RUI- Understanding the pH-dependent Reduction Potential of the Rieske Protein from Thermus thermophilus Using Site-Directed Mutagenesis and Chemical Modification

Rieske蛋白是参与呼吸和光合作用等关键生物过程的电子传递蛋白。它们有一个由2个半胱氨酸和2个组氨酸配体连接的[2Fe-2S]簇。在呼吸链中，Rieske蛋白从对苯二酚中获得一个电子和一个质子，并将其转移到细胞色素C1。三个目标将最终形成关于Rieske蛋白的pH依赖的还原潜力的详细信息。1)Rieske蛋白质家族的独特之处在于具有高度保守的三维结构，但具有广泛的还原潜力。这项工作将通过对旨在改变还原潜力的突变体进行协调的光谱和结构研究，来测试铁-硫蛋白质如何调节还原潜力。2)组氨酸配体对化学修饰剂的反应性将提供对蛋白质如何携带质子和电子的洞察。为了探测反应活性，焦碳酸二乙酯(DEPC)或其他修饰剂将与蛋白质反应，以确定修饰发生在哪里。随着改变电势的突变，反应性如何变化将测试电势的效果。3)Rieske簇对pH非常稳定，而mitoNEET簇是由1个组氨酸和3个半胱氨酸连接的[2Fe-2S]簇，在低pH条件下不稳定。改变Rieske蛋白质的MitoNEET构型将决定蛋白质折叠在簇稳定中所起的作用。广泛的影响：对于PI和三一大学化学系来说，本科生研究是有抱负的化学家和生物化学家的基本经验。学生在科学的基本问题上工作，但由于他们缺乏经验，培训需要广泛和彻底。利邦还获得了快速资助和麦克奈尔资助，以增加对科学的参与，特别是在那些通常代表不足和/或是第一代大学生的群体中。三一大学正在寻求积极参与周围社区的研究，并将代表不足的群体带入科学界。

Rieske proteins are electron transport proteins involved in key biological processes, such as respiration and photosynthesis. They have a [2Fe-2S] cluster ligated by 2 cysteine and 2 histidine ligands. In the respiratory chain, the Rieske protein picks up an electron and a proton from quinol and transfers it to cytochrom c1. Three aims will culminate in detailed information about the pH-dependent reduction potential of the Rieske protein. 1) The Rieske family of proteins is unique in that there is a highly conserved 3-dimensional structure, and yet there is a wide range of reduction potentials. Work will test how iron-sulfur proteins tune reduction potential through concerted spectroscopic and structural studies of mutants designed to alter reduction potential. 2) The reactivity of the histidine ligands to chemical modifiers will provide insight into how the protein carries both a proton and an electron. To probe the reactivity, diethyl pyrocarbonate (DEPC) or other modifiers will be reacted with the protein to establish where the modification occurs. How the reactivity changes with mutations that alter potential will tested the effect of the potential. 3) Rieske clusters are very stable to pH, yet the MitoNEET cluster, which is a [2Fe-2S] cluster ligated by 1 histidine and 3 cysteines, is not stable at low pH. Altering a Rieske protein to have a MitoNEET configuration will determine the role the protein fold plays in cluster stability.Broader Impacts: For the PI and the chemistry department at Trinity University, undergraduate research is an essential experience for aspiring chemists and biochemists. The students work on fundamental problems in science, but since they are inexperienced, the training needs to be extensive and thorough. Trinity has also been awarded a FAST grant and a McNair grant to increase participation the sciences, especially in those groups that are typically underrepresented and/or are first-generation college students. Trinity University is seeking to actively engage the surrounding communities in research and bring underrepresented groups into the sciences.