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Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases

Abstract

Haem–copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O–O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O–O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical–biological basis for the design of future oxygen-reduction catalysts.

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Figure 1: Structure and function of FeBMb variants.
Figure 2: Electrochemical, spectroscopic and kinetic investigation of FeBMb variants.

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References

  1. Ferguson-Miller, S. & Babcock, G. T. Heme/copper terminal oxidases. Chem. Rev. 96, 2889–2907 (1996).

    CAS  PubMed  Google Scholar 

  2. Namslauer, A. & Brzezinski, P. Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase. FEBS Lett. 567, 103–1110 (2004).

    CAS  PubMed  Google Scholar 

  3. Kaila, V. R. I., Verkhovsky, M. I. & Wikström, M. Proton-coupled electron transfer in cytochrome oxidase. Chem. Rev. 110, 7062–707081 (2010).

    CAS  PubMed  Google Scholar 

  4. Wikström, M. Cytochrome c oxidase: 25 years of the elusive proton pump. Biochim. Biophys. Acta 1655, 241–2247 (2004).

    PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  6. Castresana, J. & Saraste, M. Evolution of energetic metabolism: the respiration-early hypothesis. Trends Biochem. Sci. 20, 443–4448 (1995).

    CAS  PubMed  Google Scholar 

  7. Sousa, F. L. et al. The superfamily of heme–copper oxygen reductases: types and evolutionary considerations. Biochim. Biophys. Acta Bioenerg. 1817, 629–6637 (2012).

    CAS  Google Scholar 

  8. Sousa, F. L. et al. Early bioenergetic evolution. Phil. Trans. R. Soc. B 368, 2013008 (2013).

    Google Scholar 

  9. Flock, U., Watmough, N. J. & Ädelroth, P. Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen. Biochemistry 44, 10711–10719 (2005).

    CAS  PubMed  Google Scholar 

  10. Brzezinski, P. & Gennis, R. B. Cytochrome c oxidase: exciting progress and remaining mysteries. J. Bioenerg. Biomembr. 40, 521–5531 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Konstantinov, A. A. Cytochrome c oxidase: intermediates of the catalytic cycle and their energy-coupled interconversion. FEBS Lett. 586, 630–639 (2012).

    CAS  PubMed  Google Scholar 

  12. Kim, E., Chufan, E. E., Kamaraj, K. & Karlin, K. D. Synthetic models for heme–copper oxidases. Chem. Rev. 104, 1077–1133 (2004).

    CAS  PubMed  Google Scholar 

  13. Collman, J. P. & Ghosh, S. Recent applications of a synthetic model of cytochrome c oxidase: beyond functional modeling. Inorg. Chem. 49, 5798–5810 (2010).

    CAS  PubMed  Google Scholar 

  14. Hematian, S., Garcia-Bosch, I. & Karlin, K. D. Synthetic heme/copper assemblies: toward an understanding of cytochrome c oxidase interactions with dioxygen and nitrogen oxide. Acc. Chem. Res. 48, 2462–2474 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Collman, J. P., Dey, A., Yang, Y., Ghosh, S. & Decreau, R. A. O2 reduction by a functional heme/nonheme bis-iron NOR model complex. Proc. Natl Acad. Sci. USA 106, 10528–10533 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Raven, E. L. Designer haem proteins: what can we learn from protein engineering? Heteroatom Chem. 13, 501–505 (2002).

    CAS  Google Scholar 

  17. Gibney, B. R. & Tommos, C. De novo protein design in respiration and photosynthesis. Adv. Photosynth. Respir. 22, 729–751 (2005).

    CAS  Google Scholar 

  18. Korendovych, I. V. et al. Design of a switchable eliminase. Proc. Natl Acad. Sci. USA 108, 6823–6827 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Gibney, B. R. in Protein Folding and Metal Ions—Mechanisms, Biology and Disease (eds Gomes, C. & Wittung-Stafshede, P.) 227–245 (Taylor & Francis, 2011).

    Google Scholar 

  20. Zastrow, M. L. & Pecoraro, V. L. Designing functional metalloproteins: from structural to catalytic metal sites. Coord. Chem. Rev. 257, 2565–2588 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Yu, F. et al. Protein design: toward functional metalloenzymes. Chem. Rev. 114, 3495–3578 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Rufo, C. M. et al. Short peptides self-assemble to produce catalytic amyloids. Nat. Chem. 6, 303–309 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Sigman, J. A., Kwok, B. C., Gengenbach, A. & Lu, Y. Design and creation of a Cu(II)-binding site in cytochrome c peroxidase that mimics the CuB–heme center in terminal oxidases. J. Am. Chem. Soc. 121, 8949–8950 (1999).

