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.

  • Letter
  • Published:

Rational design of a structural and functional nitric oxide reductase

Nature volume 462, pages 1079–1082 (2009)Cite this article

Abstract

Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally1,2,3, it is more difficult to design functional proteins4,5,6,7,8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11,12,13,14,15,16,17,18,19,20. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

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: Crystal structure of rationally designed Fe B Mb overlays closely with minimized computer model.
Figure 2: Designed FeBMb has NO reduction activity in the presence of Fe2+.
Figure 3: Product of NO reaction with Fe B Mb is N 2 O.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Crystallographic data for FeBMb have been deposited in the Protein Data Bank with accession number 3K9Z.

References

  1. Regan, L. & DeGrado, W. F. Characterization of a helical protein designed from first principles. Science 241, 976–978 (1988)

    Article  ADS  CAS  Google Scholar 

  2. Hecht, M. H., Richardson, J. S., Richardson, D. C. & Ogden, R. C. De novo design, expression, and characterization of Felix: a four-helix bundle protein of native-like sequence. Science 249, 884–891 (1990)

    Article  ADS  CAS  Google Scholar 

  3. Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level accuracy. Science 302, 1364–1368 (2003)

    Article  ADS  CAS  Google Scholar 

  4. Bolon, D. N. & Mayo, S. L. Enzyme-like proteins by computational design. Proc. Natl Acad. Sci. USA 98, 14274–14279 (2001)

    Article  ADS  CAS  Google Scholar 

  5. Kaplan, J. & DeGrado, W. F. De novo design of catalytic proteins. Proc. Natl Acad. Sci. USA 101, 11566–11570 (2004)

    Article  ADS  CAS  Google Scholar 

  6. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science 319, 1387–1391 (2008)

    Article  ADS  CAS  Google Scholar 

  7. Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008)

    Article  ADS  Google Scholar 

  8. Lu, Y., Yeung, N., Sieracki, N. & Marshall, N. M. Design of functional metalloproteins. Nature 460, 855–862 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Shifman, J. M. & Mayo, S. L. Exploring the origins of binding specificity through the computational redesign of calmodulin. Proc. Natl Acad. Sci. USA 100, 13274–13279 (2003)

    Article  ADS  CAS  Google Scholar 

  10. Kang, S.-g. & Saven, J. G. Computational protein design: structure, function and combinatorial diversity. Curr. Opin. Chem. Biol. 11, 329–334 (2007)

    Article  CAS  Google Scholar 

  11. Robertson, D. E. et al. Design and synthesis of multi-haem proteins. Nature 368, 425–432 (1994)

    Article  ADS  CAS  Google Scholar 

  12. Hay, M., Richards, J. H. & Lu, Y. Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase. Proc. Natl Acad. Sci. USA 93, 461–464 (1996)

    Article  ADS  CAS  Google Scholar 

  13. Yeung, B. K., Wang, X., Sigman, J. A., Petillo, P. A. & Lu, Y. Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase. Chem. Biol. 4, 215–221 (1997)

    Article  CAS  Google Scholar 

  14. Sigman, J. A., Kwok, B. C. & Lu, Y. From myoglobin to heme-copper oxidase: design and engineering of a CuB center into sperm whale myoglobin. J. Am. Chem. Soc. 122, 8192–8196 (2000)

    Article  CAS  Google Scholar 

  15. Case, M. A. & McLendon, G. L. Metal-assembled modular proteins: toward functional protein design. Acc. Chem. Res. 37, 754–762 (2004)

    Article  CAS  Google Scholar 

  16. Cochran, F. V. et al. Computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor. J. Am. Chem. Soc. 127, 1346–1347 (2005)

    Article  CAS  Google Scholar 

  17. Watanabe, Y. & Hayashi, T. Functionalization of myoglobin. Prog. Inorg. Chem. 54, 449–493 (2005)

    Article  CAS  Google Scholar 

  18. Ghosh, D. & Pecoraro, V. L. Probing metal-protein interactions using a de novo design approach. Curr. Opin. Chem. Biol. 9, 97–103 (2005)

    Article  CAS  Google Scholar 

  19. Petros, A. K., Reddi, A. R., Kennedy, M. L., Hyslop, A. G. & Gibney, B. R. Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites. Inorg. Chem. 45, 9941–9958 (2006)

    Article  CAS  Google Scholar 

  20. Koder, R. L. et al. Design and engineering of an O2 transport protein. Nature 458, 305–309 (2009)

    Article  ADS  CAS  Google Scholar 

  21. Wasser, I. M., de Vries, S., Moënne-Loccoz, P., Schröder, I. & Karlin, K. D. Nitric oxide in biological denitrification: Fe/Cu metalloenzyme and metal complex NOx redox chemistry. Chem. Rev. 102, 1201–1234 (2002)

    Article  CAS  Google Scholar 

  22. van der Oost, J. et al. The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase. FEMS Microbiol. Lett. 121, 1–9 (1994)

