Structural Characterization of Highly Reactive Heme Enzyme Intermediates

知识价值：一类重要的蛋白质，存在于植物和动物王国中，在活性部位具有所谓的血红素基团，其结构和固有的化学反应性在很大程度上决定了其关键功能特性，包括通过血红蛋白和肌红蛋白运输和储存氧气，抗菌活性和清除生物体中的污染物。最后一种功能是由一组称为细胞色素P450的酶促进的。某些血红素酶，如氢过氧化物酶，负责从生命系统中消除危险的氧化物质。尽管这些过程对维持生命非常重要，但这些与氧和衍生物种的关键反应的许多细节仍然不清楚，因为反应发生得如此之快，以至于难以捕获和表征出现并实际控制反应的短暂中间体。该研究项目采用了两种强大的方法的有效组合，试图通过确定被困中间体的关键部分的结构来澄清许多未解决的问题。首先，血红素酶与氧气反应，并在非常低的温度下迅速冷冻以停止进一步的反应。然后将其暴露于高能光，即所谓的伽马射线，其导致冷冻样品中产生电子。这些电子可以迁移到氧与血红素铁结合的位置，以减少结合的双氧并产生这些通常不能被捕获的高活性物质;低温限制了进一步的反应，允许进一步研究活性中间体。在这项工作中，拉曼光谱，这是非常敏感的血红素活性部位的结构，被利用。这种整体的方法是用来表征这些关键的，但难以捉摸的物种的几个重要类型的血红素蛋白，包括肌红蛋白，过氧化物酶和细胞色素P450s.Broader影响的活性位点结构：由于血红素蛋白参与了这么多方面的植物和动物的生活，包括重要的农业和环境科学的过程，获得更好地了解这些蛋白质如何实际上执行其功能是广泛的一般和根本的重要性。显然，该项目将为研究生，本科生和高中生提供非常有效的培训机会;马奎特大学有一个蓬勃发展的本科课程，强烈鼓励化学专业的学生正式参与，并有可能获得高达6个学分的毕业。此外，通过与当地高中和MSOE（另一所当地大学）合作开展的SMART Team计划，与当地高中学生的联系和交流更加有效。该项目由分子和细胞生物科学部门的分子系统集群和化学部门的生命过程化学共同支持。

Intellectual Merit:An important class of proteins, occurring throughout both the plant and animal kingdoms, possess a so-called heme group at the active sites whose structure and inherent chemical reactivity are largely responsible for their critical functional properties, including the transport and storage of oxygen by hemoglobin and myoglobin, anti-bacterial activity and the clearing of pollutants from living organisms. This last function being facilitated by a group of enzymes known as cytochromes P450. Certain heme enzymes, such as the hydroperoxidases, are responsible for elimination of dangerous oxidizing species from living systems. Despite the great importance of such processes to maintenance of life, many of the details of these critical reactions with oxygen and derived species still remain unclear, because the reactions occur so fast that the fleeting intermediates that arise and actually control the reaction are difficult to trap and characterize. This research project employs an effective combination of two powerful methods to attempt to clarify many of these unresolved issues by determining the structure of key parts of the trapped intermediates. First, the heme enzyme is reacted with oxygen and very quickly frozen at very low temperatures to stop further reaction. It is then exposed to high energy light, so called gamma rays, which cause electrons to be generated in the frozen sample. These electrons can migrate to the site where the oxygen is bound to the heme iron to reduce the bound dioxygen and generate these highly reactive species that normally can¡¦t be trapped; the low temperatures restrict further reactions, allowing the reactive intermediates to be further studied. In this work Raman spectroscopy, which is exquisitely sensitive to the heme active site structure, is exploited. This overall approach is used to characterize the active site structure of these crucial, but elusive species for several important types of heme proteins, including myoglobins, peroxidases and the cytochrome P450s.Broader Impacts:Inasmuch as heme proteins are involved in so many aspects of plant and animal life, including processes important to agricultural and environmental sciences, gaining a better understanding of how these proteins actually perform their function is of wide general and fundamental importance. Obviously, this project will present very effective training opportunities for graduate, undergraduate and High School students; Marquette University has a thriving undergraduate program and Chemistry Majors are strongly encouraged to participate formally, with the possibility of earning up to 6 credit hours towards graduation. Also, connections and communications with local high school students is now made more efficient through a so-called SMART Team program being conducted in cooperation with local high schools and MSOE, another local college.This project is jointly supported by Molecular Systems cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes in the Division of Chemistry.