The pH- Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α- Helical Metalloprotein

Abstract

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α- helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH- induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa- coordinate thiolate heme (9- ¤pH- ¤11) and the penta- coordinate imidazole heme (6- ¤pH- ¤8.5) reproduces well the heme ligation in chloroperoxidases or cyt- P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini- heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.A pH- dependent preference for the heme ligation is achieved when using self- assembling α- helical coiled coils having Cys and His as binding sites. EPR and UV/Vis absorption spectroscopies uncovered a switch in heme axial ligand from His (6- ¤pH- ¤8.5) to Cys (9- ¤pH- ¤11), mimicking the heme sites in cyt- P450 monooxygenase, chloroperoxidase and peroxidases, all three in a single artificial protein.