This dissertation describes the design, construction, and directed evolution of artificial metalloenzymes. Transition metal catalysts and enzymes possess unique and often complementary properties that have made them important tools for chemical synthesis. In hopes of combining the selectivity and adaptability of enzymes with the reactivity of transition metal catalysts, researchers have explored artificial metalloenzymes (ArMs) in which secondary coordination sphere effects impart selectivity to metal catalysts, accelerate chemical reactions, and are systematically optimized via directed evolution. Our ultimate goal is to achieve non-directed and selective C-H functionalization. Chapter one describes unnatural amino acid (UAAs) synthesis. UAA incorporation into protein scaffolds could provide a facile method to construct ArMs and metallopeptides. However, neither the synthesis nor the incorporation of catalytically active UAAs have been reported. We developed a general approach for the synthesis of UAAs using 9-borabicyclononanyl group which enables simultaneous protection of both the amine and carboxylic acid functionalities. Both bidentate and tridentate Pd complexes were synthesized and confirmed by HRMS-ESI and NMR characterization. These UAAs catalyzed imidate rearrangements, but peptides containing these UAAs did not impart enantioselectivity to this reaction, and we were unable to achieve incorporation of the UAAs into proteins using codon suppression methods. We next pursued incorporation of UAAs with catalytically active organic side chains. Inspired by secondary amine catalyzed reactions, we designed and prepared two UAAs, prolyl-lysine and imidazolidinone-lysine, with strong resemblance to pyrrolysine. Both UAAs catalyzed aldol and Michael reactions under physiological conditions. This work was conducted in collaboration with Dr. Zhihui Zhang and Hao Yang, and was published in Organometallics, 2012, 31, 7328-7331. Chapter two describes the construction of Mn terpyridine ArMs for oxygenation. Mn terpyridine is a robust catalyst for benzylic C-H oxygenation and olefin epoxidation. We designed and synthesized a maleimide-substituted Mn-terpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization. This work was conducted in collaboration with Dr. Poonam Srivastava and Ken Ellis-Guardiola, and was published in Tetrahedron, 2014, 70, 4245-4249. Chapter three describes the construction and directed evolution of dirhodium ArMs. We first demonstrated that strain-promoted azide–alkyne cycloaddition could be used for rapid and general ArM formation. This approach was used to incorporate an alkyne-substituted dirhodium complex into a prolyl oligopeptidase (POP), and the resulting ArM catalyzed carbenoid N-H insertion with low enantioselectivity. Scaffold mutagenesis was then employed to improve the enantioselectivity of N-H insertion reactions involving a range of aryl amines and donor-acceptor carbene precursors. Despite great success in evolving natural enzymes, no example of directed evolution applied to engineering ArMs has been reported. We established a streamlined, high-throughput protocol for design, expression, purification and screening of ArM libraries. After the process was sufficiently optimized, the first round of directed evolution from an engineered parent protein, F99H328, produced the mutant G1P1_D5 which achieved 54% ee. This work was published in Chembiochem, 2014, 15, 223-227. This work was conducted in collaboration with Dr. Poonam Srivastava, Hao Yang, Hyun June Park and Ken Ellis-Guardiola.