The Novel Iron-Sulfur Clusters of Hydrogenase

该项目旨在扩展我们对生物铁硫（FeS）簇的结构、功能和化学反应性的了解，这是自然界中最普遍的电子载体。我们正在研究一种新型的FeS中心的结构和性质，称为H簇，发现在氢化酶的催化位点。在早期的研究中，研究表明，中亲氢酶中的新型H簇可能由四个铁原子组成，并具有两种类型的含氮配体，当簇与CO（一种有效的抑制剂）共价结合时，其中一种会移位。氢化酶现在已经从“超嗜热”细菌——海洋热菌（Thermotoga maritima, Tm）和一种叫做ES-4的古细菌（古细菌）中纯化出来，这种细菌生长在90摄氏度，110摄氏度是生命的上限。ES-4酶的铁含量是所有已知氢化酶中最低的。它的最佳催化温度在100℃以上，在95℃下稳定至少24小时。我们建议对这种氢化酶进行广泛的生化和光谱研究，以研究其催化FeS中心的性质。此外，还获得了ES-4氢化酶的晶体，衍射波长超过2.8 A，适于结构测定。因此，ES-4氢化酶基因将被克隆，为晶体学分析提供氨基酸序列。还将尝试获得编码超嗜热氢化酶的基因的异源表达系统。一旦从晶体学中获得结构信息，这将使提出的“超热稳定性”和FeS H簇催化机制能够通过位点定向诱变直接测试。这项研究的最终目标是在一种高温稳定的4fe -铁氧还蛋白中构建一个H簇，这种蛋白是从一种生长到105℃的古细菌中纯化出来的。在前期资助期内，通过2D-NMR测定了该铁氧还蛋白的二级结构，并在大肠杆菌中克隆表达了该基因。通过诱变对其4Fe-4S簇进行修饰，将作为超嗜热氢化酶h2活化簇的潜在模型。氢气（H2）是化学和能源工业各种过程中的重要中间体，被一些人认为是未来的燃料。许多微生物也能够消耗或产生H2，这些反应是由一种称为氢化酶的酶催化的。氢化酶已被认为是几种基于生物的系统中从可再生资源中生产H2和在化学合成中使用H2的关键催化剂。然而，传统细菌中氢化酶的不稳定性严重限制了它们的实际应用。此外，由于没有任何氢化酶的结构信息，这些酶是如何催化H2活化的尚不清楚。到目前为止，已经确定了一种称为“H”簇的不寻常类型的含铁硫中心与催化密切相关，但氢化酶H簇的结构或其作用机制尚不清楚。我们从一种生长到110℃（目前生命的上限温度）的超嗜热微生物中纯化出了氢化酶，并证明该酶在100℃下可以稳定生长24小时。此外，我们还获得了这种氢化酶的晶体，适合于结构测定。利用生化、光谱学和晶体学分析，我们的目标是在分子水平上获得超嗜热氢化酶的完整描述，包括蛋白质及其h2激活铁硫簇的结构，阐明其催化机制，并解释其显著的稳定性。这些目标将通过涉及600升发酵和大规模厌氧蛋白纯化，晶体学，各种生化，动力学，电位学和分子生物学技术以及一系列常规和发展光谱的密切合作来实现。此外，我们将尝试使用重组DNA技术构建H2激活H簇在一个小的非常稳定的蛋白质中的模型。这项研究的结果将扩展我们对生物铁硫簇的结构和作用的认识，并将首次详细了解H2是如何被酶激活的。超嗜热氢化酶的可用性及其稳定的模型也可能具有重要的实际应用。* * *

9405783 Adams This project seeks to expand upon our knowledge of the structure, function and chemical reactivity of biological iron-sulfur (FeS) clusters, the most ubiquitous electron carriers in nature. We are investigating the structure and properties of a novel type of FeS center, termed the H cluster, found at the catalytic site of hydrogenase. In earlier studies, it was shown the novel H cluster in mesophilic hydrogenases is probably comprised of four Fe atoms and has two types of nitrogenous ligands, one of which is displaced when the cluster covalently binds CO, a potent inhibitor. Hydrogenases have now been purified from the "hyperthermophilic" bacterium, Thermotoga maritima (Tm), which grows up to 90 C, and from an archaeon (archaebacterium) termed ES-4 which grows at 110 C, the upper temperature limit for life. The ES-4 enzyme has the lowest iron content of any known hydrogenase. It has an optimum temperature for catalysis above 100 C and is stable at 95 C for at least 24 hours. Extensive biochemical and spectroscopic studies of this hydrogenase are proposed to investigate the nature of its catalytic FeS center. In addition, crystals of ES-4 hydrogenase suitable for structural determination have been obtained that diffract past 2.8 A. The gene for ES-4 hydrogenase will therefore be cloned to provide the amino acid sequence for crystallographic analyses. Attempts will also be made to obtain an heterologous expression system for the gene encoding a hyperthermophilic hydrogenase. Once structural information is available from crystallography, this will enable proposed mechanisms of both "hyperthermostability" and of catalysis by the FeS H cluster to be directly tested by site-directed mutagenesis. An ultimate goal of this research is to construct an H cluster within an exceptionally thermostable 4Fe-ferredoxin that has been purified from Pyrococcus furiosus, an archaeon which grows up to 105 . In the prior funding period, the secondary structure of this ferredoxin wa s determined by 2D-NMR, and its gene was cloned and expressed in Escherichia coli. Modification of its 4Fe-4S cluster through mutagenesis will be explored as a potential model for the H2-activating cluster of the hyperthermophilic hydrogenases. %%% Hydrogen gas (H2) is an important intermediate in a variety of processes in the chemical and energy industry and is considered by some as the fuel of the future. Many microorganisms are also able to either consume or produce H2, and these reactions are catalyzed by an enzyme termed hydrogenase. Hydrogenases have been proposed as the key catalyst in several biologically-based systems for both the production of H2 from renewable resources and for the use of H2 in chemical syntheses. However, the instability of hydrogenases from conventional bacteria severely limit their practical application. In addition, it is not known how these enzymes catalyze H2 activation, as structural information is not available for any hydrogenase. So far it has been established that an unusual type of iron-sulfur-containing center termed the "H" cluster is intimately involved in catalysis, but the structure for the hydrogenase H cluster or its mechanism of action are not known. This proposal focuses on hydrogenases from so-called hyperthermophilic microorganisms, which have the remarkable property of growing near and even above 100 C. We have purified hydrogenase from an hyperthermophile which grows up to 110 C, the current upper temperature limit of life, and shown that the enzyme is stable for 24 hr at 100 C. Moreover, we have obtained crystals of this hydrogenase which are suitable for structural determination. Using biochemical, spectroscopic and crystallographic analyses, our objectives are to obtain a complete description of a hyperthermophilic hydrogenase at the molecular level, including structures for the protein and its H2-activating iron-sulfur cluster, elucidating its catalytic mechanism, and an explanation of its remarkable stability. These goals will be achieved by an intensely collaborative effort involving 600 liter fermentations and large scale anaerobic protein purifications, crystallography, a variety of biochemical, kinetic, potentiometric and molecular biology techniques, and a range of both conventional and developmental spectroscopies. In addition, we will attempt using recombinant DNA techniques to construct a model of the H2 activating H cluster in a small extremely stable protein. The results from the proposed research will extend our knowledge of the structures and roles of biological iron-sulfur clusters, and will provide the first detailed insight into how H2 is activated enzymatically. The availability of hyperthermophilic hydrogenases and stable models of them may also have important practical applications. ***