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:

Structures of protein–protein complexes involved in electron transfer

Nature volume 496, pages 123–126 (2013)Cite this article

Subjects

Abstract

Electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism1. Key to these roles is the formation of transient inter-protein electron transfer complexes. The structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic studies2,3. Inter-protein electron transfer processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 through the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one-electron reduction of nitrite to NO, a precursor to N2O, is performed by either a haem- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome4,5. Here we report the structures of the newly characterized three-domain haem-c-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01 Å resolution and its M92A and P93A mutants. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.

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: Structural organization of RpNiR.
Figure 2: Details of interaction between cytochrome and Cu binding domains.
Figure 3: Comparison of interactions between cytochrome and Cu binding domains in wild type RpNiR, P93A and M92A mutants.
Figure 4: Details of the proton pathway in RpNiR and its comparison with PhNiR (Protein Data Bank entry 2zoo).

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the crystal structures have been deposited in Protein Data Bank under accession numbers 3ziy (r3ziysf), 4ax3 (r4ax3sf), 2yqb (r2yqbsf) and 3zbm (r3zbmsf).

References

  1. Moser, C. M., Keske, M., Warnke, K., Farid, R. S. & Dutton, P. L. Nature of biological electron transfer. Nature 355, 796–802 (1992)

    Article  ADS  CAS  Google Scholar 

  2. Jeng, M.-F., Englander, S. W., Pardue, K., Rogalskyj, J. S. & McLendon, G. Structural dynamics in an electron-transfer complex. Nature Struct. Biol. 1, 234–238 (1994)

    Article  CAS  Google Scholar 

  3. Williams, P. A. et al. Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome c550 and cytochrome cd1 nitrite reductase. Nature Struct. Biol. 2, 975–982 (1995)

    Article  CAS  Google Scholar 

  4. Zumft, W. G. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Eady, R. R. & Hasnain, S. S. in Comprehensive Coordination Chemistry II Vol. 8 (eds Que, L. & Tolman, W. ), Ch. 28 759–786 (Elsevier, 2003)

    Book  Google Scholar 

  6. Nojiri, M. et al. Structural basis of inter-protein electron transfer for nitrite reduction in denitrification. Nature 462, 117–121 (2009)

    Article  ADS  CAS  Google Scholar 

  7. Merkle, A. C. & Lehnert, N. Binding and activation of nitrite and nitric oxide by copper nitrite reductase and corresponding model complexes. Dalton Trans. 41, 3355–3368 (2012)

    Article  CAS  Google Scholar 

  8. Ellis, M. J., Dodd, F. E., Sawers, G., Eady, R. R. & Hasnain, S. S. Atomic resolution structures of native copper nitrite reductase from Alcaligenes xylosoxidans and the active site mutant Asp92Glu. J. Mol. Biol. 328, 429–438 (2003)

    Article  CAS  Google Scholar 

  9. Antonyuk, S. V., Strange, R. W., Sawers, G., Eady, R. R. & Hasnain, S. S. Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism. Proc. Natl Acad. Sci. USA 102, 12041–12046 (2005)

    Article  ADS  CAS  Google Scholar 

  10. Godden, J. W. et al. The 2.3 angstrom Xray structure of nitrite reductase from Achromobacter cycloclastes. Science 253, 438–442 (1991)

    Article  ADS  CAS  Google Scholar 

  11. Tocheva, E. I., Rosell, F. I., Mauk, A. G. & Murphy, M. E. P. Side-on copper-nitrosyl coordination by nitrite reductase. Science 304, 867–870 (2004)

    Article  ADS  CAS  Google Scholar 

  12. Strange, R. W. et al. The substrate binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase. Nature Struct. Biol. 2, 287–292 (1995)

    Article  CAS  Google Scholar 

  13. Strange, R. W. et al. Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase. J. Mol. Biol. 287, 1001–1009 (1999)

    Article  CAS  Google Scholar 

  14. Wijma, H. J., Jeuken, L. J. C., Verbeet, M., Ph, Armstrong, F. A. & Canters, G. W. Protein film voltammetry of copper-containing nitrite reductase reveals reversible inactivation. J. Am. Chem. Soc. 128, 8557–8565 (2007)

