Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions.

Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP), and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation enhanced Mn(II)-binding affinity, as evidenced by a 2.8-fold decrease in the KM of Mn(II) oxidation. Second, introducing a salt bridge through Lys179Arg mutation improved Glu35 and Glu181 coordination to Mn(II), decreasing KM 2.6-fold. Third, eliminating a steric clash that prevented Glu37 from orienting toward Mn(II) resulted i...