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.
| Original language | English |
|---|---|
| Pages (from-to) | 3974-3978 |
| Number of pages | 5 |
| Journal | Angewandte Chemie - International Edition |
| Volume | 60 |
| Issue number | 8 |
| DOIs | |
| State | Published - Feb 19 2021 |
Funding
This work was funded by the US NIH (ES012236) (to V.L.P), the French National Centre for Scientific Research (CNRS/UMR 7281, Marseille), the French PACA Region (APR‐EX “LIPCAT”) and the CNRS Program for International Collaborations (PICS 07624) (to A.I). The French EPR Federation/TGE RENARD (IR3443) is also acknowledged for partial support (to A.I.). UltraScan software's development for AUC was funded by NIH grant GM120600 and data analysis by NSF/XSEDE allocation grant MCB070038‐A13 (to B.D.). T.K. acknowledges a postdoctoral fellowship (APR‐EX “LIPCAT”) and a foreign researcher allowance from the city of Marseilles. We thank Jill Harland (University of Michigan, Ann Arbor) for her assistance in preparing AUC samples and Valerie Monnier (Aix Marseille Univ, CNRS, Centrale Marseille, FSCM, Spectropole, Marseille) for detailed analysis of the ESI‐MS data. This work was funded by the US NIH (ES012236) (to V.L.P), the French National Centre for Scientific Research (CNRS/UMR 7281, Marseille), the French PACA Region (APR-EX ?LIPCAT?) and the CNRS Program for International Collaborations (PICS 07624) (to A.I). The French EPR Federation/TGE RENARD (IR3443) is also acknowledged for partial support (to A.I.). UltraScan software's development for AUC was funded by NIH grant GM120600 and data analysis by NSF/XSEDE allocation grant MCB070038-A13 (to B.D.). T.K. acknowledges a postdoctoral fellowship (APR-EX ?LIPCAT?) and a foreign researcher allowance from the city of Marseilles. We thank Jill Harland (University of Michigan, Ann Arbor) for her assistance in preparing AUC samples and Valerie Monnier (Aix Marseille Univ, CNRS, Centrale Marseille, FSCM, Spectropole, Marseille) for detailed analysis of the ESI-MS data.
| Funders | Funder number |
|---|---|
| MCB070038‐A13 | |
| GM120600, PICS 07624 | |
| ES012236 | |
| R01GM141086 | |
| University of Michigan | |
| CNRS/UMR 7281 |
Keywords
- EPR spectroscopy
- cyt P450 monooxygenase
- heme enzymes
- protein design
- thiolate ligands