Structural Characterization of Highly Reactive Heme Enzyme Intermediates

智力价值：一类重要的蛋白质，存在于动植物界，在活性部位有一个所谓的血红素基团，其结构和固有的化学反应在很大程度上决定了它们的关键功能特性，包括通过血红蛋白和肌红蛋白运输和储存氧气，抗菌活性和清除活生物体中的污染物。这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种。尽管这样的过程对维持生命非常重要，但这些与氧气和衍生物质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，以至于出现并实际控制反应的短暂中间体很难捕获和表征。这项研究项目采用了两种强有力的方法的有效组合，试图通过确定被困中间体的关键部分的结构来澄清许多这些悬而未决的问题。首先，血红素酶与氧气反应，在非常低的温度下非常迅速地冷冻，以阻止进一步的反应。然后，它被暴露在高能光线下，也就是所谓的伽马射线，这种光线会在冻结的样本中产生电子。这些电子可以迁移到氧与血红素铁结合的位置，以还原结合的氧气，并生成这些通常无法捕获的高活性物种；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，利用了对血红素活性中心结构非常敏感的拉曼光谱。这一总体方法被用来描述这些关键但难以捉摸的物种对几种重要类型的血红素蛋白的活性部位结构，包括肌红蛋白、过氧化物酶和细胞色素P450。广泛的影响：由于血红素蛋白涉及动植物生活的许多方面，包括对农业和环境科学重要的过程，因此更好地了解这些蛋白如何实际发挥其功能具有广泛的一般性和根本性的重要性。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；马奎特大学拥有蓬勃发展的本科课程，强烈鼓励化学专业的学生正式参与，毕业前有可能获得最多6个学分。此外，通过与当地高中和另一所当地大学MSOE合作开展的所谓SMART团队计划，与当地高中生的联系和交流现在变得更加有效。该项目由分子和细胞生物科学部的分子系统集群和化学部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.