Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase

Nature Chemistry volume 7, pages 696–702 (2015)Cite this article

Subjects

Abstract

Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C–H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe–S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe–S bond lengths can be understood in terms of variations in the hydrogen-bonding patterns within the ‘cys-pocket’ (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe–S bond, which enables greater electron donation from the axial thiolate ligand. This observation may in part explain P450's greater propensity for C–H bond activation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: P450 displays weaker/poorer hydrogen-bonding interactions compared with CPO.
Figure 2: Rates of substrate oxidation by CPO-I and CYP119A1-I.
Figure 3: Variable-temperature Mössbauer data.
Figure 4: Representative Fe K-edge EXAFS data and Fourier transforms for CPO-I and CYP119A1-I.

Similar content being viewed by others

References

  1. Groves, J. T. Enzymatic C–H bond activation using push to get pull. Nature Chem. 6, 89–91 (2014).

    Article  CAS  Google Scholar 

  2. Dawson, J. H. Probing structure–function relations in heme-containing oxygenases and peroxidases. Science 240, 433–439 (1988).

    Article  CAS  Google Scholar 

  3. 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 

  4. 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 

  5. 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 

  6. 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 

  7. Krest, C. M. et al. Reactive intermediates in cytochrome P450 catalysis. J. Biol. Chem. 288, 17074–17081 (2013).

    Article  CAS  Google Scholar 

  8. McQuarters, A. B., Wolf, M. W., Hunt, A. P. & Lehnert, N. 1958–2014: after 56 years of research, cytochrome P450 reactivity is finally explained. Angew. Chem. Int. Ed. 53, 4750–4752 (2014).

    Article  CAS  Google Scholar 

  9. Jung, C. The mystery of cytochrome P450 compound I: a mini-review dedicated to Klaus Ruckpaul. Biochim. Biophys. Acta. 1814, 46–57 (2011).

    Article  CAS  Google Scholar 

  10. 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 

  11. Song, W. J., Ryu, Y. O., Song, R. & Nam, W. Oxoiron(IV) porphyrin π-cation radical complexes with a chameleon behavior in cytochrome P450 model reactions. J. Biol. Inorg. Chem. 10, 294–304 (2005).

    Article  CAS  Google Scholar 

  12. Kamachi, T., Kouno, T., Nam, W. & Yoshizawa, K. How axial ligands control the reactivity of high-valent iron(IV)–oxo porphyrin π-cation radicals in alkane hydroxylation: a computational study. J. Inorg. Biochem. 100, 751–754 (2006).

    Article  CAS  Google Scholar 

  13. Kang, Y. et al. Enhanced reactivities of iron(IV)-oxo porphyrin π-cation radicals in oxygenation reactions by electron-donating axial ligands. Chem. Eur. J. 15, 10039–10046 (2009).

    Article  CAS  Google Scholar 

  14. 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 

  15. Urano, Y., Higuchi, T., Hirobe, M. & Nagano, T. A pronounced axial thiolate ligand effect on the reactivity of the high valent oxo-iron porphyrin intermediate. FASEB J. 11, A817–A817 (1997).

    Article  Google Scholar 

  16. 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 

  17. van Rantwijk, F. & Sheldon, R. A. Selective oxygen transfer catalysed by heme peroxidases: synthetic and mechanistic aspects. Curr. Opin. Biotechnol. 11, 554–564 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Stone, K. L., Behan, R. K. & Green, M. T. X-ray absorption spectroscopy of chloroperoxidase compound I: insight into the reactive intermediate of P450 chemistry. Proc. Natl Acad. Sci. USA 102, 16563–16565 (2005).

    Article  CAS  Google Scholar 

  20. Sundaramoorthy, M., Terner, J. & Poulos, T. L. The crystal structure of chloroperoxidase: a heme peroxidase–cytochrome P450 functional hybrid. Structure 3, 1367–1377 (1995).

    Article  CAS  Google Scholar 

  21. Poulos, T. L., Finzel, B. C. & Howard, A. J. High-resolution crystal-structure of cytochrome-P450cam. J. Mol. Biol. 195, 687–700 (1987).

    Article  CAS  Google Scholar 

  22. 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 

  23. Zhang, R., Nagraj, N., Lansakara, D. S. P., Hager, L. P. & Newcomb, M. Kinetics of two-electron oxidations by the compound I derivative of chloroperoxidase, a model for cytochrome P450 oxidants. Org. Lett. 8, 2731–2734 (2006).

