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Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

A Corrigendum to this article was published on 23 November 2012

This article has been updated


De novo proteins provide a unique opportunity to investigate the structure–function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling the substrate access cavity and introducing a critical third His ligand to the metal-binding cavity. Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a 106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

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Figure 1: Important structural features of amino-acid sequences for the original and redesigned DFsc proteins.
Figure 2: Folding and metal-binding characterizations of G4DFsc and 3His-G4DFsc.
Figure 3: Ferroxidase activity of G4DFsc (top) and 3His-G4DFsc (bottom) indicated by the formation of a strong oxo-to-ferric charge-transfer band near 360 nm.
Figure 4: Hydroquinone oxidase activity of G4DFsc (top) and 3His-G4DFsc (bottom).
Figure 5: Oxygenation of p-anisidine by G4DFsc (top) and 3His-G4DFsc (bottom) in the presence of two equivalents of Fe(II).
Figure 6: N-hydroxylation of p-anisidine by 3His-G4DFsc.

Change history

  • 13 November 2012

    In the version of this Article originally published, the name of the co-author Thomas Szyperski mistakenly included a middle initial G. This has now been corrected in the online versions.


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This work was supported by the National Institutes of Health (F32-GM808852 to A.J.R., F32-GM095242 to M.M.P., GM54616 to W.F.D. and U54 GM094597 to T.S.) and the National Science Foundation (MCB-0919027 to E.I.S. and a MRSEC grant, DMR-1120901, to UPenn's LRSM).

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Authors and Affiliations



J.R.C. mutated, expressed and purified the initial G4DFsc construct. J.R.C. and A.J.R. performed the characterization and kinetic studies for G4DFsc. D.W.K. performed the computational modelling studies to determine the feasibility of 3His-G4DFsc mutations. A.J.R. mutated, expressed, produced and purified the 3His-G4DFsc construct, characterized the protein and designed, conducted and analysed the kinetic assays. S.E.B. expressed, produced and purified the 3His-G4DFsc construct and performed Co(II) binding titrations. M.M.P. characterized and analysed the possible reaction products of the arylamine-oxidation reaction, and expressed and purified 3His-G4DFsc and 3His-G2DFsc and their N15/C13 labelled forms for NMR experiments. H.J. synthesized, purified and characterized possible reaction products for retention-time comparisons. Y.W. collected and, with T.S., analysed the NMR structural data. R.A.S. performed and, with E.I.S., analysed Fe(II) CD titrations. A.J.R. and W.F.D. co-wrote the manuscript in consultation with the other contributors.

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Correspondence to Amanda J. Reig or William F. DeGrado.

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Reig, A., Pires, M., Snyder, R. et al. Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins. Nature Chem 4, 900–906 (2012).

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