EAPSI: Modifying Proteins to Investigate Key Intermediates in Oxygen Binding Enzymes

Wolf Matthew, Ann Arbor MI

Investigators

Abstract

Iron-containing heme proteins catalyze a wide variety of reactions in biology, including binding and activating oxygen towards reacting with many molecules in the body. These reactions are important for respiration, metabolism of drugs and toxins, and breaking down hydrocarbons, among other functions. Although there has been much research focused on trying to understand these reactions, little is still known about the reactive "iron-oxo" intermediates that are crucial in these mechanisms. This project aims to investigate these reactive intermediates by modifying of the metal center and the protein scaffold of the stable heme protein myoglobin and binding oxygen species to the metal center. This research will be conducted in collaboration with Dr. Takashi Matsuo at the Nara Institute of Science and Technology (NAIST). Dr. Matsuo's lab has years of experience in protein modification and studying reactive oxygen intermediates, and the expertise of his lab will be invaluable to the project. By expressing the myoglobin mutants H64A and H64D, the active site pocket of myoglobin will be opened up and a potential hydrogen bonding interaction with the oxo or peroxo species can also be introduced. In addition to the active site mutations, the native heme in myoglobin will be removed and ruthenium, rhodium, and iridium porphyrins will be incorporated into the protein. Oxo and peroxo transfer reagents such as hydrogen peroxide and meta-chloroperoxybenzoic acid will be added to form high-valent metal-oxo and peroxo species. The larger transition metals may stabilize the bound oxo and peroxo species, and allow these species to be studied by a variety of spectroscopic methods. Finally, the reactivity of the oxo and peroxo species will be studied through the addition of appropriate substrates and measuring the total turnover numer and the rate of substrate oxidation or oxygenation over time. This NSF EAPSI award is funded in collaboration with the Japan Society for the Promotion of Science.