RUI: Novel Heme Chemistry of Cytochrome c'

本计画将探讨木糖氧化产碱杆菌细胞色素c'（AXCP）的新颖血红素化学，特别著重于大学生的参与。PI和同事先前报道了AXCP的前所未有的反应性，其中一氧化氮（NO）通过推定的二亚硝酰基中间体与近端血红素面配位。这种替代血红素-NO结合模式，涉及远端和近端血红素面，是挑战以前的假设血红素蛋白如何与NO反应，并提出了新的机制，用于调节配体相互作用，可能是相关的血红素为基础的传感器，如可溶性鸟苷酸环化酶。AXCP的新血红素-NO化学在以下报道中具有进一步的意义：细胞色素c'可以作为NO还原酶或可逆的NO转运蛋白保护细菌免受NO毒性。PI组将结合动力学、光谱学、诱变、高分辨率X射线晶体学和电化学，对近端和远端血红素囊进行详细的基于结构反应性的解剖。具体目标是（一）了解近端NO结合的机制（二）确定血红素-NO释放的近端和远端口袋的作用，以及（三）调查血红素氧化还原reactivity.The更广泛的影响，这个项目包括在东俄勒冈州大学（EOU）-一个小的，公共文科学院-本科生的特殊机会，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，以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蓬勃发展的本科研究文化（以内部的东俄勒冈州科学杂志为例），并将建立在PI以前的成功基础上，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.