RUI: Novel Heme Chemistry of Cytochrome c'

本项目将研究Alcaligenes xylosoxidans细胞色素c' （AXCP）的新型血红素化学，并特别关注本科生的参与。PI及其同事先前报道了AXCP的前所未有的反应性，其中一氧化氮（NO）通过假定的二硝基中间体与近端血红素面协调。这种可选择的血红素-一氧化氮结合模式，涉及远端和近端血红素面，挑战了以前关于血红素蛋白如何与一氧化氮反应的假设，并提出了调节配体相互作用的新机制，可能与血红素传感器有关，如可溶性鸟苷酸环化酶。鉴于细胞色素c'可以作为NO还原酶或作为可逆NO转运体保护细菌免受NO毒性的报道，AXCP的新血红素-NO化学具有进一步的意义。pi小组将使用动力学、光谱学、诱变、高分辨率x射线晶体学和电化学相结合的方法，对近端和远端血红素袋进行详细的结构反应性解剖。具体目标是：(i)了解近端NO结合机制；（ii）确定近端和远端口袋对血红素-NO释放的作用；（iii）研究血红素氧化还原反应性。这个项目更广泛的影响包括为东俄勒冈大学（EOU）的本科生提供特殊的机会，这是一所小型的公立文理学院，从事前沿研究。除了在EOU进行蛋白质纯化和动力学测量外，学生还将前往合作实验室获得蛋白质表征方面的额外经验，以及对研究生研究环境的见解。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），并将在PI之前的成功基础上鼓励学生在研究生阶段追求科学。

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.