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:

Type-zero copper proteins

Nature Chemistry volume 1, pages 711–715 (2009)Cite this article

Abstract

Many proteins contain copper in a range of coordination environments, where it has various biological roles, such as transferring electrons or activating dioxygen. These copper sites can be classified by their function or spectroscopic properties. Those with a single copper atom are either type 1, with an intense absorption band near 600 nm, or type 2, with weak absorption in the visible region. We have built a novel copper(ii) binding site within structurally modified Pseudomonas aeruginosa azurins that does not resemble either existing type, which we therefore call ‘type zero’. X-ray crystallographic analysis shows that these sites adopt distorted tetrahedral geometries, with an unusually short Cu–O (G45 carbonyl) bond. Relatively weak absorption near 800 nm and narrow parallel hyperfine splittings in electron paramagnetic resonance spectra are the spectroscopic signatures of type zero copper. Cyclic voltammetric experiments demonstrate that the electron transfer reactivities of type-zero azurins are enhanced relative to that of the corresponding type 2 (C112D) protein.

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: The distorted tetrahedral coordination sphere of C112D/M121X (X = L,F,I) azurins features a relatively short Cu–O(G45 carbonyl) bond.
Figure 2: The position of D112 shifts among the proteins, leading to variations in hydrogen bonding to the carboxylate.
Figure 3: XANES of C112D (black) and C112D/M121X (X = L, green; F, orange; I, purple) azurins with focus on the 8,979 eV and 8,987 eV pre-edge features.
Figure 4: Cu(ii/i)-electrode electron-transfer rates (ks) obtained for C112D (black), C112D/M121L (green), and C112D/M121I (purple) azurins from analysis of cyclic voltammetry data (Au–SAM electrochemistry).

Similar content being viewed by others

References

  1. Gray, H. B. Biological inorganic chemistry at the beginning of the 21st century. Proc. Natl Acad. Sci. USA 100, 3563–3568 (2003).

    Article  CAS  Google Scholar 

  2. Gradinaru, C., Crane, B. R., Abrahamsson, M. L. & Gray, H. B. Electron transfer in metalloproteins (blue copper azurin). Biophys. J. 86, 473A (2004).

    Article  Google Scholar 

  3. Roberts, S. A. et al. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proc. Natl Acad. Sci. USA 99, 2766–2771 (2002).

    Article  CAS  Google Scholar 

  4. Palmer, A. E. et al. Spectroscopic characterization and O-2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p. Biochemistry 41, 6438–6448 (2002).

    Article  CAS  Google Scholar 

  5. Solomon, E. I., Szilagyi, R. K., DeBeer George, S. & Basumallick, L. Electronic structures of metal sites in proteins and models: Contributions to function in blue copper proteins. Chem. Rev. 104, 419–458 (2004).

    Article  CAS  Google Scholar 

  6. Solomon, E. I. Spectroscopic methods in bioinorganic chemistry: Blue to green to red copper sites. Inorg. Chem. 45, 8012–8025 (2006).

    Article  CAS  Google Scholar 

  7. Lee, S. K. et al. Nature of the intermediate formed in the reduction of O2 to H2O at the trinuclear copper cluster active site in native laccase. J. Am. Chem. Soc. 124, 6180–6193 (2002).

    Article  CAS  Google Scholar 

  8. 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  CAS  Google Scholar 

  9. Mizoguchi, T. J., Di Bilio, A. J., Gray, H. B. & Richards, J. H. Blue to type 2 binding. Copper(ii) and cobalt (ii) derivatives of a Cys112Asp mutant of Pseudomonas aeruginosa azurin. J. Am. Chem. Soc. 114, 10076–10078 (1992).

    Article  CAS  Google Scholar 

  10. Mizoguchi, T. J. Probing the role of the active site cysteine of azurin by site-directed mutagenesis. PhD thesis, California Institute of Technology (1996).

