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:

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site

Nature Structural Biology volume 7, pages 910–917 (2000)Cite this article

Abstract

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.

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
Figure 2: Electron density maps of ubiquinol oxidase from E. coli.
Figure 3: Overall structures of ubiquinol oxidase from E. coli and cytochrome c oxidase from P. denitrificans.
Figure 4: The binuclear center and the possible proton pathways, the D- and K- channels, in subunit I.
Figure 5: The extrinsic domain of subunit II.
Figure 6: A possible ubiquinone binding site in ubiquinol oxidase.
Figure 7: Sequence alignment of transmembrane helices I and II in subunit I displaying the conserved polar residues of cytochrome bo3.
Figure 8: Charge translocation in reconstituted ubiquinol oxidase proteoliposomes.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Babcock, G.T. & Wikström, M. Oxygen activation and the conservation of energy in cell respiration. Nature 356, 301–309 (1992).

    Article  CAS  Google Scholar 

  2. Iwata, S. Structure and function of bacterial cytochrome c oxidase. J. Biochem. 123, 369–375 (1998).

    Article  CAS  Google Scholar 

  3. Garcia-Horseman, J.A., Barquera, B., Rumbley, J., Ma, J. & Gennis, R.B. The superfamily of heme-copper respiratory oxidases. J. Bacteriol. 176, 5587–5600 (1994).

    Article  Google Scholar 

  4. Puustinen, A., Finel, M., Haltia, T., Gennis, R.B. & Wikström, M. Properties of the two terminal oxidases of Escherichia coli. Biochemistry 30, 3936–3942 (1991).

    Article  CAS  Google Scholar 

  5. Lemieux, L.J., Calhoun, M.W., Thomas, J.W., Ingledew, W.J. & Gennis, R.B. Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis. J. Biol. Chem. 267, 2105–2113 (1992).

    CAS  PubMed  Google Scholar 

  6. Wilmanns, M., Lappalainen, P., Kelly, M., Sauer-Eriksson, E. & Saraste, M. Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center. Proc. Natl. Acad. Sci. USA 92, 11955–11959 (1995).

    Article  CAS  Google Scholar 

  7. Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 376, 660–669 (1995).

    Article  CAS  Google Scholar 

  8. Navaza, J. AmoRe: an automated package for molecular replacement. Acta Crystallogr. D 50, 1157–1163 (1994).

    Google Scholar 

  9. Cowtan, K. Joint CCP4 and ESF-EACBM newsletter on protein crystallography 31, 34–38 (1994).

  10. Chepuri, V. & Gennis, R.B. The use of gene fusions to determine the topology of all subunits of the cytochrome o terminal oxidase complex of Escherichia coli. J. Biol. Chem. 265, 12978–12986 (1990).

    CAS  PubMed  Google Scholar 

  11. Yoshikawa, S., et al. Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase, Science 280, 1723–1729 (1998).

    Article  CAS  Google Scholar 

  12. Tsukihara, T. et al. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. Science 272, 1136–1144 (1996).

    Article  CAS  Google Scholar 

  13. Ostermeier, C., Harrenga, A., Ermler, U. & Michel, H. Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment. Proc. Natl. Acad. Sci. USA 94, 10547–10553 (1997).

    Article  CAS  Google Scholar 

  14. Tsukihara T. et al. Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 Å. Science 269,1069–1074 (1995).

    Article  CAS  Google Scholar 

  15. Sato-Watanabe, M., et al. Identification of a novel quinone-binding site in the cytochrome bo complex from Escherichia coli. J. Biol. Chem. 269, 28908–28912 (1994).

    CAS  PubMed  Google Scholar 

  16. Puustinen, A., Verkhovsky, M.I., Morgan, J.E., Belevich, N.P. & Wikström, M. Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen. Proc. Natl. Acad. Sci. USA 93, 1545–1548 (1996).

    Article  CAS  Google Scholar 

  17. Tsatsos, P.H. et al. Using matrix-assisted laser desorption ionization mass spectrometry to map the quinol binding site of cytochrome bo3 from Escherichia coli. Biochemistry 37, 9884–9888 (1998).

    Article  CAS  Google Scholar 

  18. Sato-Watanabe, M., Mogi, T., Sakamoto, K., Miyoshi, H. & Anraku, Y. Isolation and characterizations of quinone analogue-resistant mutants of bo-type ubiquinol oxidase from Escherichia coli. Biochemistry 37, 12744–12752 (1998).

    Article  CAS  Google Scholar 

  19. Ma, J. et al. Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase. Biochemistry 38,15150–15156 (1999).

    Article  CAS  Google Scholar 

  20. Iwata, S. et al. Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science 281, 64–71 (1998).

    Article  CAS  Google Scholar 

  21. Lancaster, C.R.D. & Michel, H. The coupling of light-induced electron transfer and proton uptake as derived from crystal structures of reaction centres from Rhodopseudomonas viridis modified at the binding site of the secondary quinone, QB . Structure 5, 1339–13359 (1997).

