Artificial metalloenzymes (ArMs) combine the broad reactivity of transition metals with the exquisite selectivity of enzymes, expanding the scope of biocatalysis beyond natural enzymatic functions. The biotin-streptavidin (Sav) system offers a versatile scaffold for integrating synthetic metal cofactors into protein frameworks with high precision. This thesis explores the design of ArMs with multiple catalytic functions, advancing the development of multifunctional biocatalysts.

Chapter 1 introduces ArMs and highlights the Sav technology as a robust platform for embedding abiotic catalytic centers into proteins. ArMs benefit from the precision of protein engineering and the catalytic versatility of transition metals, and have found broad applications in asymmetric transformations. This sets the foundation for developing Sav-based ArMs for enantioselective and multi-step catalysis.

Chapter 2 describes the creation of the first base-metal artificial transfer hydrogenase (ATHase) using a biotinylated Mn(I) complex. While previous ATHases relied on precious metals, this work shows that Mn(I) can also function effectively in ArMs. Through chemo-genetic optimization, we engineered Mn-based ATHases that demonstrate high activity, broad substrate scope, and good functional group tolerance, underlining their potential for sustainable and cost-effective catalysis.

Chapter 3 tackles the challenge of creating ArMs with dual catalytic functions. We developed a system that incorporates two distinct biotinylated cofactors into Sav: one photoactive and one metal-based. This design enables tandem enantioselective transformations, including a photoinduced Câ H activation followed by asymmetric catalysis. The work highlights how ArMs can facilitate multi-step, integrated reaction cascades.

Chapter 4 advances dual-cofactor ArMs by focusing on their spatial arrangement within adjacent binding sites of Sav. By combining a biotinylated nickel complex with a peptide-derived amine catalyst, we achieved synergistic catalysis in an enantioselective Michael addition. This demonstrates how precise cofactor positioning can improve both activity and selectivity, offering new strategies for the design of cooperative catalytic systems.

In summary, this thesis presents significant progress in ArM design via the biotin-streptavidin platform. By integrating diverse and complementary cofactors, we created artificial enzymes with enhanced reactivity, selectivity, and substrate scope. The introduction of dual cofactor systems enables tandem and synergistic catalysis, showcasing new directions for engineering multifunctional ArMs with promising applications in green chemistry, synthetic biology, and sustainable synthesis.