Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(II) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol. Solution NMR spectroscopy in conjunction with molecular dynamics simulations show that the substrate binds in the active site pocket where it is stabilized by metal–ligand interactions as well as by burial of its hydrophobic groups. Spectrochemical redox titrations show that the protein stabilized the semiquinone by reducing the electrochemical midpoint potential for its formation via the one-electron oxidation of the catechol by approximately 400 mV (9 kcal mol−1). Therefore, the inherent chemical properties of the radical were changed drastically by harnessing its binding energy to the metalloprotein. This model sets the basis for designed enzymes with radical cofactors to tackle challenging chemistry.
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We thank R. Cooke and N. Naber for access to the EPR instrument, and for valuable discussions and advice. We thank M. Bhate for help in the initial NMR data collections, and M. Stenta for useful advice on setting up the molecular modelling. This work was supported in part by grant No. GM54616 and grant No. GM071628 from the National Institutes of Health to W.F.D. We also acknowledge support from the National Science Foundation (NSF) grant CHE 1413295 and the Materials Research Science and Engineering Centers program of the NSF, grant DMR-1120901. T.L. acknowledges support from the Swiss National Foundation of Science Fellowship 148914.
The authors declare no competing financial interests.
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Ulas, G., Lemmin, T., Wu, Y. et al. Designed metalloprotein stabilizes a semiquinone radical. Nature Chem 8, 354–359 (2016). https://doi.org/10.1038/nchem.2453
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