Mechanisms of Iron-Containing Enzymes

Project Summary/Abstract Enzymes that utilize iron-containing cofactors catalyze many different, often very difficult, chemical reactions that are fundamentally important to central life processes, such as DNA biosynthesis and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of numerous compounds with antibacterial and antifungal activities. Dysfunction of these enzymes is often associated with the onset of severe diseases, e.g. cancer, cardiovascular diseases, and diabetes. Strategies to harness the synthetic potential of these enzymes and to treat diseases associated with their dysfunction involves the rational manipulation of these processes on a molecular level, which requires a detailed knowledge of the reaction mechanisms, in particular how the enzymes control the outcome of their reactions. The Bollinger/Krebs joint group specializes in combining transient-state rapid kinetic experiments with various spectroscopic (e.g. stopped-flow absorption, freeze-quench EPR and Mössbauer) and analytical (LC/MS) methods to monitor metalloenzyme reactions. In the last 20 years, our group has successfully studied many mononuclear (MNHI) and dinuclear non-heme-iron (DNHI) enzymes by trapping and characterizing key reaction intermediates in their catalytic cycles. We trapped and characterized high-spin Fe(IV)-oxo (ferryl) intermediates in various MNHI enzymes. The ferryl intermediate initiates substrate oxidation, typically by cleavage of an aliphatic C-H bond. The outcome of these reactions is diverse and includes hydroxylation (the default outcome), halogenation, desaturation, epimerization, heterocyclization, and endoperoxidation reactions. The current focus of our research aims at deciphering the factors that result in the diverse outcomes. The long-term goal of this research is to lay the foundation for the rational manipulation of these enzymes for biotechnological applications. In collaboration with the Boal group, the B/K group has identified the heme-oxygenase diiron oxygenase (HDO) enzyme superfamily and currently studies several HDOs. The PI also has a long-standing collaboration with the Booker group on mechanistic studies of radical S- adenosylmethionine (RS) enzymes that catalyze formation of new C-S bonds. These enzymes use their [Fe4S4]RS cluster to generate the canonical 5’-deoxy-adenos-5’-yl radical that initiates substrate oxidation by cleavage of aliphatic C-H bonds and their auxiliary [Fe4S4] clusters as sulfur source. The current focus of the collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of various RS enzymes involved in formation of new C-S bonds.