Crystallographic Studies of Flavoreductases in Electron Transport Systems of Liver Microsomes

我们对位于两条电子传递途径的两种黄素还原酶（NADH-细胞色素b_5还原酶和HADH-细胞色素P450还原酶）进行了X射线晶体结构分析，其中NADH和HADH分别作为初始电子供体。我们寻找了两个好的重原子衍生物，以获得电子密度图的基础上的多同构置换程序。然而，尽管做了许多尝试，我们只能找到一个导数。因此，我们计算的电子密度图在5A分辨率的单一同质同晶置换。该电子密度图质量良好，使我们能够区分蛋白质和溶剂区域。此外，我们收集了更高分辨率的强度数据，并使用同步辐射测量精确的强度，包括异常色散效应。并根据这些数据绘制了电子密度图。我们试图解释这些图谱，然后我们估计了分子形状的轮廓，并粗略地追踪了这种酶的折叠。我们还估计了FAD的结合位置，这是本项目中最重要的关键点之一。然而，为了进行更精确的讨论，我们需要更高质量的电子密度图。因此，提高密度图的质量是今后研究的重点。我们尝试了HADPH-细胞色素P450还原酶的结晶方法。最初，我们寻找对这种酶有效的沉淀剂，并成功地找到了一些试剂。我们使用这些沉淀剂尝试了许多结晶条件。但是，我们还没有得到任何适合于X射线衍射工作的好晶体。

We have carried out X-ray crystal structure analyses of two flavoreductases (NADH-cytochrome b_5 reductase and HADPH-cytochrome P450 reductase) located in two electron transport pathways in liver microsomes where NADH and HADPH function as initial electron donors, respectively.NADH-cytochrome b_5 reductase has so far been crystallized by ourselves. We searched two good heavy-atom derivatives in order to obtain the electron density map based on the multiple isomorphous replacement procedure. In spite of many attempts, however, we could find only one derivative. Therefore, we calculated the electron density map at 5A resolution by the single isomorphous replacement. This electron density map was in good quality which enabled us to distinguish between the protein and solvent regions. Furthermore, we collected intensity data at the higher resolutions and used synchrotron radiation to measure precise intensities including anomalous dispersion effects. We also drawn the electron density map based on these data. We tried to interpret these maps, and then we estimated the outline of the molecular shape and roughly traced the folding of this enzyme. We also estimated the FAD binding position which is one of the most important key points in this projects. However, to make more precise discussion, we need better quality electron density maps. It is the most important problem in the near future to improve the quality of these density maps.We have tried to crystallize HADPH-cytochrome P450 reductase. Initially we searched precipitants effective to this enzyme and were successful in finding a few reagents. We tried many crystallization conditions using these precipitants. But, we could not yet obtained any good crystals suitable for X-ray diffraction works.