Spectroscopic Studies of Molybdoenzymes & Models

There exist fundamental gaps in the knowledge base regarding the mechanism of Mo cofactor sulfurase C- terminal (MOSC) domain enzymes and how they differ from sulfite oxidase (SO) family enzymes, how DMSO reductase (DMSOR) family enzyme geometric structure contributes to the wide range of complex chemical transformations catalyzed, and how the electronic structure of pyranopterin dithiolene (PDT) component of the Mo cofactor (Moco) modulates Mo-PDT covalency and contributes to catalysis in all pyranopterin Mo enzymes. Our long-term goal is to understand how molybdoenzyme geometric and electronic structure contribute to reactivity and function to provide a positive impact on the quality of human health. Our overall objective is to determine the mechanism of molybdoenzymes critical to human health and how the PDT contributes to catalysis by employing a combined spectroscopic approach augmented by detailed bonding, spectroscopic, and reaction coordinate computations. The central hypothesis is that specific geometric and electronic structure modifications of protein-bound Moco define the unique enzymatic reactions catalyzed. The rationale for this research is that a comprehensive understanding of mARC mediated transformations, the role of DMSOR family enzymes in supporting virulence and the transformation of small molecules into metabolites, and the complex interplay between Mo and the PDT ligand in all molybdoenzymes will provide new insights into disease states and have a positive impact on human health. We will test our central hypothesis in order to accomplish the objectives of this proposal through the successful pursuit of three Specific Aims 1) Understand mARC and related MOSC family enzymes, 2) Determine active site contributions to catalysis in DMSOR family enzymes, 3) Determine PDT electronic structure contributions to reactivity. The proposed research is innovative in its approach because it 1) utilizes a combination of spectroscopy, electronic structure computations, and kinetic data to interrogate proteins that lack additional redox chromophores, coupled with variants and models that have not been studied in detail, to derive insight into mechanism and protein scaffold constraints on geometry, 2) directly addresses prior challenges related to spectroscopically probing the PDT using new small molecule analogs that possess a PDT ligand and 3) develops S K-edge HERFD X-ray emission (HERFD-XES) as a probe of S→Mo charge transfer states. This has led to new insight into long-standing questions in the molybdoenzyme field, effectively opening new horizons for future work in this area. The proposed research is significant since it will reveal how active site electronic and geometric structure control reactivity related to the role of molybdoenzymes in disease states, drug metabolism, and gut microbial metabolism.