Project Summary The goal of the proposed research is to elucidate fundamental physical-chemical principles that govern catalysis in enzymes, with a focus on radical catalysis in coenzyme B12 (adenosylcobalamin) –dependent enzymes. This goal will be enacted through a program of biochemical and physical studies of representatives of all three classes of B12 enzymes: methylmalonyl-CoA mutase (MCM, Class I; human and Methylobacter extorquens), ethanolamine ammonia- lyase (EAL, Class II; Salmonella typhimurium), and lysine-5,6-aminomutase (Class III; LAM, Clostridium stricklandii). The approaches will be extended to address molecular mechanism in the intracellular cobalamin (B12) trafficking pathway in humans by the CblC and CblD proteins, and to the assembly and function of the bacterial microcompartment (BMC), an in vivo context of the EAL enzyme. Innovative methods, sofware and hardware for high-resolution pulsed- electron paramagnetic resonance (EPR) spectroscopy, in parallel with time- resolved, single-step reaction kinetics and steady-state solvent dynamics from continuous-wave EPR methods, applied in unique, low-temperature systems, will enable comprehensive characterization of the structural and dynamical contributions of solvent-protein-reaction coupling to radical rearrangement catalysis in the B12 enzymes. Significant biomedical and human health outcomes include: (i) fundamental knowledge about the role of protein and coupled solvent configurational states in enzyme function, latent at physiological temperatures, that contribute new tools, models and targets to the developing roadmap for leveraging these states in drug development and enzyme engineering, (ii) characterization of EAL, a contributor to the role of the gut microbiome in human health and disease progression, including links with inflammatory bowel disease, obesity, and diabetes, (iii) characterization of the assembly and function of the ethanolamine utilization (eut) BMC, one of a recently-recognized, widespread class of primitive, protein-encased bacterial organelles, (iv) insights into the molecular mechanistic basis of human metabolic disorders identified with the cbl gene cluster, and in particular, the role of the human CblC (MMACHC, methylmalonic aciduria type C and homocystinuria) and CblD proteins.