN和FAD的硫代磷酸类似物和 大鼠肝还原酶基因定点突变产物的研究 确定检测到的对FMN-和NADPH结合结构域的影响 通过线条加宽和/或化学位移；2)互补和 激光共振拉曼光谱的补充研究 还原酶的核磁共振样品与FMN的等价物 在异四氧氮环上被13C和15N取代，并在 突变的还原酶来探测黄素(氢)的环境 成键效应)；3)完整结晶的研究 和用于X射线结晶学的蛋白水解酶 研究；以及4)还原酶结合性质的测定 磷(结合磷脂？)以及它的职能作用。这 技术的结合将允许全面和， 希望，对结构-功能性质的总结研究 这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.