RESPONSES TO OXYGEN TOXICITY BY ANAEROBIC MICROORGANISMS

分子氧在有氧生物体中扮演着一个矛盾的角色，因为它既是产生能量的必需品，也是非常有毒的。O2在正常代谢过程中会产生极具活性的衍生物，会对包括DNA、脂质和蛋白质在内的各种细胞成分造成相当大的损害。事实上，这些所谓的蛋白质与多种人类疾病有关，包括癌症、糖尿病、阿尔茨海默氏症和关节炎。需氧菌对ROS的防御系统已经建立得很好，其中包括超氧化物歧化酶和过氧化氢酶等。相比之下，几种厌氧菌的基因组序列显示它们不包含这些酶，这表明它们有替代的途径。了解厌氧菌中O2反应的分子基础对厌氧病原体引起的疾病的治疗具有直接的指导意义。预计这些生物体的生存能力关键取决于它们对ROS的防御效率。因此，厌氧菌和需氧菌在清除这三个物种的途径上的差异可能被用作药物开发的高度特异性靶标。这项研究的总体目标是阐明这些途径在高温厌氧菌，狂热球菌，它在100摄氏度生长的最佳。从该生物体中分离纯化出一种新的非血红素铁蛋白，命名为neelaredosin，它具有超氧化物还原酶而不是歧化酶活性。编码该蛋白的基因已被克隆和测序，重组蛋白(从大肠杆菌中获得)的晶体结构已被解析为1.7A。在所检测的所有厌氧菌中都发现了奈拉雷还蛋白的同源物。P.Furiosus蛋白是一种同源四聚体，每个亚基(14 KDa)含有一个铁原子，它以前所未有的配位几何构型与蛋白质结合，涉及天然蛋白和活性中心残基发生变化的突变体的四态和稳态前动力学。在没有外源配体和存在外源配体的情况下，该蛋白质及其突变体中金属中心的结构、振动、磁、电子和氧化还原性质将使用一系列补充光谱技术进行表征：电子顺磁共振、光吸收、变温磁圆二色谱、共振拉曼和红外傅立叶变换。奈拉雷还蛋白、突变体和带有结合的外源配体的衍生物的结构将用结晶学确定。最后，将利用基因组和蛋白质组学方法分析在不同胁迫条件下生长的P.Furiosus的蛋白质模式和基因转录，以表征参与O2解毒途径的其他蛋白质。

Molecular O2 plays a paradoxical role in aerobic organisms in that it is both essential for energy generation as well as very toxic. Extremely reactive derivatives of O2 are produced during normal metabolism and can cause considerable damage to various cellular components, including DNA, lipids and proteins. In fact, these so-called been implicated in a wide variety of human diseases including cancer, diabetes, Alzheimer's and arthritis. The defense systems against ROS in aerobes have been well established and involve, among others, the enzyme superoxide dismutase and catalase. In contrast, the genome sequences of several anaerobes reveal that they do not contain these enzymes, which suggest that they have alternative pathways. Understanding the molecular basis of the O2 response in anaerobes has direct ramifications for the treatment of diseases caused by anaerobic pathogens. It is expected that the viability of these organisms is crucially dependent on the efficiency of their defense against ROS. Differences between anaerobes and aerobes in the pathways used to scavenger thee species might therefore be exploited as highly specific targets for drug development. The overall objective of this research is to elucidate these pathways in the hyperthermophilic anaerobe, Pyrococcus furiosus, which grows optimally at 100 degrees Centigrade. A novel non-heme iron protein termed neelaredoxin which has superoxide reductase rather than dismutase activity has been purified from this organism. The gene encoding it has been cloned and sequenced, and the crystal structure of the recombinant protein (obtained from Escherichia coli) has been solved to 1.7 A. Homologs of neelaredoxin have been found in all anaerobes examined. The P. furiosus protein is a homotetramer and contains one iron atom per subunit (of 14 kDa), which is bound to the protein in an unprecedented coordination geometry, involving four state and pre-steady state kinetics of the native protein and of mutants in which active site residues have been changed. The structural, vibrational, magnetic, electronic and redox properties of the metal center in this protein and mutants thereof, in the absence and presence of exogenous ligands, will be characterized using a range of complimentary spectroscopic techniques: electron paramagnetic resonance, optical absorption, and variable temperature magnetic circular dichroism, resonance Raman and Fourier transform infra-red. The structure of neelaredoxin, mutants and derivatives with bound exogenous ligands will be determined using crystallography. Finally, genomic and proteomic methods will be used to analyze protein patterns and gene transcripts in P. furiosus grown under various stress conditions to characterize other proteins involved in the O2 detoxification pathway.