The Novel Iron-Sulfur Clusters of Hydrogenase

9405783亚当斯本项目旨在扩大我们对生物铁硫(FeS)团簇的结构、功能和化学反应能力的了解，FeS团簇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，发现于氢酶的催化位置。早期的研究表明，中温氢酶中新的H簇可能由四个Fe原子组成，并且有两种含氮配体，当该簇与有效的抑制剂CO共价结合时，其中一种被取代。氢酶现在已经从“超高温”细菌Thermotoga maritima(TM)和一种名为ES-4的考古菌(考古菌)中纯化出来，ES-4生长在110摄氏度，这是生命的上限温度。ES-4酶是所有已知氢酶中含铁量最低的。它具有100℃以上的最佳催化温度，并在95℃下至少稳定24小时。对该氢酶进行了广泛的生化和光谱研究，以探讨其催化FeS中心的性质。此外，还获得了衍射峰大于2.8A的适合于结构测定的ES-4氢酶晶体。因此，ES-4氢酶基因将被克隆以提供用于结晶学分析的氨基酸序列。还将尝试获得编码超高温氢酶的基因的异源表达系统。一旦从结晶学获得结构信息，这将使所提出的“超热稳定性”和FeS H簇催化的机制能够通过定点突变直接测试。这项研究的最终目标是在从狂热杆菌中纯化的异常耐热的4Fe-铁氧还蛋白中构建一个H簇，这是一种可生长到105岁的古细菌。在之前的资助期间，该铁氧还蛋白Wa-S的二级结构通过2D-核磁共振确定，其基因已在大肠杆菌中克隆和表达。通过诱变对其4Fe-4S簇进行修饰，将成为高温氢酶氢激活簇的潜在模型。氢气(H2)是化工和能源工业中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氢，这些反应是由一种名为氢酶的酶催化的。氢酶已经被认为是几个基于生物的系统中的关键催化剂，这些系统既可以从可再生资源中生产氢气，也可以在化学合成中使用氢气。然而，传统细菌产生的氢酶的不稳定性严重限制了其实际应用。此外，这些酶如何催化氢气活化尚不清楚，因为没有任何氢酶的结构信息。到目前为止，人们已经证实，一种不寻常的被称为H簇的铁硫中心与催化密切相关，但氢酶H簇的结构及其作用机制尚不清楚。我们从一个生长到110℃的高温微生物中提纯了氢酶，证明了该酶在100℃下24小时的稳定性，并且得到了适合于结构测定的氢酶晶体。通过生化、光谱分析和结晶学分析，我们的目标是在分子水平上对高温氢酶进行完整的描述，包括蛋白质及其氢活化铁硫簇的结构，阐明其催化机理，并解释其显著的稳定性。这些目标将通过密切合作实现，包括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，以及一系列传统和发展光谱。此外，我们将尝试使用重组DNA技术来构建一个模型，该模型将在一个非常稳定的小蛋白中激活H2的H簇。拟议中的研究结果将扩大我们对生物铁-硫簇的结构和作用的知识，并将首次详细了解氢是如何被酶激活的。高温氢酶及其稳定模型的可获得性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. ***