Structure and assembly of the bacterial module for the respiration of nitrous oxide

该项目包括研究利用温室气体一氧化二氮的斯图兹氏假单胞菌厌氧呼吸模块。人们正在寻找一种需要能量的多聚体复合体的结构见解，包括一种组装一氧化二氮还原酶所必需的ATPase。可溶性成分，即细胞质ATPase和周质金属插入酶将被过表达、纯化、生化特征和结晶研究。该项目包括与ATPase的结合研究，ATPase和插入酶的定点突变，以确定必要的残基和获得功能洞察力。后者也是通过建立基于分离的ATPase、插入酶和过表达的膜结合转导或载体蛋白的体外囊泡系统来寻求的。该项目包括从不同来源分离一氧化二氮还原酶，目的是获得其结构。将通过诱变的方法对一氧化二氮还原酶的催化部位进行结构研究，并对突变的蛋白质进行光谱表征。我们还旨在从一株假单胞菌中获得有关呼吸硝酸还原酶的结构信息，作为通过氧化还原环机制催化质子转运的呼吸复合体的例子。

The project comprises the investigation of the anaerobic respiratory module of Pseudomonas stutzeri utilizing the greenhouse gas nitrous oxide. Structural insights are being sought for an energy-requiring multimeric complex, including an ATPase which is necessary for the assembly of nitrous oxide reductase. The soluble components, i.e. the cytoplasmic ATPase and a periplasmic metal insertase will be overexpressed, purified, biochemically characterized and subjected to crystallization studies. The project involves binding studies with the ATPase, site-directed mutagenesis of the ATPase and insertase to identify essential residues and gain functional insights. The latter is also sought by establishing a vesicular in vitro system based on isolated ATPase, insertase, and the overexpressed membrane-bound transducer or carrier protein. The project comprises the isolation of nitrous oxide reductase from different sources with the objective of obtaining its structure. Structural investigation of the catalytic site of nitrous oxide reductase by means of mutagenesis and spectroscopic characterization of the mutant proteins will be done. We aim also at obtaining structural information on the respiratory nitrate reductase from a pseudomonad as an example of a respiratory complex that catalyzes proton translocation by a redox loop mechanism.