Mechanistic Studies of Catalytic Multifunctional Globins Using Advanced Structural Methods

该奖项将资助北卡罗来纳州立大学的Reza GHiladi博士和Stefan Franzen博士研究海洋环境中的含铁蛋白质，特别是血红素-铁蛋白，以此作为了解它们在哺乳动物系统中功能的模型。血红素-铁蛋白在自然界中普遍存在，它们发挥着重要的生物学作用，几乎是所有生命系统的基础。血红素-铁蛋白的显著例子包括血液中的氧气载体、血红蛋白和在肝脏中发现的称为细胞色素P450的解毒酶。虽然血红素蛋白的重要性已经被化学和生物界认识到多年，但应用最新的新兴技术来研究它们的功能是具有挑战性的。使用相关但更简单的海洋血红蛋白为开发这些方法提供了一条途径，同时也更好地了解了血红素蛋白在生物学和化学中的不同作用。拟议的实验程序有助于在原子水平上表征分子如何与这些重要的蛋白质结合并改变它们的功能。这使得研究生可以在高分辨率X射线和中子结晶学以及时间分辨结构方法方面获得专门的培训。快速时间刻度方法是尖端技术，它突破了研究酶如何在生命系统中执行任务的实验可获得的极限。该项目与“造星”推广计划相结合，为农村中学科学课堂提供参观、演示、讲座和涵盖化学/生活界面的课程计划。该项目还邀请国内(橡树岭国家实验室)和国际(埃塞克斯大学和波兰科学院)合作伙伴进行数据收集和分析。该研究项目旨在确定底物与脱卤素过氧化物酶结合时发生的一组独特的结构相互作用，从而控制血红素中间体的动力学和反应活性，并最终区分结合底物特有的酶活性。采用快速冷冻-猝灭、笼式O2载体光解和CO闪光光解技术，以捕获中间体，并启动时间分辨研究，以前所未有的细节表征这些与催化周转相关的机械上重要的中间体。脱氢过氧化物酶与其他血红蛋白、过氧化物酶和过氧化物酶的关系建立了与多功能蛋白质相关的蛋白质结构-功能关系的新范式，补充了已建立的单功能酶的结构-功能关系。这项研究的信息有可能促进对催化球蛋白的理解，确定导致血红素蛋白质(以及更广泛的金属酶)之间活性差异的结构特征，并能够比较海洋来源的酶与陆地来源或细菌来源的酶的结构-功能相关性。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Reza Ghiladi and Stefan Franzen from North Carolina State University to investigate iron containing proteins, specifically heme-iron proteins, from marine environments as models for understanding their function in mammalian systems. Heme-iron proteins are found universally throughout nature, and they perform important biological roles that are the foundation for nearly all living systems. Notable examples of heme-iron proteins include the oxygen carrier in blood, hemoglobin, and the detoxifying enzymes known as cytochromes P450 found in the liver. While the importance of heme proteins has been recognized by the chemical and biological communities for many years, it is challenging to apply the latest emerging techniques to study their function. The use of related but simpler marine hemoglobin provides an avenue to develop these methods while also gaining a better understanding the diverse roles of heme proteins in biology and chemistry. The proposed experimental procedures help characterize at the atomic level how molecules will bind to these important proteins and alter their function. This pursuit allows graduate students to acquire specialized training in both high resolution X-ray and neutron crystallography, as well as in time-resolved structural methods. The fast timescale methods are cutting-edge techniques that push the limits of what is experimentally obtainable for studying how enzymes perform their tasks within living systems. This project is integrated with the "Creating STARS" outreach program to provide rural middle school science classrooms with visits, demonstrations, lectures, and lessons plans covering the chemistry/life interface. The project is also engaging both domestic (Oak Ridge National Laboratory) and international (University of Essex and the Polish Academy of Science) partners in data acquisition and analysis.This research project seeks to identify a unique set of structural interactions that occur upon substrate binding to the enzyme dehaloperoxidase, thereby controlling the kinetics and reactivity of the heme intermediates, and ultimately a discrimination in enzymatic activity specific to the bound substrate. Rapid-freeze-quench, caged O2-carrier photolysis, and CO flash photolysis techniques are to be conducted to enable the trapping of intermediates and to initiate time-resolved studies to characterize in unprecedented detail these mechanistically important intermediates relevant to catalytic turnover. The relationship of dehaloperoxidase to other hemoglobins, peroxidases and peroxygenases establishes new paradigms of protein structure-function relationships relevant to multifunctional proteins, complementing those established for monofunctional enzymes. Information from this study has the potential to advance the understanding of catalytic globins, determine the structural features that lead to activity differentiation across heme proteins (and across metalloenzymes more generally), and enable the comparison of the structure-function correlation in enzymes of marine origin in relation to those from terrestrial or bacterial sources.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.