The molybdenum cofactor (Moco) is an essential biomolecule for all forms of life. Numerous enzymes rely on the proper functioning of Moco for normal physiological functions. Failures to biosynthesize Moco cause severe physiological distress and even childhood death. Outside the protein environment, this cofactor is unstable and has not yet been chemically synthesized. The Moco has several redox active components, and together, these components can carry out up to ten-electron chemistry, while in most cases, the substrate transformation requires only two electrons. To understand how different components stabilize the electronic structure that supports reactivity, we will investigate discrete molecules with defined features. The work outlined in this proposal seeks to complete the synthesis of the closest chemical mimic of Moco. While in the reduced state, Moco is unstable outside the protein environment, and how it is stabilized within a protein is not clear. The proposal also seeks to understand the geometric and electronic properties of Moco in the DMSO reductase family of enzymes using periplasmic nitrate reductase (NapA) and its variants as a model through biochemical and biophysical studies. The specific aims of the proposal are: 1) to synthesize pterin Mo-centers and Mo=X (X=S, O) complexes allowing investigation of their geometric and electronic structures in relation to reactivity; 2) to understand the geometric, electronic, and functional properties of the Mo-center in the DMSOR family using NapA as a model. Overall, new important molecular-level knowledge will be generated about the structure-function of a class of enzymes important to human health.