Structure/Function Studies on Flavoproteins

描述（由申请人提供）：拟议工作的目的是使用结构，生物化学和遗传方法的组合来研究黄素蛋白的结构/功能关系。这项工作将集中在两个结构同源的FAD含酶，putidaredoxin还原酶从恶臭假单胞菌和凋亡诱导因子从小鼠。Putidaredoxin还原酶在细胞色素P450 cam单加氧酶中催化电子从NADH转移到铁硫蛋白putidaredoxin。putidaredoxin还原酶和putidaredoxin之间的复合物形成和电子转移的机制还不清楚。我们最近已经证明，putidaredoxin还原酶具有氧化还原活性的半胱氨酸，可以作为二硫醇/二硫化物氧化还原酶。由于没有推定的二硫键氧化还原中心编码的CysXXCys或CysXXXXCys基序特征的传统二硫键还原酶，二硫醇/二硫键氧化还原的机制putidaredoxin还原酶催化似乎是独特的，需要阐明。细胞凋亡诱导因子是一种非半胱天冬酶依赖性的哺乳动物死亡效应因子，在细胞凋亡诱导后，其从其正常定位（线粒体膜间隙）易位至细胞核，在细胞核中其引起染色质浓缩和DNA片段化。该蛋白诱导细胞凋亡的生理功能和机制尚不清楚。 我们的研究将解决的问题是如何结构的putidaredoxin还原酶和 凋亡诱导因子与其催化功能有关。putidaredoxin还原酶和凋亡诱导因子的晶体结构将被解决，比较，并与酶的催化性能。从结构的解释，这些关系将进一步探讨和修改的基因工程和特定的结构变化对蛋白质的催化功能的影响将进行评估。本研究将解释putidaredoxin还原酶催化的电子传递和二硫键/二硫键氧化还原的机制，并为深入了解凋亡诱导因子的作用机制和功能提供理论依据。

DESCRIPTION (provided by applicant): The aim of the proposed work is to use a combination of structural, biochemical and genetic approaches to study structure/function relationships in flavoproteins. The work will focus on two structurally homologous FAD-containing enzymes, putidaredoxin reductase from Pseudomonas putida and apoptosis inducing factor from mice. Putidaredoxin reductase catalyzes electron transfer from NADH to an iron-sulfur protein, putidaredoxin, in cytochrome P450cam monooxygenase. The mechanism of complex formation and electron transfer between putidaredoxin reductase and putidaredoxin is not well understood. We have recently demonstrated that putidaredoxin reductase has redox active cysteines and can function as dithiol/disulfide oxidoreductase. Since there are no putative disulfide redox centers encoded by CysXXCys or CysXXXXCys motifs characteristic for traditional disulfide reductases, the mechanism of dithiol/disulfide oxidoreduction catalyzed by putidaredoxin reductase seems to be unique and needs to be elucidated. Apoptosis inducing factor is a phylogenetically old mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus where it causes chromatin condensation and DNA fragmentation. Neither physiological function nor mechanism of apoptosis induced by this protein is known. Our research will address the question of how the structures of putidaredoxin reductase and apoptosis inducing factor are related to their catalytic function. Crystal structures of putidaredoxin reductase and apoptosis inducing factor will be solved, compared, and related to the catalytic properties of the enzymes. From structural interpretations, these relationships will be further probed and modified by genetic engineering and the effect of specific structural changes on catalytic function of the proteins will be assessed. The study will explain the mechanisms of electron transfer and dithiol/disulfide oxidoreduction catalyzed by putidaredoxin reductase and will provide an insight into the mechanism and function of apoptosis inducing factor.