    Google Scholar 

  11. Lancaster, K. M., Yokoyama, K., Richards, J. H., Winkler, J. R. & Gray, H. B. High-potential C112D/M121X (X = M, E, H, L) Pseudomonas aeruginosa azurins. Inorg. Chem. 48, 1278–1280 (2009).

    Article  CAS  Google Scholar 

  12. 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 

  13. Vaguine, A. A., Richelle, J. & Wodak, S. J. SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr. D 55, 191–205 (1999).

    Article  CAS  Google Scholar 

  14. Engman, K. C., Sandberg, A., Leckner, J. & Karlsson, B. G. Probing the influence on folding behavior of structurally conserved core residues in P-aeruginosa apo-azurin. Protein Sci. 13, 2706–2715 (2004).

    Article  CAS  Google Scholar 

  15. Faham, S. et al. Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein. J. Biol. Inorg. Chem. 2, 464–469 (1997).

    Article  CAS  Google Scholar 

  16. Gray, H. B., Malmstrom, B. G. & Williams, R. J. P. Copper coordination in blue proteins. J. Biol. Inorg. Chem. 5, 551–559 (2000).

    Article  CAS  Google Scholar 

  17. DeBeer, S. et al. X-ray absorption spectra of the oxidized and reduced forms of C112D azurin from Pseudomonas aeruginosa. Inorg. Chem. 38, 433–438 (1999).

    Article  CAS  Google Scholar 

  18. Hitchman, M. A., Olson, C. D. & Belford, R. L. Behavior of in-plane g-tensor in low-symmetry d1 and d9 systems with application to copper and vanadyl chelates. J. Chem. Phys. 50, 1195–1203 (1969).

    Article  CAS  Google Scholar 

  19. Gewirth, A. A., Cohen, S. L., Schugar, H. J. & Solomon, E. I. Spectroscopic and theoretical studies of the unusual electron-paramagnetic-resonance parameters of distorted tetrahedral cupric sites - correlations to X-ray spectral features of core levels. Inorg. Chem. 26, 1133–1146 (1987).

    Article  CAS  Google Scholar 

  20. Mabbs, F. E. & Machin, D. J. Magnetism and Transition Metal Complexes (Dover Publications, 2008).

    Google Scholar 

  21. Stevens, K. W. H. On The magnetic properties of covalent XY6 complexes. Proc. R. Soc. Lond. A 219, 542–555 (1953).

    Article  CAS  Google Scholar 

  22. Griffiths, J. H. E. & Owen, J. Complex hyperfine structures in microwave spectra of covalent iridium compounds. Proc. R. Soc. Lond. A 226, 96–111 (1954).

    Article  CAS  Google Scholar 

  23. Fujita, K. et al. Mimicking protein-protein electron transfer: Voltammetry of Pseudomonas aeruginosa azurin and the Thermus thermophilus Cu-A domain at omega-derivatized self-assembled-monolayer gold electrodes. J. Am. Chem. Soc. 126, 13954–13961 (2004).

    Article  CAS  Google Scholar 

  24. Ghosh, S., Xie, X., Dey, A., Sun, Y., Scholes, C. P. & Solomon, E. I. Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function. Proc. Natl Acad. Sci. USA 106, 4969–4974 (2009).

    Article  CAS  Google Scholar 

  25. Miura, Y. et al. Direct electrochemistry of CueO and its mutants at residues to and near Type I Cu for oxygen-reducing biocathode. Fuel Cells 9, 70–78 (2009).

    Article  CAS  Google Scholar 

  26. Tsujimura, S., Miura, Y. & Kano, K. CueO-immobilized porous carbon electrode exhibiting improved performance of electrochemical reduction of dioxygen to water. Electrochimica Acta 53, 5716–5720 (2008).

    Article  CAS  Google Scholar 

  27. Miura, Y. et al. Bioelectrocatalytic reduction of O-2 catalyzed by CueO from Escherichia coli adsorbed on a highly oriented pyrolytic graphite electrode. Chem. Lett. 36, 132–133 (2007).

