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、生化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上同源的含有FAD的酶，恶臭假单胞菌的恶臭还蛋白还原酶和小鼠的凋亡诱导因子。在细胞色素P450cam单加氧酶中，Putidaredosin还原酶催化NADH到铁硫蛋白Putidaredosin的电子传递。恶臭还蛋白还原酶和恶臭还蛋白之间形成复合体和电子转移的机制还不是很清楚。我们最近证明了恶臭还蛋白还原酶具有氧化还原活性的半胱氨酸，并且可以作为二硫醇/二硫代氧化还原酶发挥作用。由于传统的二硫键还原酶不存在由CysXXCys或CysXXXXCys基序编码的二硫键氧化还原中心，因此Putidaredosin还原酶催化二硫醇/二硫键氧化还原的机制似乎是独特的，需要进一步研究。凋亡诱导因子是一种古老的哺乳动物死亡效应因子，不依赖于半胱氨酸天冬氨酸氨基转移酶(Caspase)，在诱导细胞凋亡时，它从正常定位的线粒体膜间隙移位到细胞核，在那里导致染色质凝聚和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。 我们的研究将解决这个问题，即腐烂还蛋白还原酶和 细胞凋亡诱导因子与其催化功能有关。我们将解析、比较和研究恶臭还蛋白还原酶和细胞凋亡诱导因子的晶体结构，并将其与酶的催化性能联系起来。通过结构解释，这些关系将被基因工程进一步探索和修改，并将评估特定结构变化对蛋白质催化功能的影响。这项研究将解释Putidaredosin还原酶催化的电子传递和二硫醇/二硫键氧化还原的机制，并将为深入了解细胞凋亡诱导因子的机制和功能提供依据。

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.