Structural Characterization of Highly Reactive Heme Enzyme Intermediates

Marquette University, Milwaukee WI

Investigators

Abstract

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.