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The Levinson Emerging Scholars Program
Margaux Pinney - Biochemistry, Chemistry
Margaux is a senior in Biochemistry (BS), Chemistry (BS) and Mathematics (minor) with Departmental Honors in Chemistry and Biochemistry. After transferring to UW from Green River Community College, Margaux began doing research in the lab of Professor Jim Mayer. In the Mayer Group, she investigated the potential for proton-coupled electron transfer at synthetic iron-sulfer clusters, before moving on to her current, independent project. She now investigates the reversibility of Compound I formation in horseradish peroxidase. Compound I is also an intermediate in the catalytic cycle of cytochrome P450s. Since approximately 70% of xenobiotics, such as drugs or chemicals from the environment that enter the body, are oxidized by P450s, this research has pharmaceutical and toxicological implications. This research could also be applied to synthetic processes, due to the unique mechanism of oxidation. Based on her experience with undergraduate research at the University of Washington, Margaux will be pursuing her PhD. in biochemistry after graduation. Margaux is also involved with several groups on campus, such as the Tolo Chapter of Moratar Board, a senior honors society, as well as the Undergraduate Research Leaders, and is particularly interested in getting students, particularly women, involved in undergraduate research.
Mentor: Jim Mayer, Chemistry
Project Title: Reversibility of Compound I Formation by Horseradish Peroxidase
Abstract: Horseradish peroxidase (HRP) is part of a family of heme-containing metalloenzymes that reduce the O-O bond of peroxides while oxidizing a wide variety of substrates; in the case of HRP, hydrogen peroxide is reduced to water. In the presence of an oxidant and the absence of a substrate to oxidize, hydrogen peroxide coordinates to the iron cofactor of HRP, forming a ferric-hydroperoxide intermediate known as Compound 0, and subsequent O-O bond cleavage forms water and a very oxidizing ferryl-oxo porphyrin radical cation intermediate, known as Compound I. Compound I is of particular interest because it is also formed by cytochrome P450s, a family of enzymes that react with almost all foreign substances entering the body, including drugs and other chemicals in the environment. HRP is an excellent enzyme to test this reversibility because, unlike P450s, it is extremely robust and easy to work with. The goal of this project is to probe the potential reversibility of Compound I formation, a step traditionally thought to be irreversible. A key experiment will react HRP with excess unlabeled H2O2 in 18O-labeled water, and determine whether mixed-labeled H2O2 is created, which indicates that the step that forms Compound I is reversible. The enzyme will be removed by protein filtration and the existence of mixed-labeled H2O2 will be probed by adding a water soluble phosphine to the filtrate, which will readily react with excess H2O2 and be oxidized. 31P NMR spectroscopy will be used to determine whether the phosphine oxide is labeled because the NMR shifts of 16O-phosphine oxide and 18O-phosphine oxide are distinct. If this reaction proves to be reversible, this knowledge could then be used to describe cytochrome P450s. This research would be immensely important to pharmaceutical and toxicological research, as well as synthetic processes.