    Article  CAS  Google Scholar 

  24. Ueyama, N., Nishikawa, N., Yamada, Y., Okamura, T. & Nakamura, A. Cytochrome P-450 model (porphinato)(thiolato)iron(III) complexes with single and double NH···S hydrogen bonds at the thiolate site. J. Am. Chem. Soc. 118, 12826–12827 (1996).

    Article  CAS  Google Scholar 

  25. Ueyama, N. et al. Synthesis and properties of octaethylporphinato(arenethiolato)iron(III) complexes with intramolecular NH–S hydrogen bond: chemical function of the hydrogen bond. Inorg. Chem. 37, 2415–2421 (1998).

    Article  CAS  Google Scholar 

  26. Suzuki, N. et al. Novel iron porphyrin–alkanethiolate complex with intramolecular NH···S hydrogen bond: synthesis, spectroscopy, and reactivity. J. Am. Chem. Soc. 121, 11571–11572 (1999).

    Article  CAS  Google Scholar 

  27. Ueno, T. et al. Role of the invariant peptide fragment forming NH···S hydrogen bonds in the active site of cytochrome P-450 and chloroperoxidase: synthesis and properties of Cys-containing peptide Fe(III) and Ga(III) (octaethylporphinato) complexes as models. Inorg. Chem. 38, 1199–1210 (1999).

    Article  CAS  Google Scholar 

  28. Yoshioka, S. et al. Roles of the proximal hydrogen bonding network in cytochrome P450(cam)-catalyzed oxygenation. J. Am. Chem. Soc. 124, 14571–14579 (2002).

    Article  CAS  Google Scholar 

  29. Dey, A. et al. Sulfur K-edge XAS and DFT calculations on P450 model complexes: effects of hydrogen bonding on electronic structure and redox potentials. J. Am. Chem. Soc. 127, 12046–12053 (2005).

    Article  CAS  Google Scholar 

  30. Matsumura, H. et al. Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand. Biochemistry 47, 4834–4842 (2008).

    Article  CAS  Google Scholar 

  31. Dey, A. et al. S K-edge XAS and DFT calculations on cytochrome P450: covalent and ionic contributions to the cysteine-Fe bond and their contribution to reactivity. J. Am. Chem. Soc. 131, 7869–7878 (2009).

    Article  CAS  Google Scholar 

  32. Galinato, M. G. I., Spolitak, T., Ballou, D. P. & Lehnert, N. Elucidating the role of the proximal cysteine hydrogen-bonding network in ferric cytochrome P450cam and corresponding mutants using magnetic circular dichroism spectroscopy. Biochemistry 50, 1053–1069 (2011).

    Article  CAS  Google Scholar 

  33. Lehnert, N. Elucidating second coordination sphere effects in heme proteins using low-temperature magnetic circular dichroism spectroscopy. J. Inorg. Biochem. 110, 83–93 (2012).

    Article  CAS  Google Scholar 

  34. Park, S. Y. et al. Thermophilic cytochrome P450 (CYP119) from Sulfolobus solfataricus: high resolution structure and functional properties. J. Inorg. Biochem. 91, 491–501 (2002).

    Article  CAS  Google Scholar 

  35. Beitlich, T., Kuhnel, K., Schulze-Briese, C., Shoeman, R. L. & Schlichting, I. Cryoradiolytic reduction of crystalline heme proteins: analysis by UV–vis spectroscopy and X-ray crystallography. J. Synchrotron Radiat. 14, 11–23 (2007).

    Article  CAS  Google Scholar 

  36. Green, M. T. Imidazole-ligated compound I intermediates: the effects of hydrogen bonding. J. Am. Chem. Soc. 122, 9495–9499 (2000).

    Article  CAS  Google Scholar 

  37. Green, M. T. The structure and spin coupling of catalase compound I: a study of noncovalent effects. J. Am. Chem. Soc. 123, 9218–9219 (2001).

    Article  CAS  Google Scholar 

  38. Weiss, R. et al. Delocalization over the heme and the axial ligands of one of the two oxidizing equivalents stored above the ferric state in the peroxidase and catalase compound-I intermediates: indirect participation of the proximal axial ligand of iron in the oxidation reactions catalyzed by heme-based peroxidases and catalases? J. Biol. Inorg. Chem. 1, 377–383 (1996).

