Peroxidase activity of a myoglobin mutant with three distal histidines forming a metal-binding site: Implications for the cross-reactivity of cytochrome c oxidase

Rationally designed biosynthetic models provide fantastic advantages for addressing important issues in chemistry and biology. Previously, two distal histidines were introduced in the heme pocket of myoglobin (Mb) at positions 29 and 43. The resultant His29 and His43, together with the native His64, formed a metal-binding site in the designed L29H/F43H Mb mutant, which closely mimics the heterobinuclear center of native cytochrome c oxidase (CcO). Instead of studying the oxidase activity, we herein investigated the cross-reactivity, i.e., the peroxidase activity of this mutant, as well as the effect of metal ions binding to the designed metal-binding site on protein reactivity. It was found that the introduced two distal histidines with no bound metal ion serve to improve the peroxidase activity of L29H/F43H Mb compared with that of WT Mb containing a single distal histidine, whereas the binding of metal ions such as Cu(II) and Zn(II) to L29H/F43H Mb inhibits the catalytic activity to a different extent. These findings provide valuable insights into the peroxidase activity observed for the native CcO, as well as the role of metal ion in fine-tuning the protein cross-reactivity.