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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Kolberg, M., Strand, K. R., Graff, P. & Andersson, K. K. Structure, function, and mechanism of ribonucleotide reductases. BBA-Proteins Proteom. 1699, 1–34 (2004).
Shanklin, J., Guy, J. E., Mishra, G. & Lindqvist, Y. Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes. J. Biol. Chem. 284, 18559–18563 (2009).
Berthold, D. A. & Stenmark, P. Membrane-bound diiron carboxylate proteins. Annu. Rev. Plant Biol. 54, 497–517 (2003).
Solomon, E. I. et al. Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem. Rev. 100, 235–350 (2000).
Lippard, S. J. Hydroxylation of C–H bonds at carboxylate-bridged diiron centres. Phil. Trans. R. Soc. A 363, 861–877 (2005).
Peters, J. W., Lanzilotta, W. N., Lemon, B. J. & Seefeldt, L. C. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282, 1853–1858 (1998).
Iyer, R. B., Silaghi-Dumitrescu, R., Kurtz, D. M. & Lanzilotta, W. N. High-resolution crystal structures of Desulfovibrio vulgaris (Hildenborough) nigerythrin: facile, redox-dependent iron movement, domain interface variability, and peroxidase activity in the rubrerythrins. J. Biol. Inorg. Chem. 10, 407–416 (2005).
Jin, S., Kurtz, D. M., Liu, Z-J., Rose, J. & Wang, B-C. X-ray crystal structures of reduced rubrerythrin and its azide adduct: a structure-based mechanism for a non-heme diiron peroxidase. J. Am. Chem. Soc. 124, 9845–9855 (2002).
Tempel, W. et al. Structural genomics of Pyrococcus furiosus: X-ray crystallography reveals 3D domain swapping in rubrerythrin. Proteins 57, 878–882 (2004).
Johnson, D. C., Dean, D. R., Smith, A. D. & Johnson, M. K. Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74, 247–281 (2005).
Yu, C-A., Wen, X., Xiao, K., Xia, D. & Yu, L. Inter- and intra-molecular electron transfer in the cytochrome bc(1) complex. Biochim. Biophys. Acta 1555, 65–70 (2002).
Silaghi-Dumitrescu, R., Kurtz, D. M., Ljungdahl, L. G. & Lanzilotta, W. N. X-ray crystal structures of Moorella thermoacetica FprA. Novel diiron site structure and mechanistic insights into a scavenging nitric oxide reductase. Biochemistry 44, 6492–6501 (2005).
Calhoun, J. R. et al. Artificial diiron proteins: From structure to function. Peptide Sci. 80, 264–278 (2005).
Maglio, O. et al. Diiron-containing metalloproteins: developing functional models. C.R. Chim. 10, 703–720 (2007).
Andreini, C., Bertini, I., Cavallaro, G., Najmanovich, R. J. & Thornton, J. M. Structural analysis of metal sites in proteins: non-heme iron sites as a case study. J. Mol. Biol. 388, 356–380 (2009).
Lombardi, A. et al. Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins. Proc. Natl Acad. Sci. USA 97, 6298–6305 (2000).
Faiella, M. et al. An artificial di-iron oxo-protein with phenol oxidase activity. Nature Chem. Biol. 5, 882–884 (2009).
Kaplan, J. & DeGrado, W. F. De novo design of catalytic proteins. Proc. Natl Acad. Sci. USA 101, 11566–11570 (2004).
Lee, D. & Lippard, S. J. in Comprehensive Coordination Chemistry II 2nd edn, 8 (eds McCleverty, J. A. & Meyer, T. J.) Section II 309–342 (Elsevier, 2003).
Bou-Abdallah, F. The iron redox and hydrolysis chemistry of the ferritins. Biochem. Biophys. Acta 1800, 719–731 (2010).
Calhoun, J. R. et al. Computational design and characterization of a monomeric helical dinuclear metalloprotein. J. Mol. Biol. 334, 1101–1115 (2003).
Bell, C. B. et al. Spectroscopic definition of the biferrous and biferric sites in de novo designed four-helix bundle DFsc peptides: implications for O2 reactivity of binuclear non-heme iron enzymes. Biochemistry 48, 59–73 (2009).
Calhoun, J. R. et al. Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the ‘intermediate’ and reaction mechanism. J. Am. Chem. Soc. 130, 9188–9189 (2008).
Zocher, G., Winkler, R., Hertweck, C. & Schulz, G. E. Structure and action of the N-oxygenase AurF from Streptomyces thioluteus. J. Mol. Biol. 373, 65–74 (2007).
