PROJECT SUMMARY Enzymatic reactions comprise the basic building blocks essential to the biochemical processes of cellular metabolism. While most enzymatic reactions are reversible, certain enzymes accelerate a reaction in one direction significantly differently than the reverse direction. The mechanistic determinants of this phenomenon – often referred to as “catalytic bias” – are generally unknown. Our long-term goal of this project is to identify the fundamental principles adopted by redox enzymes (enzymes involved in oxidation-reduction reactions) that provide molecular level control of the catalytic bias. In doing so, we will provide a greater fundamental understanding of the factors that control metabolic processes in all life. Specifically, the objective of this proposal is to delineate determinants that influence the directional reactivity of model Fe containing hydrogenases that are structurally related but exhibit large differences in catalytic bias for the specific reversible hydrogen (H2) oxidation reaction from electron and protons. The central hypothesis is that catalytic bias commonly in redox enzymes results from the relative differential stabilization and destabilization of oxidation states of the respective redox cofactors critical to determining the rate limiting step of the catalytic cycle. The rationale underlying the proposal is that the implementation will result in defining mechanistic determinants of the proposed model system. The proposed work will culminate in elucidating universal, mechanistic factors of catalytic bias that govern a large number of, if not all, redox enzymes. The central hypothesis will be tested by pursuing three specific aims that examine how 1) the reduction potential of the active site proximal accessory redox clusters; 2) the characteristics of the active site cluster protein environment; and 3) the secondary coordination sphere structural dynamics can control reactivity and catalytic bias in cofactor-based oxidation reduction catalysis. To pursue these aims, we will employ an innovative strategy integrating biochemical, structural, spectroscopic, and computational approaches on a unique, one-of-a-kind model hydrogenase platform. The proposed research is significant, because it will delineate molecular determinants that influence the fine-tuning of redox cofactors through the relative stabilization or destabilization of oxidation states to afford certain reactivity fundamental to directing energy and matter in cells. The work will provide additional foundational resources in the form of a blueprint for integrating multiple biophysical and computational approaches.