Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6‐arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non‐biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein‐derived ligands. By introduction of ruthenium cofactors into a 4‐helical bundle, transfer hydrogenation catalysts were generated that displayed a 35‐fold rate increase when compared to the respective small molecule reaction in solution. A ruthenium organometallic complex is transformed into an effective transfer hydrogenation catalyst upon exchanging ligands with a naïve protein. The direct coordination of protein sidechains to the metal is an underutilised feature in artificial metalloenzymes.