This dissertation describes the preparation, characterization, and application of artificial metalloenzymes for the selective functionalization of organic molecules. These catalysts combine a catalytic metal complex and a protein scaffold, allowing one to exploit the reactivity of the transition metal catalyst while taking advantage of the selectivity and evolvability of proteins. The work described herein is specifically focused on the application of Pyrococcus furiosus prolyl oligopeptidase as an enabling protein scaffold for the generation of exceptionally robust and selective artificial metalloenzymes.,Chapter One introduces the concept of catalyst control of selectivity and the challenges surrounding it, particularly in the context of C-H functionalization. Site-selective C-H-functionalization, the process whereby a specific C-H bond is converted to a desired functional group, remains a challenging problem for small molecule catalysis. An overview of methods to achieve site-selective catalysis is described, encompassing strategies employing small-molecule transition metal catalysis and enzyme catalysis. A third class of catalyst, artificial metalloenzymes, is introduced as potentially combining the advantages of small molecule catalysts (reactivity) with enzymes (evolvability/selectivity). The many methods that have been employed to generate artificial metalloenzymes is then discussed in detail, with a particular focus on challenges in the characterization of these catalysts.,Chapter Two describes the formation of a wide variety of artificial metalloenzymes formed using serine hydrolase scaffolds. Electrophilic phosphonate-tethered metal catalysts have been covalently bioconjugated to serine hydrolases, exploiting the native mechanism of this class of enzymes. The synthetic procedures to generate such metal cofactors are described. A panel of serine hydrolase scaffolds is explored using these catalysts. A number of the resulting artificial metalloenzymes have been applied successfully for C-H oxygenation, epoxidation, and cyclopropanation, albeit with limited selectivity. Strategies to enhance these selectivities are also elaborated in this chapter.,Chapter Three introduces the application of Pyrococcus furiosus prolyl oligopeptidase as an ideal protein scaffold for artificial metalloenzyme formation. Methodology for the expression of this scaffold has been optimized, and the stability of the scaffold has been investigated. Methods for the routine characterization of artificial metalloenzymes using this scaffold are described, including native activity assays, mass spectrometry, and LC/MS/MS. Finally, the Pyrococcus furiosus prolyl oligopeptidase crystal structure has been solved and a detailed description of its structural features is presented. A number of discoveries pertinent to the native activity of the enzyme and its application as an artificial metalloenzyme scaffold are elaborated.,Chapter Four describes the formation of dirhodium artificial metalloenzymes using Pyrococcus furiosus prolyl oligopeptidase. Enantioselective cyclopropanation has been catalyzed with these systems and catalyst performance has been optimized through rational mutagenesis and directed evolution. Biophysical studies into the origin of the observed selectivity were conducted, with a hypothetical histidine-rhodium interactions at the center of investigation. Finally, a detailed digestion LC/MS/MS study into artificial metalloenzyme self-modification with diazo- substrates is described. This revealed that self-modification occurs during catalysis, potentially impacting the selectivity of the catalyzed reaction. The methods described lay the groundwork for future detailed studies.