    Article  CAS  Google Scholar 

  39. Green, M. T. Evidence for sulfur-based radicals in thiolate compound I intermediates. J. Am. Chem. Soc. 121, 7939–7940 (1999).

    Article  CAS  Google Scholar 

  40. Schulz, C. E., Nyman, P. & Debrunner, P. G. Spin fluctuations of paramagnetic iron centers in proteins and model complexes: Mössbauer and EPR results. J. Chem. Phys. 87, 5077–5091 (1987).

    Article  CAS  Google Scholar 

  41. Winkler, H., Schulz, C. & Debrunner, P. G. Spin fluctuation rates from Mössbauer spectra of high-spin ferrous rubredoxin. Phys. Lett. A 69, 360–363 (1979).

    Article  Google Scholar 

  42. Chandrasekaran, P. et al. Prediction of high-valent iron K-edge absorption spectra by time-dependent density functional theory. Dalton Trans. 40, 11070–11079 (2011).

    Article  CAS  Google Scholar 

  43. Westre, T. E. et al. A multiplet analysis of Fe K-edge 1s→ 3d pre-edge features of iron complexes. J. Am. Chem. Soc. 119, 6297–6314 (1997).

    Article  CAS  Google Scholar 

  44. Spiro, T. G. & Wasbotten, I. H. CO as a vibrational probe of heme protein active sites. J. Inorg. Biochem. 99, 34–44 (2005).

    Article  CAS  Google Scholar 

  45. Mak, P. J., Yang, Y. T., Im, S., Waskell, L. A. & Kincaid, J. R. Experimental documentation of the structural consequences of hydrogen-bonding interactions to the proximal cysteine of a cytochrome P450. Angew. Chem. Int. Ed. 51, 10403–10407 (2012).

    Article  CAS  Google Scholar 

  46. Hollenberg, P. F., Hager, L. P., Blumberg, W. E. & Peisach, J. An electron paramagnetic resonance study of the high and low spin forms of chloroperoxidase. J. Biol. Chem. 255, 4801–4807 (1980).

    CAS  PubMed  Google Scholar 

  47. Paulat, F. & Lehnert, N. Electronic structure of ferric heme nitrosyl complexes with thiolate coordination. Inorg. Chem. 46, 1547–1549 (2007).

    Article  CAS  Google Scholar 

  48. Williams-Smith, D. L., Bray, R. C., Barber, M. J., Tsopanakis, A. D. & Vincent, S. P. Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy. Biochem. J. 167, 593–600 (1977).

    Article  CAS  Google Scholar 

  49. George, G. N. EXAFSPAK; http://ssrl.slac.stanford.edu/exafspak.html (2000).

Download references

Acknowledgements

This work was supported by the National Institutes of Health (NIH, R01-GM101390). The authors thank M. Latimer and E. Nelson for on-site assistance at the synchrotron. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-76SF00515). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH, National Institute of General Medical Sciences (including P41GM103393).

Author information

Author notes

  1. Courtney M. Krest

    Present address: Present address: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory Menlo Park, California 94025, USA,

Authors and Affiliations

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

    Courtney M. Krest, Alexey Silakov, Jonathan Rittle, Timothy H. Yosca, Elizabeth L. Onderko, Julio C. Calixto & Michael T. Green

Authors
  1. Courtney M. Krest
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexey Silakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jonathan Rittle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Timothy H. Yosca
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Elizabeth L. Onderko
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julio C. Calixto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael T. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.T.G. designed the experiments. M.T.G. wrote the manuscript with input from the other authors. C.M.K., T.H.Y., E.L.O. and J.C.C. were responsible for EXAFS data collection and analyses. C.M.K. and A.S. were responsible for the VTM measurements. A.S. performed the VTM analyses. J.R. collected and analysed the kinetic data. C.M.K., T.H.Y. and E.L.O. were responsible for sample preparation.

Corresponding author

Correspondence to Michael T. Green.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1013 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Krest, C., Silakov, A., Rittle, J. et al. Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase. Nature Chem 7, 696–702 (2015). https://doi.org/10.1038/nchem.2306

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2306

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing