Structure and Function of Iron-Hydrogenases in a Hyperthermophilic Bacterium and of an Analogous Protein in Higher Eukaryotes

亚当斯这个项目寻求扩展我们对生物铁硫(FeS)团簇的结构、功能和组装的知识，FeS团簇是自然界中最普遍的电子载体。这项研究涉及氢酶的性质和作用，氢酶是一种独特的催化氢氧化和产生氢气的酶。这种酶将从超嗜热菌Thermotoga maritima(TM)中获得，该细菌在90摄氏度下生长，是已知的两种最嗜热菌之一。其质量为160 kDa的异三聚体(ABG)氢酶被认为含有11个铁-硫簇。其中包括催化中心，即所谓的H团簇，它由六个铁原子组成。已经克隆和测序的编码这三个亚基的基因将在大肠杆菌中表达，并将对这些蛋白质进行单独和组合的鉴定，包括结构测定。两种被认为参与氢酶在体内组装和激活的酶，氢酶激活蛋白(HAP)和氰化物二水合酶，将被纯化和鉴定，它们的基因将与氢酶的基因共表达。最近完成的TM基因组将用于鉴定参与氢酶合成的其他辅助蛋白，以及那些参与TM末端电子转移途径的辅助蛋白。这些不同的蛋白质将被识别，并将使用蛋白质组学和转录分析来研究它们的调节。最后，包括人和果蝇在内的真核生物含有细菌铁氢酶的类似物，并将以酵母(Saccharmyces Cerevisiae)为模型系统进行纯化和表征。因此，本研究的总体目标是分别确定氢酶和相关酶中分子内和分子间电子转移的途径，以确定所涉及的金属中心的性质和作用，以及参与氢气活化的新簇是如何组装的。这些目标将通过包括大规模(600升)发酵和大规模厌氧蛋白质纯化、结晶学、各种生化、动力学、电位和分子生物学技术以及基因组和蛋白质组分析的技术组合来实现。该项目将提供关于一种酶的基本信息，该酶催化最简单的反应、氢气的产生和消耗、具有基本化学和生物意义的反应。这将包括酶的新的含铁中心如何能够进行这些反应，以及蛋白质如何在细胞内组装。在包括人类在内的高等生物体中发现了一种类似氢酶的蛋白质，但这种生物体尚不清楚是否能代谢氢气。这项研究将有助于理解这种耐人寻味的蛋白质的性质，这种蛋白质可能在感知一氧化碳、一氧化氮或氰化物等氢以外的小分子方面发挥很好的作用。

Adams This project seeks to expand upon our knowledge of the structure, function and assembly of biological iron-sulfur (FeS) clusters, the most ubiquitous electron carriers in nature. The research concerns the properties and role of hydrogenase, an enzyme that has the unique ability to catalyze H2 oxidation and H2 production. The enzyme will be obtained from the hyperthermophilic bacterium, Thermotoga maritima (Tm), an organism that grows at 90 degrees C and is one of the two most thermophilic bacteria known. Its heterotrimeric (abg) hydrogenase of mass 160 kDa is proposed to contain eleven iron-sulfur clusters. These include the catalytic site, the so-called H cluster, which consists of six iron atoms. The genes encoding the three subunits, which have already been cloned and sequenced, will be expressed in Escherichia coli and the proteins will be characterized individually and in combination, including structural determinations. Two enzymes thought to be involved in the in vivo assembly and activation of hydrogenase, the hydrogenase-activating protein (HAP) and cyanide dihydratase, will be purified and characterized, and their genes will be coexpressed with those of hydrogenase. The recently completed genome of Tm will be used to identify other accessory proteins involved in hydrogenase synthesis, as well as those that participate in the terminal electron transfer pathway of Tm. These various proteins will be identified and their regulation will be studied using proteomics and transcriptional analyses. Finally, eukaryotes including human and Drosophila contain an analog of bacterial Fe-hydrogenase, and this will be purified and characterized using yeast (Saccharomyces cerevisiae) as the model system. The overall objectives of this research are, therefore, to define the pathways of intra- and intermolecular electron transfer in hydrogenase and associated enzymes, respectively, to determine the nature and role of the metal centers involved, and how novel clusters such as that involved in H2 activation are assembled. These goals will be achieved by a combination of techniques including large scale (600 liter) fermentations and large scale anaerobic protein purifications, crystallography, various biochemical, kinetic, potentiometric and molecular biology techniques, together with genomic and proteomic analyses. This project will provide fundamental information on an enzyme which catalyzes the simplest of reactions, the production and consumption of hydrogen gas, reactions of fundamental chemical and biological significance. This will include how the enzyme's novel iron-containing center is able to carry out these reactions, and how the protein is assembled inside the cell. A protein analogous to hydrogenase is found in higher organisms, including humans, but such organisms are not known to metabolize hydrogen gas. This research will lead to an understanding of the nature of this intriguing protein, which may well function in sensing small molecules other than hydrogen, such as carbon monoxide, nitric oxide or cyanide.