    Article  CAS  Google Scholar 

  28. Blanford, C. F., Foster, C. E., Heath, R. S. & Armstrong, F. A. Efficient electrocatalytic oxygen reduction by the ‘blue’ copper oxidase, laccase, directly attached to chemically modified carbons. Faraday Discuss. 140, 319–335 (2008).

    Article  CAS  Google Scholar 

  29. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 26 (1992).

  30. CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  31. Cohen, S. X. et al. ARP/wARP and molecular replacement: the next generation. Acta Crystallogr. D 64, 49–60 (2008).

    Article  CAS  Google Scholar 

  32. Golombek, A. P. & Hendrich, M. P. Quantitative analysis of dinuclear manganese(ii) EPR spectra. J. Magn. Reson. 165, 33–48 (2003).

    Article  CAS  Google Scholar 

  33. George, G. N. EXAFSPAK (Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Stanford University).

  34. Tenderhold, A. PySpline (Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Stanford University).

  35. DeLeon, J. M., Rehr, J. J., Zabinsky, S. I. & Albers, R. C. Abinitio curved-wave X-ray-absorption fine-structure. Phys. Rev. B 44, 4146–4156 (1991).

    Article  Google Scholar 

  36. Rehr, J. J., DeLeon, J. M., Zabinsky, S. I. & Albers, R. C. Theoretical X-ray absorption fine-structure standards. J. Am. Chem. Soc. 113, 5135–5140 (1991).

    Article  CAS  Google Scholar 

  37. Yokoyama, K. et al. Electron tunneling through Pseudomonas aeruginosa azurins on SAM gold electrodes. Inorg. Chim. Acta 361, 1095–1099 (2008).

    Article  CAS  Google Scholar 

  38. Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J. Electroanal. Chem. 101, 19–28 (1979).

    Article  CAS  Google Scholar 

  39. Pascher, T., Karlsson, B. G., Nordling, M., Malmstrom, B. G. & Vanngard, T. Reduction potentials and their pH dependence in site-directed-mutant forms of azurin from Pseudomonas aeruginosa. Eur. J. Biochem. 212, 289–296 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Brunschwig for assistance with Fourier transform infrared spectroscopy, Z. Gates and L. Thomas for assistance with X-ray diffraction data collection, and M. Day and J. Kaiser for discussions of crystal structural analyses. We thank E. Solomon for helpful comments on electronic structural formulations, and Y. Sheng for assistance with protein expression and purification. Stanford Synchrotron Radiation Lightsource operations are funded by DOE(BES). The Structural Molecular Biology program is supported by NIH (NCRR BMTP) (Grant Number 5 P41 RR001209)N and DOE(BER). This work was supported by NIH DK019038(HBG), Stanford GCEP, and NSF CHE-0802907. The Caltech Molecular Observatory is supported by the Gordon and Betty Moore Foundation.

Author information

Authors and Affiliations

  1. Beckman Institute, California Institute of Technology, Pasadena, 91125, California, USA

    Kyle M. Lancaster, Keiko Yokoyama, John H. Richards & Harry B. Gray

  2. Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, 94025, California, USA

    Serena DeBeer George

  3. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, 14853, New York, USA

    Serena DeBeer George

Authors
  1. Kyle M. Lancaster
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Serena DeBeer George
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keiko Yokoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John H. Richards
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Harry B. Gray
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.M.L. and H.B.G. conceived and designed the experiments; K.M.L., S.D.G., and K.Y. performed the experiments; K.M.L., S.D.G., K.Y., and H.B.G. analysed the data, K.M.L., S.D.G., J.H.R., and H.B.G. co-wrote the paper.

Corresponding authors

Correspondence to John H. Richards or Harry B. Gray.

Supplementary information

Supplementary information

Supplementary information (PDF 2637 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lancaster, K., George, S., Yokoyama, K. et al. Type-zero copper proteins. Nature Chem 1, 711–715 (2009). https://doi.org/10.1038/nchem.412

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.412

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