Coordination-site engineering in mononuclear non-heme metalloenzymes: From evolutionary motifs to programmable metal centers

Non-heme metalloenzymes are one of the most diverse classes of biological catalysts, performing a wide range of oxidative, hydrolytic, and radical-mediated transformations through metal centers that are directly coordinated by protein-derived amino acid ligands. Unlike heme enzymes whose coordination environments are constrained by the porphyrin macrocycle, non-heme metal centers display remarkable diversity in metal identity, ligand composition, coordination geometry, and electronic structure. This coordination diversity underlies the functional versatility of natural metalloenzymes and the expanding scope of artificial metalloenzyme design. In this review, we examined mononuclear non-heme metalloenzymes from the perspective of coordination chemistry. We first summarize the representative coordination motifs found in iron, manganese, nickel, copper, and zinc enzymes, highlighting how combinations of metal ions and coordinating amino acids generated a broad evolutionary coordination landscape. We also discuss metal promiscuity, cambialistic enzymes, and metal-switching systems, which reveal the inherent adaptability of biological metal-binding sites. Building on these principles, we review recent advances in coordination-site engineering, including the redesign of native coordination spheres, metal substitution, grafting of natural metal-binding motifs, incorporation of non-canonical ligands through genetic code expansion, incorporation of abiological metals, and de novo metalloprotein design. We focus particularly on emerging evidence showing that many non-heme metalloenzymes operate through dynamic coordination environments rather than single static active-site structures. We propose that the deliberate control of dynamic metal centers represents a promising design principle for next-generation artificial metalloenzymes and programmable catalytic systems.