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Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity

Nature Chemistry volume 9, pages 623–628 (2017)Cite this article

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Abstract

Strong electron-donation from the axial thiolate ligand of cytochrome P450 has been proposed to increase the reactivity of compound I with respect to C–H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we have prepared a selenolate-ligated cytochrome P450 compound I intermediate. This isoelectronic perturbation allows for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepared with meta-chloroperbenzoic acid and characterized by UV–visible, Mössbauer and electron paramagnetic resonance spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we find that the selenolate-ligated compound I cleaves C–H bonds more rapidly than the wild-type intermediate.

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Figure 1: The selenolate-ligated compound I intermediate, a ferryl radical species.
Figure 2: Preparation of SeCYP119 compound I.
Figure 3: CW EPR and Mössbauer spectra of SeCYP119-I.
Figure 4: Target testing for the accumulation of compound I in the reaction of m-CPBA with premixed enzyme-substrate solutions.

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References

  1. Rittle, J. & Green, M. T. Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science 330, 933–937 (2010).

    Article  CAS  Google Scholar 

  2. Yosca, T. H. et al. Iron(IV)hydroxide pKa and the role of thiolate ligation in C–H bond activation by cytochrome P450. Science 342, 825–829 (2013).

    Article  CAS  Google Scholar 

  3. Dunford, H. B. Heme Peroxidases (Wiley-VCH, 1999).

    Google Scholar 

  4. Peter, S. et al. Selective hydroxylation of alkanes by an extracellular fungal peroxygenase. FEBS J. 278, 3667–3675 (2011).

    Article  CAS  Google Scholar 

  5. Wang, X., Peter, S., Kinne, M., Hofrichter, M. & Groves, J. T. Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I. J. Am. Chem. Soc. 134, 12897–12900 (2012).

    Article  CAS  Google Scholar 

  6. Hu, S. H. & Hager, L. P. Highly enantioselective propargylic hydroxylations catalyzed by chloroperoxidase. J. Am. Chem. Soc. 121, 872–873 (1999).

    Article  CAS  Google Scholar 

  7. Zaks, A. & Dodds, D. R. Chloroperoxidase-catalyzed asymmetric oxidations: substrate specificity and mechanistic study. J. Am. Chem. Soc. 117, 10419–10424 (1995).

    Article  CAS  Google Scholar 

  8. Yosca, T. H. et al. Setting an upper limit on the myoglobin iron(IV)hydroxide pKa: insight into axial ligand tuning in heme protein catalysis. J. Am. Chem. Soc. 136, 9124–9131 (2014).

    Article  CAS  Google Scholar 

  9. Green, M. T., Dawson, J. H. & Gray, H. B. Oxoiron(IV) in chloroperoxidase compound II is basic: implications for P450 chemistry. Science 304, 1653–1656 (2004).

    Article  CAS  Google Scholar 

  10. Wang, X., Ullrich, R., Hofrichter, M. & Grove, J. T. Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive. Proc. Natl Acad. Sci. USA 112, 3686–3691 (2015).

    CAS  PubMed  Google Scholar 

  11. Krest, C. M. et al. Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase. Nat. Chem. 7, 696–702 (2015).

    Article  CAS  Google Scholar 

  12. Takahashi, A., Kurahashi, T. & Fujii, H. Effect of imidazole and phenolate axial ligands on the electronic structure and reactivity of oxoiron(IV) porphyrin π-cation radical complexes: drastic increase in oxo-transfer and hydrogen abstraction reactivities. Inorg. Chem. 48, 2614–2625 (2009).

    Article  CAS  Google Scholar 

  13. Prokop, K. A., de Visser, S. P. & Goldberg, D. P. Unprecedented rate enhancements of hydrogen-atom transfer to a manganese(V)-oxo corrolazine complex. Angew. Chem. Int. Ed. 49, 5091–5095 (2010).

    Article  CAS  Google Scholar 

  14. Gross, Z. & Nimri, S. A pronounced axial ligand effect on the reactivity of oxoiron(IV) porphyrin cation radicals. Inorg. Chem. 33, 1731–1732 (1994).

    Article  CAS  Google Scholar 

  15. Ohno, T. et al. Remarkable axial thiolate ligand effect on the oxidation of hydrocarbons by active intermediate of iron porphyrin and cytochrome P450. J. Inorg. Biochem. 82, 123–125 (2000).

    Article  CAS  Google Scholar 

  16. Adachi, S., Nagano, S., Watanabe, Y., Ishimori, K. & Morishima, I. Alteration of human myoglobin proximal histidine to cysteine or tyrosine by site-directed mutagenesis: characterization and their catalytic activities. Biochem. Biophys. Res. Commun. 180, 138–144 (1991).

    Article  CAS  Google Scholar 

  17. Adachi, S. et al. Roles of proximal ligand in heme-proteins: replacement of proximal histidine of human myoglobin with cysteine and tyrosine by site-directed mutagenesis as models for P-450, chloroperoxidase, and catalase. Biochemistry 32, 241–252 (1993).

    Article  CAS  Google Scholar 

  18. Hildebrand, D. P., Ferrer, J. C., Tang, H. L., Smith, M. & Mauk, A. G. Trans effects on cysteine ligation in the proximal his93cys variant of horse heart myoglobin. Biochemistry 34, 11598–11605 (1995).

    Article  CAS  Google Scholar 

  19. Sigman, J. A., Pond, A. E., Dawson, J. H. & Lu, Y. Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation. Biochemistry 38, 11122–11129 (1999).

    Article  CAS  Google Scholar 

  20. Murugan, R. & Mazumdar, S. Structure and redox properties of the haem centre on the C357M mutant of cytochrome P450cam. ChemBioChem 6, 1204–1211 (2005).

