Metalloproteins catalyze some of Nature’s most amazing and difficult chemical transformations. One such transformation, of interest to our laboratory, involves the use of a Rieske Oxygenase to facilitate the functionalization of a traditionally inert C-H bond. This chemistry permits microorganisms to catabolize pollutants that are deposited into the environment and is also important to the biosynthetic pathways of natural products that showcase industrial and medicinal properties. However, despite the value of known Rieske Oxygenase transformations, exploiting an enzyme in an industrial application often requires substantial engineering and an extensive understanding of how protein structure relates to its function. Therefore, in this presentation, I will describe our recent endeavors to improve the practical applicability of Rieske Oxygenases using biochemical and biophysical methods to molecularly detail the structure–function relationships in these remarkable catalysts.