SITE-SPECIFIC PROPERTIES OF A UNIQUE IRON-SULFUR PROTEIN

含有非血红素铁和无机硫簇的蛋白质是 自然界中最普遍的电子载体。他们密切地参与其中 在呼吸、光合作用等基本生物过程中， 发酵和固氮。它最近得到了认可， 然而，铁-硫团簇实现了除简单功能以外的功能 电子转移：它们参与结合小分子底物 并催化化学反应。现已知有几种金属酶 它们含有不寻常的铁-硫团簇作为其催化位置的一部分。 包括固氮酶、氢酶、一氧化碳脱氢酶、乌头酸酶和 各种水解酶。因此，这里提出的研究旨在 阐明了传统的铁-硫团簇在 氧化还原蛋白已经被“功能化”，使它们能够 参与金属酶的基本生物反应。一个假设 这已经从对活性中心的描述中获得了信任 金属酶和类似物的合成是部分或全部 这些“催化铁-硫团簇”是基于立方烷类型的[Fe4S4] 结构单元，通过非半胱氨酸基连接而“功能化” 或通过用另一种转变取代一种铁 本提案的总体目标是检验这一假说。 通过形成和研究光谱和配基结合 新化合物[MFe3S4]团簇(M=Fe，Ni，Mo，V，W)的性质 从极端嗜热菌--热球菌中获得的铁氧还蛋白 狂犬病，一种在100摄氏度下生长最佳的有机体。 呋喃假单胞菌铁氧还蛋白(Mr=7,500)含有单一的[Fe4S4]簇。这个 蛋白质的非凡之处在于其极高的热稳定性(它是稳定的 对于95%的温度，持续至少12小时)，并且作为4Fe的唯一例子- 铁还蛋白具有一个铁原子的非半胱氨基连接，如 [Fe4S4]2+1+团簇的新光谱性质及其易性 定量去除这个铁原子，得到常规的[Fe3S4]1+0 集群。这种铁氧还蛋白的性质使其成为 研究铁硫团簇如何官能化。vbl.使用 生化和重组DNA技术与一系列 光谱学，例如电子顺磁共振，磁环 二向色性、共振拉曼、穆斯堡尔、电子核双共振 和X射线吸收，这项提议的具体目的是调查 A)外源配体与其[Fe4S4]簇结合，b)形成和 混合金属[MFe3S4]团簇的表征，c)结构 特定氨基酸残基突变的后果，以及d) 导致蛋白质“超热稳定性”的因素。在……里面 除了提供对结构、功能和化学成分的洞察 铁-S团簇在多种金属酶中的性质 该提案还解决了金属簇-蛋白质的根本问题 相互作用和蛋白质-蛋白质相互作用的稳定性和 反应性。

Proteins containing clusters of non-heme iron and inorganic sulfur are the most ubiquitous electron carriers in nature. They are intimately involved in such fundamental biological processes as respiration, photosynthesis, fermentation, and nitrogen fixation. It has recently been recognized, however, that iron-sulfur clusters fulfill functions other than simple electron transfer: they participate in binding small molecule substrates and catalyzing chemical reactions. Several metalloenzymes are now known that contain unusual iron-sulfur clusters as part of their catalytic sites. These include nitrogenase, hydrogenase, CO dehydrogenase, aconitase, and various hydrolases. The research proposed here is therefore designed to elucidate the mechanisms by which conventional iron-sulfur clusters in redox proteins have been "functionalized" such that they are able to take part in fundamental biological reactions in metalloenzymes. One hypothesis that has gained credence from the characterization of the active sites of metalloenzymes and the synthesis of analog compounds is that some or all of these "catalytic iron-sulfur clusters" are based on a cubane-type [Fe4S4] structural unit, that has been "functionalized" by non-cysteinyl ligation of a specific Fe site or by replacement of one Fe by another transition metal M. The overall objective of this proposal is to test this hypothesis by forming and investigating the spectroscopic and ligand binding properties of a [MFe3S4] cluster (where M = Fe, Ni, Mo, V, W) in a novel ferredoxin obtained from the extremely thermophilic bacterium, Pyrococcus furiosus, an organism that grows optimally at 100oC. P. furiosus ferredoxin (Mr = 7,500) contains a single [Fe4S4] cluster. The protein is remarkable both for its extreme thermal stability (it is stable for 95%C for at least 12 hours) and in being the only example of a 4Fe- ferredoxin that has non-cysteinyl ligation of one Fe atom, as evidenced by the novel spectroscopic properties of the [Fe4S4]2+1+ cluster and the ease of quantitative removal of this Fe atom to yield a conventional [Fe3S4]1+0 cluster. The properties of this ferredoxin make it the ideal system for investigating how iron-sulfur clusters may be functionalized. Using biochemical and recombinant DNA techniques in conjunction with an array of spectroscopies, e.g. electron paramagnetic resonance, magnetic circular dichroism, resonance Raman, Mossbauer, electron nuclear double resonance and X-ray absorption, the specific aims of this proposal are to investigate a) exogenous ligand binding to its [Fe4S4] cluster, b) the formation and characterization of mixed metal [MFe3S4] clusters, c) the structural consequences of mutations of specific amino acid residues, and d) the factors responsible for the "hyperthermostability" of the protein. In addition to providing insight into the structure, function and chemical properties of the Fe-S clusters in a broad range of metalloenzymes, the proposal also addresses the fundamental problems of metal cluster-protein interactions and protein-protein interactions in terms of stability and reactivity.