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

亚当斯这个项目旨在扩大我们的知识的结构，功能和组装的生物铁硫（FeS）集群，最普遍的电子载体在自然界中。 该研究涉及氢化酶的性质和作用，氢化酶是一种具有催化H2氧化和H2产生的独特能力的酶。 该酶将从极端嗜热细菌Thermotoga maritima（Tm）中获得，Thermotoga maritima（Tm）是一种在90摄氏度下生长的生物体，是已知的两种最嗜热细菌之一。 它的异三聚体（abg）氢化酶的质量160 kDa的建议含有11个铁硫簇。 这些包括催化位点，即所谓的H簇，由六个铁原子组成。 已经克隆和测序的编码三个亚基的基因将在大肠杆菌中表达，蛋白质将单独和组合进行表征，包括结构测定。 两种酶被认为是参与在体内组装和激活的氢化酶，氢化酶激活蛋白（HAP）和氰化物二水合酶，将被纯化和表征，它们的基因将与氢化酶的基因共表达。 最近完成的基因组的Tm将被用来确定其他辅助蛋白参与氢化酶的合成，以及那些参与终端电子转移途径的Tm。 这些不同的蛋白质将被确定，并将使用蛋白质组学和转录分析研究其调控。 最后，包括人类和果蝇在内的真核生物含有细菌Fe-氢化酶的类似物，这将使用酵母（酿酒酵母）作为模型系统进行纯化和表征。 本研究的总体目标是，因此，定义氢化酶和相关酶的分子内和分子间电子转移的途径，分别确定所涉及的金属中心的性质和作用，以及如何组装新的簇，如参与H2活化。这些目标将通过包括大规模（600升）发酵和大规模厌氧蛋白纯化、结晶学、各种生物化学、动力学、电位和分子生物学技术以及基因组学和蛋白质组学分析的技术组合来实现。该项目将提供有关酶的基本信息，该酶催化最简单的反应，氢气的生产和消耗，具有基本化学和生物学意义的反应。 这将包括酶的新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于氢化酶的蛋白质，但这些生物体并不知道会代谢氢气。 这项研究将有助于了解这种有趣的蛋白质的性质，这种蛋白质可能在感知氢以外的小分子（如一氧化碳、一氧化氮或氰化物）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.