EAPSI: Modifying Proteins to Investigate Key Intermediates in Oxygen Binding Enzymes

EAPSI：修饰蛋白质以研究氧结合酶的关键中间体

• 批准号: 1514852
• 负责人: Matthew Wolf
• 金额: $ 0.51万
• 依托单位: Wolf Matthew
• 依托单位国家: 美国
• 项目类别: Fellowship Award
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1514852&HistoricalAwards=false
• 关键词: EAPSI; Modifying; Proteins; Investigate; Key

Iron-containing heme proteins catalyze a wide variety of reactions in biology, including binding and activating oxygen towards reacting with many molecules in the body. These reactions are important for respiration, metabolism of drugs and toxins, and breaking down hydrocarbons, among other functions. Although there has been much research focused on trying to understand these reactions, little is still known about the reactive "iron-oxo" intermediates that are crucial in these mechanisms. This project aims to investigate these reactive intermediates by modifying of the metal center and the protein scaffold of the stable heme protein myoglobin and binding oxygen species to the metal center. This research will be conducted in collaboration with Dr. Takashi Matsuo at the Nara Institute of Science and Technology (NAIST). Dr. Matsuo's lab has years of experience in protein modification and studying reactive oxygen intermediates, and the expertise of his lab will be invaluable to the project. By expressing the myoglobin mutants H64A and H64D, the active site pocket of myoglobin will be opened up and a potential hydrogen bonding interaction with the oxo or peroxo species can also be introduced. In addition to the active site mutations, the native heme in myoglobin will be removed and ruthenium, rhodium, and iridium porphyrins will be incorporated into the protein. Oxo and peroxo transfer reagents such as hydrogen peroxide and meta-chloroperoxybenzoic acid will be added to form high-valent metal-oxo and peroxo species. The larger transition metals may stabilize the bound oxo and peroxo species, and allow these species to be studied by a variety of spectroscopic methods. Finally, the reactivity of the oxo and peroxo species will be studied through the addition of appropriate substrates and measuring the total turnover numer and the rate of substrate oxidation or oxygenation over time. This NSF EAPSI award is funded in collaboration with the Japan Society for the Promotion of Science.

含铁血红素蛋白在生物学中催化各种各样的反应，包括结合和激活氧与体内许多分子反应。这些反应对于呼吸、药物和毒素的代谢以及分解碳氢化合物等功能都很重要。虽然已经有很多研究集中在试图了解这些反应，仍然很少有人知道的反应性“铁氧”中间体是至关重要的，在这些机制。本研究旨在通过修饰血红素蛋白肌红蛋白的金属中心和蛋白质支架，并将氧物种结合到金属中心来研究这些活性中间体。这项研究将与奈良科学技术研究所（NAIST）的松尾隆博士合作进行。Matsuo博士的实验室在蛋白质修饰和活性氧中间体研究方面拥有多年的经验，他的实验室的专业知识对该项目将是无价的。通过表达肌红蛋白突变体H64 A和H64 D，肌红蛋白的活性位点口袋将被打开，并且还可以引入与氧代或过氧物种的潜在氢键相互作用。除了活性位点突变，肌红蛋白中的天然血红素将被去除，钌、铑和铱卟啉将被掺入蛋白质中。将添加氧代和过氧转移试剂如过氧化氢和间氯过氧苯甲酸以形成高价金属-氧代和过氧物质。较大的过渡金属可以稳定结合的氧和过氧物种，并允许这些物种通过各种光谱方法进行研究。最后，将通过添加适当的底物并测量总周转数和底物氧化或氧化速率来研究氧代和过氧物质的反应性。这个NSF EAPSI奖是与日本科学促进协会合作资助的。