    Article  CAS  Google Scholar 

  21. Yoshioka, S., Takahashi, S., Hori, H., Ishimori, K. & Morishima, I. Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam . Eur. J. Biochem. 268, 252–259 (2001).

    Article  CAS  Google Scholar 

  22. Auclair, K., Moënne-Loccoz, P. & de Montellano, P. R. O. Roles of the proximal heme thiolate ligand in cytochrome P450cam . J. Am. Chem. Soc. 123, 4877–4885 (2001).

    Article  CAS  Google Scholar 

  23. Coelho, P. S. et al. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol. 9, 485–487 (2013).

    Article  CAS  Google Scholar 

  24. Vatsis, K. P., Peng, H. M. & Coon, M. J. Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity. J. Inorg. Biochem. 91, 542–553 (2002).

    Article  CAS  Google Scholar 

  25. Vatsis, K. P., Peng, H. M. & Coon, M. J. Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4. Arch. Biochem. Biophys. 434, 128–138 (2005).

    Article  CAS  Google Scholar 

  26. Moroder, L. Isosteric replacement of sulfur with other chalcogens in peptides and proteins. J. Pept. Sci. 11, 187–214 (2005).

    Article  CAS  Google Scholar 

  27. Huber, R. E. & Criddle, R. S. Comparison of chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch. Biochem. Biophys. 122, 164–173 (1967).

    Article  CAS  Google Scholar 

  28. Jiang, Y. Y. et al. Calculated and experimental spin state of seleno cytochrome P450. Angew. Chem. Int. Ed. 48, 7193–7195 (2009).

    Article  CAS  Google Scholar 

  29. Aldag, C. et al. Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine. Proc. Natl Acad. Sci. USA 106, 5481–5486 (2009).

    Article  CAS  Google Scholar 

  30. Sivaramakrishnan, S. et al. A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid. Biochemistry 50, 3014–3024 (2011).

    Article  CAS  Google Scholar 

  31. Sivaramakrishnan, S. et al. Proximal ligand electron donation and reactivity of the cytochrome P450 ferric-peroxo anion. J. Am. Chem. Soc. 134, 6673–6684 (2012).

    Article  CAS  Google Scholar 

  32. Vandemeulebroucke, A., Aldag, C., Stiebritz, M. T., Reiher, M. & Hilvert, D. Kinetic consequences of introducing a proximal selenocysteine ligand into cytochrome P450cam. Biochemistry 54, 6692–6703 (2015).

    Article  CAS  Google Scholar 

  33. Cohen, S., Kumar, D. & Shaik, S. In silico design of a mutant of cytochrome P450 containing selenocysteine. J. Am. Chem. Soc. 128, 2649–2653 (2006).

    Article  CAS  Google Scholar 

  34. Yosca, T. H. & Green, M. T. Preparation of compound I in P450cam: the prototypical P450. Isr. J. Chem. 56, 834–840 (2016).

    Article  CAS  Google Scholar 

  35. Müller, S. et al. The formation of diselenide bridges in proteins by incorporation of selenocysteine residues: biosynthesis and characterization of (Se)2-thioredoxin. Biochemistry 33, 3404–3412 (1994).

    Article  Google Scholar 

  36. Sanchez, J. F., Hoh, F., Strub, M. P., Aumelas, A. & Dumas, C. Structure of the cathelicidin motif of protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein. Structure 10, 1363–1370 (2002).

    Article  CAS  Google Scholar 

  37. Strub, M. P. et al. Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing. Structure 11, 1359–1367 (2003).

    Article  CAS  Google Scholar 

  38. Rutter, R. et al. Chloroperoxidase compound-I: electron paramagnetic resonance and Mössbauer studies. Biochemistry 23, 6809–6816 (1984).

    Article  CAS  Google Scholar 

  39. Rittle, J., Younker, J. M. & Green, M. T. Cytochrome P450: the active oxidant and its spectrum. Inorg. Chem. 49, 3610–3617 (2010).

    Article  CAS  Google Scholar 

  40. Groves, J. T., McClusky, G. A., White, R. E. & Coon, M. J. Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. evidence for a carbon radical intermediate. Biochem. Biophys. Res. Commun. 81, 154–160 (1978).

    Article  CAS  Google Scholar 

  41. Mayer, J. M. Hydrogen atom abstraction by metal-oxo complexes: understanding the analogy with organic radical reactions. Acc. Chem. Res. 31, 441–450 (1998).

    Article  CAS  Google Scholar 

  42. Green, M. T. C–H bond activation in heme proteins the role of thiolate ligation in cytochrome P450. Curr. Opin. Chem. Biol. 13, 84–88 (2009).

    Article  CAS  Google Scholar 

  43. Epel, B. & Silakov, A. Kazan Viewer Version 2.1 (2006); https://sites.google.com/site/silakovalexey/kazan-viewer

  44. Stoll, S. & Schweiger, A. Easyspin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by NIH (R01-GM101390).

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Authors and Affiliations

  1. Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, 16802, USA

    Elizabeth L. Onderko & Alexey Silakov

  2. Department of Chemistry, University of California, Irvine, 92697, California, USA

    Timothy H. Yosca & Michael T. Green

  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, 92697, California, USA

    Timothy H. Yosca & Michael T. Green

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  1. Elizabeth L. Onderko
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  2. Alexey Silakov
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Contributions

E.O. and M.G. wrote the manuscript and designed the experiments. E.O. prepared the samples, collected and analysed stopped-flow, Mössbauer and EPR data. A.S. collected and analysed EPR data. T.Y. provided input on and assisted with experiments.

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Correspondence to Michael T. Green.

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Onderko, E., Silakov, A., Yosca, T. et al. Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity. Nature Chem 9, 623–628 (2017). https://doi.org/10.1038/nchem.2781

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