Choi, Y. S., Zhang, H., Brunzelle, J. S., Nair, S. K. & Zhao, H. In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis. Proc. Natl Acad. Sci. USA 105, 6858–6863 (2008).
DeGrado, W. F., Summa, C. M., Pavone, V., Nastri, F. & Lombardi, A. De novo design and structural characterization of proteins and metalloproteins. Annu. Rev. Biochem. 68, 779–819 (1999).
Di Costanzo, L. et al. Toward the de novo design of a catalytically active helix bundle: a substrate-accessible carboxylate-bridged dinuclear metal center. J. Am. Chem. Soc. 123, 12749–12757 (2001).
Calhoun, J. R. et al. Solution NMR structure of a designed metalloprotein and complementary molecular dynamics refinement. Structure 16, 210–215 (2008).
Kulp, D. W. et al. Structural informatics, modeling and design using a new open-source Molecular Software Library (MSL). J. Comp. Chem. 33, 1645–1661 (2012).
Bertini, L. & Luchinat, C. High spin cobalt(II) as a probe for the investigation of metalloproteins. Adv. Inorg. Biochem. 6, 71–111 (1984).
Solomon, E. I., Pavel, E. G., Loeb, K. E. & Campochiaro, C. Magnetic circular dichroism spectroscopy as a probe of the geometric and electronic structure of non-heme ferrous enzymes. Coord. Chem. Rev. 144, 369–460 (1995).
Brown, C. A., Remar, G. J., Musselman, R. L. & Solomon, E. I. Spectroscopic and electronic structure studies of met-hemerythrin model complexes: a description of the ferric-oxo dimer bond. Inorg. Chem. 34, 688–717 (1995).
Yang, X., Chen-Barrett, Y., Arosio, P. & Chasteen, N. D. Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins. Biochemistry 37, 9743–9750 (1998).
Yang, X., Le Brun, N. E., Thomson, A. J., Moore, G. R. & Chasteen, N. D. The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin. Biochemistry 39, 4915–4923 (2000).
Fox, B. G., Shanklin, J., Ai, J., Loehr, T. M. & Sanders-Loehr, J. Resonance Raman evidence for an Fe–O–Fe center in stearoyl-ACP desaturase. Primary sequence identity with other diiron-oxo proteins. Biochemistry 33, 12776–12786 (1994).
Bollinger, J. et al. Mechanism of assembly of the tyrosyl radical–dinuclear iron cluster cofactor of ribonucleotide reductase. Science 253, 292–298 (1991).
Vincent, J. B., Olivier-Lilley, G. L. & Averill, B. A. Proteins containing oxo-bridged dinuclear iron centers: a bioinorganic perspective. Chem. Rev. 90, 1447–1467 (1990).
Korboukh, V. K., Li, N., Barr, E. W., Bollinger, J. M. & Krebs, C. A long-lived, substrate-hydroxylating peroxodiiron(III/III) intermediate in the amine oxygenase, AurF, from Streptomyces thioluteus. J. Am. Chem. Soc. 131, 13608–13609 (2009).
Corbett, J. F. Benzoquinone imines. Part V. Mechanism and kinetics of the reaction of p-benzoquinone monoimines with m-phenylenediamines. J. Chem. Soc., B: 823–826 (1969).
Corbett, J. F. & Gamson, E. P. Benzoquinone imines. Part XI. Mechanism and kinetics of the reaction of p-benzoquinone di-imines with aniline and its derivatives. J. Chem. Soc. Perkin Trans. 2 1531–1537 (1972).
Simurdiak, M., Lee, J. & Zhao, H. A new class of arylamine oxygenases: evidence that p-aminobenzoate N-oxygenase (AurF) is a di-iron enzyme and further mechanistic studies. ChemBioChem 7, 1169–1172 (2006).
Li, N., Korboukh, V. K., Krebs, C. & Bollinger, J. M. Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus. Proc. Natl Acad. Sci. USA 107, 15722–15727 (2010).
Fries, A., Bretschneider, T., Winkler, R. & Hertweck, C. A ribonucleotide reductase-like electron transfer system in the nitroaryl-forming N-oxygenase AurF. ChemBioChem 12, 1832–1835 (2011).
Johnson, B. H. & Hecht, M. H. Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Nature Biotech. 12, 1357–1360 (1994).
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).
The authors declare no competing financial interests.
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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). https://doi.org/10.1038/nchem.1454
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