RESPONSES TO OXYGEN TOXICITY BY ANAEROBIC MICROORGANISMS

分子O2在有氧生物中起着矛盾的作用，因为它既是能量产生所必需的，也是非常有毒的。在正常代谢过程中会产生活性极高的O2衍生物，并对各种细胞成分（包括DNA、脂质和蛋白质）造成相当大的损害。事实上，这些所谓的被牵连在各种各样的人类疾病，包括癌症，糖尿病，阿尔茨海默氏症和关节炎。在需氧菌中针对ROS的防御系统已经很好地建立，并且涉及酶超氧化物歧化酶和过氧化氢酶等。相反，几种厌氧菌的基因组序列显示它们不含这些酶，这表明它们有替代途径。了解厌氧菌中O2反应的分子基础对于治疗由厌氧病原体引起的疾病具有直接影响。 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。厌氧菌和需氧菌之间的差异，用于清除这些物种的途径，因此可能会被开发为高度具体的药物开发的目标。这项研究的总体目标是阐明这些途径中的超嗜热厌氧菌，激烈火球菌，最佳生长在100摄氏度。一种新的非血红素铁蛋白称为neelaredoxin，它具有超氧化物还原酶，而不是歧化酶的活性已被纯化，从这种生物体。编码它的基因已被克隆和测序，重组蛋白（从大肠杆菌中获得）的晶体结构已解为1.7A。neelaredoxin的同源物已被发现在所有的厌氧菌检查。激烈毕赤酵母蛋白质是同源四聚体，每个亚基（14 kDa）含有一个铁原子，其以前所未有的配位几何结构与蛋白质结合，涉及天然蛋白质和活性位点残基已改变的突变体的四态和前稳态动力学。在不存在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、磁性、电子和氧化还原性质将使用一系列互补光谱技术表征：电子顺磁共振、光学吸收和变温磁性圆二色谱、共振拉曼和傅立叶变换红外。将使用晶体学确定neelaredoxin、突变体和具有结合的外源配体的衍生物的结构。最后，基因组学和蛋白质组学方法将用于分析在各种胁迫条件下生长的P. furiosus中的蛋白质模式和基因转录本，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.