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。 他们密切参与 在呼吸作用、光合作用 发酵和固氮。 最近人们认识到， 然而，铁-硫簇完成了除了简单功能之外的功能 电子转移：它们参与结合小分子底物 催化化学反应。 现在已知有几种金属酶 含有不寻常的铁硫簇作为其催化位点的一部分。 这些酶包括固氮酶、氢化酶、CO脱氢酶、顺乌头酸酶和 各种水解酶。 因此，本文提出的研究旨在 阐明了传统的铁硫簇合物在 氧化还原蛋白已经被“功能化”，使得它们能够 参与金属酶的基本生物反应。 一个假设 这已经从活性位点的特征中得到了证实， 金属酶和类似物化合物的合成的一个重要特征是， 这些“催化铁硫簇合物”是基于立方烷型[Fe 4S 4] 结构单元，其已通过非半胱氨酰连接“官能化” 或通过用另一个过渡取代一个Fe 金属M. 本提案的总体目标是检验这一假设 通过形成和研究光谱和配体结合， [MFe_3S_4]团簇（M = Fe，Ni，Mo，V，W）在一种新的 从极端嗜热细菌火球菌中获得的铁氧还蛋白 furiosus，一种在100摄氏度下生长最佳的生物。 P. furiosus铁氧还蛋白（Mr = 7，500）含有单个[Fe 4S 4]簇。 的 蛋白质是显着的，因为它的极端热稳定性（它是稳定的， 在95%C下持续至少12小时），并且是4Fe-2Fe的唯一实例。 铁氧还蛋白具有一个Fe原子的非半胱氨酰连接，如由 [Fe_4S_4]~（2+）~（1+）簇合物的光谱性质及其在离子交换中的应用 定量去除该Fe原子以产生常规的[Fe 3S 4]1+0 集群 这种铁氧还蛋白的性质使其成为用于 研究铁硫簇如何被官能化。 使用 生物化学和重组DNA技术结合一系列 光谱学，例如电子顺磁共振、磁性圆 二向色性，共振拉曼，穆斯堡尔，电子核双共振 和X射线吸收，本提案的具体目的是调查 a）外源配体与其[Fe 4 S 4]簇结合，B）形成和 混合金属[MFe 3S 4]簇的表征，c）结构 特定氨基酸残基突变的后果，和d） 导致蛋白质“超热稳定性”的因素。 在 除了提供对结构、功能和化学性质的深入了解外， Fe-S簇在广泛的金属酶中的性质， 该提案还解决了金属簇蛋白质的基本问题 相互作用和蛋白质-蛋白质相互作用的稳定性， 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.