RUI: Novel Heme Chemistry of Cytochrome c'

在许多对生命至关重要的过程中(如呼吸、气体传感/信号和毒性防御)，血红素蛋白必须与特定的双原子气体结合，例如氧气(O2)、一氧化碳(CO)或一氧化氮(NO)。为了调节血红素的反应性(并实现选择性的气体响应)，蛋白质利用了血红素口袋环境的结构和化学性质。该项目的一个主要目标是调查血红素铁站点的空间环境如何显著影响它们与气体的反应性。这一知识对于理解动物、植物和细菌中的基本生化反应以及合理设计具有独特性能的血红素气体传感器非常重要。本科生研究是该项目不可或缺的一部分，为学生和当地高中教师提供激励经验和未来科学努力的动力。该项目将专注于细菌细胞色素c‘的新的血红素活性，其中空间限制显著不利于O2、CO和NO的远端配位，同时促进不寻常的远端至近端的血红素-NO转化。NO在血红素两面(远端和近端)的配位代表了一种新型的血红素蛋白活性，该活性可能与细胞色素c‘的NO结合功能(S)有关，也可能与气敏蛋白中的NO选择性反应有关。通过鉴定木氧碱杆菌细胞色素c‘(AXCP)的末端口袋变异体以及其他细胞色素c’，总的目标是确定末端空间限制、结构扰动和血红素活性之间的联系，包括末端与近端的无配位。将检验“负载弹簧”假设，即拥挤的末端口袋和双原子气体(XO)配体之间的空间碰撞导致结构扰动的高能血红素-XO络合物的反应性改变。该项目将利用光谱学(紫外-可见吸收、共振拉曼和电子顺磁共振)、动力学(停流和闪光光解)和其他技术(X射线结晶学和电位法)来研究野生型和变异型细胞色素c的反应性随远端空间体积和气体特性的变化。本科生和高中教师将接受这些技巧的培训，并参与解释结果。

In many processes vital for life (e.g. respiration, gas sensing/signaling, and toxicity defense) heme proteins must bind a specific diatomic gas, e.g. oxygen (O2), carbon monoxide (CO), or nitric oxide (NO). To modulate heme reactivity (and achieve a selective gas response), proteins utilize the structural and chemical properties of the heme pocket environments. A major goal of this project is to investigate how the spatial environments of heme iron sites can dramatically affect their reactivity with gases. This knowledge is important for understanding fundamental biochemical reactions in animals, plants, and bacteria, as well as for the rational design of heme-gas sensors with unique properties. Undergraduate research is an integral part of the project, providing students and local high-school teachers with stimulating experiences and motivation for future scientific endeavors.The project will focus on the novel heme reactivity of bacterial cytochromes c' in which steric constraints significantly disfavor the distal coordination of O2, CO, and NO, while promoting an unusual distal to proximal heme-NO conversion. The coordination of NO at both heme faces (distal and proximal) represents a new type of heme protein reactivity that may be relevant to the proposed NO-binding function(s) of cytochromes c', as well as to NO-selective responses in gas-sensing proteins. By characterizing distal pocket variants of Alcaligenes xylosoxidans cytochrome c' (AXCP), as well as other cytochromes c', the overall goal is to define the connection between distal steric constraints, structural perturbations, and heme reactivity, including distal vs proximal NO coordination. A "loaded spring" hypothesis will be tested in which steric clashes between the crowded distal pocket and diatomic gas (XO) ligands lead to structurally perturbed high-energy heme-XO complexes with altered reactivity. The project will utilize spectroscopy (UV-visible absorption, resonance Raman, and Electron Paramagnetic Resonance), kinetics (stopped-flow and flash photolysis) and other techniques (X-ray crystallography and potentiometry) to study wild-type and variant cytochrome c' reactivity as function of distal steric bulk and gas identity. Undergraduates and high school teachers will receive training in these techniques and be involved in interpreting the results.