    Article  Google Scholar 

  15. Bertini, I. I. & Cavallaro, G. G. Cytochrome c: occurrence and functions. Chem. Rev. 106, 90–115 (2006)

    Article  CAS  Google Scholar 

  16. Ellis, M. J., Grossmann, J. G., Eady, R. R. & Hasnain, S. S. Genomic analysis reveals widespread occurrence of new classes of copper nitrite reductases. J. Biol. Inorg. Chem. 12, 1119–1127 (2007)

    Article  CAS  Google Scholar 

  17. Nojiri, M. et al. Structure and function of a hexameric copper-containing nitrite reductase. Proc. Natl Acad. Sci. USA 104, 4315–4320 (2007)

    Article  ADS  CAS  Google Scholar 

  18. Yamaguchi, K. et al. Characterization of two type 1 Cu sites of Hyphomicrobium denitrificans nitrite reductase: a new class of copper-containing nitrite reductases. Biochemistry 43, 14180–14188 (2004)

    Article  CAS  Google Scholar 

  19. Han, C. et al. Characterization of a novel copper heme c dissimilatory nitrite reductase from Ralstonia pickettii. Biochem. J. 444, 219–226 (2012)

    Article  CAS  Google Scholar 

  20. Lyons, J. A. et al. Structural insights into electron transfer in caa3-type cytochrome oxidase. Nature 487, 514–518 (2012)

    Article  ADS  CAS  Google Scholar 

  21. de la Lande, A., Babcock, N. S., Rezac, J., Sanders, B. C. & Salahub, D. R. Surface residues dynamically organize water bridges to enhance electron transfer between proteins. Proc. Natl Acad. Sci. USA 107, 11799–11804 (2010)

    Article  ADS  CAS  Google Scholar 

  22. Lin, J., Balabin, A. & Beratan, D. N. The nature of aqueous tunneling pathways between electron-transfer proteins. Science 310, 1311–1313 (2005)

    Article  ADS  CAS  Google Scholar 

  23. Hough, M. A., Eady, R. R. & Hasnain, S. S. Identification of the proton channel to the active site type 2 Cu centre of nitrite reductase: structural and enzymatic properties of His254Phe and Asn90Ser mutants. Biochemistry 47, 13547–13553 (2008)

    Article  CAS  Google Scholar 

  24. Boulanger, M. J., Kukimoto, M., Nishiyama, M., Horinouchi, S. & Murphy, M. E. Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase. J. Biol. Chem. 275, 23957–23964 (2000)

    Article  CAS  Google Scholar 

  25. Otwinowski, Z. & Minor, W. in Methods in Enzymology: Macromolecular Crystallography A (eds Carter, C. W. & Sweet, R. M. ) 307–326 (Academic Press, 1997)

    Book  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Collaborative Computational Project, Number 4. The CCP4 Suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  28. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  29. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  30. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: A program to check the stereochemical quality of protein structures. J. App. Crystallogr. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  31. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council, UK (grant number BB/G005869/1 (to S.S.H. & R.R.E.)). S.V.A. acknowledges the support from the Wellcome Trust (grant number 097826/Z/11/Z). We thank the staff and management of SOLEIL and Diamond for the provision of crystallographic facilities at their synchrotron centres. We thank the members of the molecular biophysics groups, particularly R. Strange and G. Wright, for their help.

Author information

Author notes

  1. Svetlana V. Antonyuk and Cong Han: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZX, UK,

    Svetlana V. Antonyuk, Cong Han, Robert R. Eady & S. Samar Hasnain

Authors
  1. Svetlana V. Antonyuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cong Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert R. Eady
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. Samar Hasnain
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.V.A., R.R.E. and S.S.H. conceived and designed the project; C.H. cloned, expressed and purified proteins; S.V.A. and C.H. crystallized the proteins; S.V.A. did data processing, structure determination and refinement; S.V.A., R.R.E. and S.S.H. wrote the manuscript.

Corresponding authors

Correspondence to Svetlana V. Antonyuk or S. Samar Hasnain.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures 1-4. (PDF 2567 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Antonyuk, S., Han, C., Eady, R. et al. Structures of protein–protein complexes involved in electron transfer. Nature 496, 123–126 (2013). https://doi.org/10.1038/nature11996

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11996

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