Structure-property-function Studies Of Myoglobin Mutants With Two Or Three Distal Histidines

Rationally designed biosynthetic models provide fantastic advantages foraddressing important issues in chemistry and biology. By introduction of aseconddistal histidine (Leu29to His29mutation or Phe43to His43mutation) in theheme pocket of sperm whale myoglobin (Mb), L29H Mb and F43H Mb mutantsexhibit enhanced peroxidase activity with two cooperative distal histidines using H2O2as an oxidant.We further studied that when L29H Mb was immobilized inindidecyldimethylammonium bromide (DDAB) film on graphic electrode, it canefficiently reduce O2in aerobic buffer solution to H2O2, followed by electrode-drivenoxidation of peroxidase substrate such as guaiacol, which is more effective than thatof WT Mb with a single distal His64. Moreover, the peroxidase-like activity ofimmobilized L29H Mb was found to be affected by pH values, which suggests thatthe distal histidines play a key role in reductive activation of molecular oxygen byproviding protons. Since there is no native peroxidase with two distal histidines andalso peroxidases normally use H2O2as an oxidant, this study thus provides new cluesfor creating functional enzymes beyond native peroxidases.In additon, two distal histidines were introduced in the heme pocket ofmyoglobin (Mb) at positions29and43. The resultant His29and His43, together withthe 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). We herein investigated the peroxidase activity of this mutant, as well as theeffect of metal ions binding to the designed metal-binding site on protein reactivity. Itwas found that the introduced two distal histidines with no bound metal ion serve toimprove the peroxidase activity of L29H/F43H Mb compared with that of WT Mbcontaining 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. Thesefindings provide valuable insights into the peroxidase activity observed for the nativeCcO, as well as the role of metal ion in fine-tuning the protein cross-reactivity.Taken together, these studies provide us important information for understandingthe delicate structure-property-reactivity-function relationship of heme proteins.