Artificial Metalloenzymes (ArMs) containing non-natural organometallic cofactors have commanded increasing attention for their ability to catalyse transition-metal dependent transformations with the selectivity and mild conditions associated with enzymes. This work investigates novel ArMs where an organometallic cofactor has been introduced via ligand exchange reactions, thus ensuring an intimate link between the first and second coordination-sphere, with the latter being dictated by the protein fold. Further, the development of a high-throughput microfluidic screening platform is presented, with the aim to facilitate efficient directed evolution.

The first chapter gives a theoretical description of the matter covered by this thesis and reviews the relevant fields, including considerations for the formation of ArMs, in vitro directed evolution and a brief discussion of transition metal catalysis, focusing on ruthenium. The second chapter covers the practical aspects of the work conducted, including detailed descriptions of synthetic processes as well as biochemical and microfluidic techniques.

The third chapter details the design, synthetic challenges and characterisation of a range of substrate molecules used for the establishment of fluorescence assays for different ArM activities. Particular focus is placed on the synthesis and use of charged substrates that do not leak from the aqueous phase when used in water-in-oil droplet microfluidics.

The fourth chapter focuses on the formation of ArMs from organometallic complexes. A systematic study is presented, describing how the steric and electronic properties of a ruthenium piano-stool complex, carrying a key bipyridine ligand, led to various speciation of cytochrome b₅₆₂. Applying findings from this study, the scope of organometallic fragments that could be introduced was further expanded to include new metal centres and ligands. The resulting conjugates were characterised and investigated for catalytic activity. Finally, a method for evolving cofactor recognition using pre-fluorescent complexes is presented.

The fifth chapter details the development of a microfluidic workflow compatible with the ArMs used in this thesis, aiming to rapidly encapsulate, express and sort large genetic libraries of ArMs and thus enable directed evolution. Coupling of the genotype and phenotype is achieved by covalent attachment to hydrogel beads, mitigating the possibility of catalytic poisoning when using an in vivo approach.

The concluding chapter summarises the key results of this work and gives perspective on potential future developments.