    Article  CAS  Google Scholar 

  23. Girsch, P. & de Vries, S. Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans . Biochim. Biophys. Acta 1318, 202–216 (1997)

    Article  CAS  Google Scholar 

  24. Hendriks, J., Gohlke, U. & Saraste, M. From NO to OO: nitric oxide and dioxygen in bacterial respiration. J. Bioenerg. Biomembr. 30, 15–24 (1998)

    Article  CAS  Google Scholar 

  25. Watmough, N. J. et al. Nitric oxide in bacteria: synthesis and consumption. Biochim. Biophys. Acta 1411, 456–474 (1999)

    Article  CAS  Google Scholar 

  26. Wasser, I. M., Huang, H.-w., Moënne-Loccoz, P. & Karlin, K. D. Heme/non-heme diiron(II) complexes and O2, CO, and NO adducts as reduced and substrate-bound models for the active site of bacterial nitric oxide reductase. J. Am. Chem. Soc. 127, 3310–3320 (2005)

    Article  CAS  Google Scholar 

  27. Collman, J. P. et al. A functional nitric oxide reductase model. Proc. Natl Acad. Sci. USA 105, 15660–15665 (2008)

    Article  ADS  CAS  Google Scholar 

  28. Moënne-Loccoz, P. et al. Nitric oxide reductase from Paracoccus denitrificans contains an oxo-bridged heme/non-heme diiron center. J. Am. Chem. Soc. 122, 9344–9345 (2000)

    Article  Google Scholar 

  29. Zumft, W. G. Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme-copper oxidase type. J. Inorg. Biochem. 99, 194–215 (2005)

    Article  CAS  Google Scholar 

  30. Zhao, X., Yeung, N., Wang, Z., Guo, Z. & Lu, Y. Effects of metal ions in the CuB center on the redox properties of heme in heme-copper oxidases: spectroelectrochemical studies of an engineered heme-copper center in myoglobin. Biochemistry 44, 1210–1214 (2005)

    Article  CAS  Google Scholar 

  31. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

    Article  CAS  Google Scholar 

  32. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005)

    Article  CAS  Google Scholar 

  33. Zhao, X., Yeung, N., Russell, B. S., Garner, D. K. & Lu, Y. Catalytic reduction of NO to N2O by a designed heme-copper center in myoglobin: implications for the role of metal ions. J. Am. Chem. Soc. 128, 6766–6767 (2006)

    Article  CAS  Google Scholar 

  34. Taboy, C. H., Bonaventura, C. & Crumbliss, A. L. Anaerobic oxidations of myoglobin and hemoglobin by spectroelectrochemistry. Methods Enzymol. 353, 187–209 (2002)

    Article  CAS  Google Scholar 

  35. Bonner, F. T. Nitric oxide gas. Methods Enzymol. 268, 50–57 (1996)

    Article  CAS  Google Scholar 

  36. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  37. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997)

    Article  CAS  Google Scholar 

  38. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  39. Sheldrick, G. M. & Schneider, T. R. SHELXL: high-resolution refinement. Methods Enzymol. 277, 319–343 (1997)

    Article  CAS  Google Scholar 

  40. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  41. Giuffrè, A. et al. The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications. Proc. Natl Acad. Sci. USA 96, 14718–14723 (1999)

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We thank M. J. Nilges for help with EPR analysis, S. L. Mullen and F. Sun for aiding in GC/MS data collection, E. Lee for help with computational modelling, N. M. Marshall for providing Azurin protein, J. R. Askim for help in FeBMb expression and purification, and T. Hayashi and P. Moënne-Loccoz for suggestions regarding N2O detection in solution. This work was supported by the US National Institutes of Health (GM062211).

Author Contributions N.Y. and Y.-W.L. performed most of the experimentation and wrote most of the manuscript. B.S.R. helped with the initial design of mutants and experiments. X.Z. and L.L. assisted in experimentation. K.D.M. performed computational modelling. Y.-G.G. guided crystallization and refined the crystal structure. H.R. collected crystal diffraction data. Y.L. designed, guided the project and edited the manuscript.

Author information

Authors and Affiliations

  1. Department of Chemistry,,

    Natasha Yeung, Ying-Wu Lin, Xuan Zhao, Brandy S. Russell, Lanyu Lei & Yi Lu

  2. George L. Clark X-Ray Facility and 3M Materials Laboratory,,

    Yi-Gui Gao

  3. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,

    Kyle D. Miner & Yi Lu

  4. Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA,

    Howard Robinson

Authors
  1. Natasha Yeung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying-Wu Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yi-Gui Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xuan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brandy S. Russell
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lanyu Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kyle D. Miner
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Howard Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yi Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yi Lu.

Supplementary information

Supplementary Information

This file contains supplementary Methods and Notes, Supplementary Figures S1-S8 with Legends, Supplementary Table S1 and Supplementary References. (PDF 934 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yeung, N., Lin, YW., Gao, YG. et al. Rational design of a structural and functional nitric oxide reductase . Nature 462, 1079–1082 (2009). https://doi.org/10.1038/nature08620

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08620

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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