    Article  CAS  Google Scholar 

  22. Deisenhofer, J., Epp, O., Sinning, I. & Michel, H. Crystallographic refinement at 2.3 Å resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis. J. Mol. Biol. 246, 429–457 (1995).

    Article  CAS  Google Scholar 

  23. Iverson, T.M., Luna-Chavez, C., Cecchini, G. & Rees, D.C. Structure of the Escherichia coli fumarate reductase respiratory complex. Science 284, 1961–1966 (1999).

    Article  CAS  Google Scholar 

  24. Jasaitis, A., Verkhovsky, M.I., Morgan, J.E., Verkhovskaya, M.L. & Wikström, M. Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen. Biochemistry 38, 2697–2706 (1999).

    Article  CAS  Google Scholar 

  25. Sato-Watanabe, M., Mogi, T., Miyoshi, H. & Anraku, Y. Characterization and functional role of the QH site of bo-type ubiquinol oxidase from Escherichia coli. Biochemistry 37, 5356–5361 (1998).

    Article  CAS  Google Scholar 

  26. Murray, L., Pires, R.H., Hastings, S.F. & Ingledew, W.J. Models for structure and function in quinone-binding sites: the Escherichia coli quinol oxidase, cytochrome bo3 . Trans. Biochem. Soc. 27, 581–585 (1999).

    Article  CAS  Google Scholar 

  27. Rich, P. & Fisher, N. Generic features of quinone-binding sites. Trans. Biochem. Soc. 27, 561–565 (1999).

    Article  CAS  Google Scholar 

  28. Ding, H., Robertson, D.E., Daldal, F. & Dutton, P.L. Cytochrome bc 1 complex [2Fe-2S] cluster and its interaction with ubiquinone and ubihydroquinone at the Qo site: a double-occupancy Qo site model. Biochemistry 31, 3144–3158 (1992).

    Article  CAS  Google Scholar 

  29. Ingledew, W.J., Ohnishi, T. & Salerno, J.C. Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo. Eur. J. Biochem. 227, 903–908 (1995).

    Article  CAS  Google Scholar 

  30. Rumbley, J.N., Nickels, E.F. & Gennis, R.B. One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase. Biochim. Biophys. Acta 1340, 131–142 (1997).

    Article  CAS  Google Scholar 

  31. Abramson, J., et al. Purification, crystallization and preliminary crystallographic studies on an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli. Acta Crystallogr. D 56 1076–1078 (2000).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  33. The CPP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

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

    Article  Google Scholar 

  35. Verkhovskaya, M.L., et al. Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation. Proc. Natl. Acad. Sci. USA 94, 10128–10131 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Adams, P.D., Pannu, N.S., Read, R.J. & Brunger, A.T. Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement. Proc. Natl. Acad. Sci. USA 94, 5018–5023 (1997).

    Article  CAS  Google Scholar 

  38. Esnouf, R.M. An extensively modified version of molscript that includes greatly enhanced coloring capabilities. J. Mol. Graph. 15, 132–134 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the EU Biotechnology Program and grants from the Academy of Finland, University of Helsinki (to M.W.), and the Swedish Research Councils, NFR and MFR (to S.I.). We are grateful to J. Hajdu and M.I. Verkhovsky for helpful discussions, B. Byrne and M. Nervall for figure preparation; and to A. Garcia-Horsman for contributions at an early stage of this work. We would also like to thank Hoffman-LaRoche, Switzerland, for the kind gift of ubiquinone-1.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Uppsala University, Biomedical Center Box 576, Uppsala, S-75123, Sweden

    Jeff Abramson, Gisela Larsson, Margareta Svensson-Ek & So Iwata

  2. Department of Medical Chemistry, Helsinki Bioenergetics Group, Institute of Biomedical Sciences and Biocentrum Helsinki, P.O. Box 8, FI-00014 University of Helsinki, Helsinki, Finland

    Sirpa Riistama, Audrius Jasaitis, Liisa Laakkonen, Anne Puustinen & Mårten Wikström

  3. Department of Biochemistry and Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London, SW7 2AY, UK

    So Iwata

Authors
  1. Jeff Abramson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sirpa Riistama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gisela Larsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Audrius Jasaitis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Margareta Svensson-Ek
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Liisa Laakkonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anne Puustinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. So Iwata
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mårten Wikström
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to So Iwata or Mårten Wikström.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Abramson, J., Riistama, S., Larsson, G. et al. The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site. Nat Struct Mol Biol 7, 910–917 (2000). https://doi.org/10.1038/82824

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/82824

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