RUI: Novel Heme Chemistry of Cytochrome c'

RUI：细胞色素 c 的新型血红素化学

• 批准号: 0745035
• 负责人: Colin Andrew
• 金额: $ 39.51万
• 依托单位: Eastern Oregon University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0745035&HistoricalAwards=false
• 关键词: RUI; Novel; Heme; Chemistry; Cytochrome

This project will investigate the novel heme chemistry of Alcaligenes xylosoxidans cytochrome c' (AXCP) with a particular focus on undergraduate involvement. The PI and coworkers have previously reported the unprecedented reactivity of AXCP, whereby nitric oxide (NO) coordinates to the proximal heme face by way of a putative dinitrosyl intermediate. This alternative heme-NO binding mode, involving both distal and proximal heme faces, is challenging previous assumptions of how heme proteins react with NO, and suggests novel mechanisms for modulating ligand interactions that may be relevant to heme-based sensors, such as soluble guanylate cyclase. The novel heme-NO chemistry of AXCP takes on further significance in the light of reports that cytochrome c' may protect bacteria against NO toxicity, either as an NO reductase, or else as a reversible NO-transporter. The PIs group will perform a detailed structure reactivity based dissection of the proximal and distal heme pockets using a combination of kinetics, spectroscopy, mutagenesis, high-resolution x-ray crystallography, and electrochemistry. Specific aims are to (i) understand the mechanism of proximal NO binding (ii) determine the role of the proximal and distal pockets on heme-NO release, and (iii) investigate heme redox reactivity.The broader impacts of this project include the exceptional opportunities for undergraduate students at Eastern Oregon University (EOU)--a small, public liberal arts college--to be engaged in cutting-edge research. In addition to carrying out protein purification and kinetic measurements at EOU, students will travel to collaborating laboratories to gain additional experience in protein characterization, as well as insights into graduate research environments. Overall, the project will support the thriving undergraduate research culture at EOU (exemplified by the in-house Eastern Oregon Science Journal) and will build on the previous successes of the PI in encouraging students to pursue science at the graduate level.

本项目将研究木氧碱性碱杆菌细胞色素c‘(AXCP)的新的血红素化学，特别关注本科生的参与。PI和他的同事之前已经报道了AXCP前所未有的反应活性，通过一氧化氮(NO)通过推测的二亚硝基中间体的方式与近端的血红素配位。这种可供选择的血红素-NO结合模式，同时涉及远端和近端的血红素表面，挑战了以前关于血红素蛋白如何与NO反应的假设，并提出了调节配基相互作用的新机制，这些机制可能与基于血红素的传感器有关，例如可溶性鸟苷环化酶。AXCP的新的血红素-NO化学具有进一步的意义，因为有报道称，细胞色素c‘可能作为NO还原酶或作为可逆的NO转运体保护细菌免受NO毒性。PIS小组将使用动力学、光谱、突变、高分辨率X射线结晶学和电化学相结合的方法，对近端和远端的血红素口袋进行基于结构反应性的详细解剖。具体目的是(I)了解近端NO结合的机制(Ii)确定近端和远端口袋对血红素-NO释放的作用，以及(Iii)调查血红素的氧化还原反应。该项目的更广泛的影响包括东俄勒冈大学(EOU)--一所小型的公立文理学院--的本科生从事前沿研究的特殊机会。除了在EOU进行蛋白质纯化和动力学测量外，学生们还将前往合作实验室，以获得更多蛋白质表征方面的经验，以及对研究生研究环境的洞察。总体而言，该项目将支持EOU蓬勃发展的本科生研究文化(以内部的《东俄勒冈科学杂志》为例)，并将建立在PI之前鼓励学生在研究生水平上追求科学的成功基础上。