Synthetic and Spectroscopic Investigations of Sulfur Oxidation Reactions by Nonheme Iron Enzymes - Project Summary In addition to its role as an amino acid, L-cysteine (Cys) is the precursor of numerous sulfur-containing biomolecules that function as metabolites, micronutrients, and pharmaceuticals. The formation and degradation of these compounds is often performed by mononuclear nonheme iron (MNHI) enzymes capable of harnessing the oxidative power of molecular oxygen (O2). The proposed research aims to advance our understanding of biological O2-activation by developing synthetic models of three types of enzymes that oxidize Cys-derived compounds: (i) sulfoxide synthases, (ii) ergothioneine dioxygenase (ETDO), and (iii) persulfide dioxygenase (PDO, also known as ETHE1). Sulfoxide synthases, found in some bacteria and fungi, catalyze the oxidative formation of a C-S bond in the biosynthesis of S-containing natural products with therapeutic and antioxidative properties. Two MNHI sulfoxide synthases have been reported to date, EgtB and OvoA, which perform key steps in the biosyntheses of ergothioneine (Egt) and ovothiol, respectively. In some bacteria, the degradation of Egt is initiated by a MNHI enzyme (ETDO) that catalyzes the S-dioxygenation of the thione moiety. ETDO is closely related to the better-known family of MNHI enzymes known as thiol dioxygenases. Finally, persulfide dioxygenase is required for mitochondrial degradation of the gaseous signaling agent H2S, and loss of function is responsible for the human disease ethylmalonic encephalopathy. For all three classes of enzymes, major questions remain regarding the nature of the catalytic mechanisms and the role of active- site pocket in controlling the outcomes of O2-dependent reactions. The research described in this application utilizes a bio-inspired approach that elucidates nonheme iron catalysis through the development of structural and functional active-site models, the isolation and characterization of reactive intermediates, and spectroscopic studies of synthetic and enzymatic species. The specific aims of the proposed research are to: (i) gain insights into sulfoxide synthase activity through the synthesis and characterization iron complexes that mimic the active-site structures and reactivity of EgtB and OvoA, (ii) use synthetic models of PDO and ETDO to explore the O2 reaction mechanisms of enzymes that catalyze the S-dioxygenation of non-thiol substrates, and (iii) probe the geometric and electronic structures of OvoA through parallel spectroscopic studies of enzymatic and synthetic species. These interdisciplinary studies will be carried out by a team of undergraduate students at Marquette University, who will be supervised, mentored, and assisted by the PI and graduate students. All students involved in the project will receive exceptional training in inorganic synthesis, crystallography, spectroscopic techniques, theoretical methods, and the preparation of enzyme samples.
