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.

Catalysis and Electron Transfer in De Novo Designed Metalloproteins

“…132,133 The Ivancich and Pecoraro laboratories investigated His and Cys as competing ligands to the same cofactor by presenting both within a single de novo protein. 134 This study used the GRAND family of peptides, which has been extensively studied for the incorporation of transition metal or heavy binding sites using His and Cys residues. 135−138 Free hemin was titrated into GRAND L16C L30H (GRL16CL30H), where the 16th helical position was mutated to Cys and the 30th position mutated to His.…”

“…The versatility of heme protein reactivity relies on variations in the iron coordinating ligands (His, Tyr, Cys, or Met) and modification of the heme binding environment through secondary sphere effects. While there are many examples of designed heme proteins that use His ligation, examples that use axial Cys ligation are relegated solely to small molecule mimics of heme proteins. , The Ivancich and Pecoraro laboratories investigated His and Cys as competing ligands to the same cofactor by presenting both within a single de novo protein . This study used the GRAND family of peptides, which has been extensively studied for the incorporation of transition metal or heavy binding sites using His and Cys residues. − …”

“…(A) Optical absorption and (B) 9-GHz EPR spectra of GRW-L16CL30H mini-heme protein at various pH conditions compared to the absorption spectrum of hemin in buffer (pH 7, dash-dotted curve) and the EPR spectrum of histidine bound hemin (pH 6, dash-dotted curve). Reprinted with permission from ref . Copyright 2021 John Wiley & Sons, Inc.…”

“…pH dependent coordination modes: (A) His-pentacoordinated heme (pH 7), (B) Cys-pentacoordinated heme (pH 9.0), and (C) Cys/hydroxy-hexacoordinated heme (pH 10.5) derived from spectroscopic characterization. Reprinted with permission from ref . Copyright 2021 John Wiley & Sons, Inc.…”

Axial Ligand-Engineered Single-Atom Catalysts with Boosted Enzyme-Like Activity for Sensitive Immunoassay

“…Recently, single-atom catalysts (SACs) with atomically dispersed metal sites can vividly mimic the active centers of enzymes. − Taking advantage of the maximized atomic utilization efficiency and strong metal–support interactions, the catalytic activity and selectivity of proposed SACs are remarkably higher than those of traditional nanomaterials. − Moreover, well-defined active metal sites are in favor of an in-depth understanding of the structure–property relationship, which provides potential guidance for a rational design of advanced enzyme-like catalysts at an atomic scale. , Inspired by the active iron sites of natural HRP, Fe–N–C-based SACs with heme-like structures have attracted great attention as the mimics of POD. − However, their intrinsic activities are still far away from the level of HRP, which does not meet the requirements of high sensitivity of biosensing. With an in-depth exploration of the catalytic mechanism of HRP, the enhanced catalytic activity derives from the pentacoordinated heme iron active sites and the special three-dimensional structure. , The axial coordination ligand (such as histidine or cysteine) is believed to play a crucial role in controlling the electronic structures of the active sites and even dictating their catalytic properties. − Specifically, a strong donated group of the axial ligand on metal active centers can increase the electron density on the macrocycle ring by donating electrons toward the unoccupied d-orbitals of the metal ion. This interaction has been named “push effect”, which significantly promotes the formation of a protonated ferryl (IV) porphyrin (Compound I) reactive intermediate and enhances the instinct catalytic activity. − Taking inspiration from the push effect, the introduction of axial ligands to construct a pentacoordinated environment may present a great opportunity for vividly mimicking natural HRP and further achieving the development of high-performance enzyme-like SACs.…”