    CAS  Google Scholar 

  24. Yeung, N. et al. Rational design of a structural and functional nitric oxide reductase. Nature 462, 1079–1082 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Yang, Y. et al. Direct EPR observation of a tyrosyl radical in a functional oxidase model in myoglobin during both H2O2 and O2 reactions. J. Am. Chem. Soc. 136, 1174–1177 (2014).

    Google Scholar 

  26. Yang, Y. et al. Defining the role of tyrosine and rational tuning of oxidase activity by genetic incorporation of unnatural tyrosine analog. J. Am. Chem. Soc. 137, 4594–4597 (2015).

    Google Scholar 

  27. Yang, Y. et al. A designed metalloenzyme achieving the catalytic rate of a native enzyme. J. Am. Chem. Soc. 137, 11570–11573 (2015).

    Google Scholar 

  28. Mukherjee, S. et al. A biosynthetic model of cytochrome c oxidase as an electrocatalyst for O2 reduction. Nat. Commun. 6, 8467 (2015).

    CAS  PubMed  Google Scholar 

  29. Lin, Y.-W. et al. Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin. Proc. Natl Acad. Sci. USA 107, 8581–8586 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Chakraborty, S. et al. Recent advances in biosynthetic modeling of nitric oxide reductases and insights gained from nuclear resonance vibrational and other spectroscopic studies. Inorg. Chem. 54, 9317–9329 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Miner, K. D. et al. A designed functional metalloenzyme that reduces O2 to H2O with over one thousand turnovers. Angew. Chem. Int. Ed. 51, 5589–5592 (2012).

    CAS  Google Scholar 

  32. Brantley, R. E. Jr, Smerdon, S. J., Wilkinson, A. J., Singleton, E. W. & Olson, J. S. The mechanism of autooxidation of myoglobin. J. Biol. Chem. 268, 6995–7010 (1993).

    CAS  PubMed  Google Scholar 

  33. Bhagi-Damodaran, A., Petrik, I. D., Marshall, N. M., Robinson, H. & Lu, Y. Systematic tuning of heme redox potentials and its effects on O2 reduction rates in a designed oxidase in myoglobin. J. Am. Chem. Soc. 136, 11882–11885 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Reedy, C. J. & Gibney, B. R. Heme protein assemblies. Chem. Rev. 104, 617–649 (2004).

    CAS  PubMed  Google Scholar 

  35. Chakraborty, S. et al. Spectroscopic and computational study of a nonheme iron nitrosyl center in a biosynthetic model of nitric oxide reductase. Angew. Chem. Int. Ed. 126, 2449–2453 (2014).

    Google Scholar 

  36. Butland, G., Spiro, S., Watmough, N. J. & Richardson, D. J. Two conserved glutamates in the bacterial nitric oxide reductase are essential for activity but not assembly of the enzyme. J. Bacteriol. 183, 189–199 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Bolgiano, B., Salmon, I., Ingledew, W. J. & Poole, R. K. Redox analysis of the cytochrome o-type quinol oxidase complex of Escherichia coli reveals three redox components. Biochem. J. 274, 723–730 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Ellis, W. R., Wang, H., Blair, D. F., Gray, H. B. & Chan, S. I. Spectroelectrochemical study of the cytochrome a site in carbon monoxide inhibited cytochrome c oxidase. Biochemistry 25, 161–167 (1986).

    CAS  PubMed  Google Scholar 

  39. Ibrahim, M., Denisov, I. G., Makris, T. M., Kincaid, J. R. & Sligar, S. G. Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures. J. Am. Chem. Soc. 125, 13714–13718 (2003).

    CAS  PubMed  Google Scholar 

  40. Chen, H., Ikeda-Saito, M. & Shaik, S. Nature of the Fe–O2 bonding in oxy-myoglobin: effect of the protein. J. Am. Chem. Soc. 130, 14778–14790 (2008).

    CAS  PubMed  Google Scholar 

  41. Unno, M., Chen, H., Kusama, S., Shaik, S. & Ikeda-Saito, M. Structural characterization of the fleeting ferric peroxo species in myoglobin: experiment and theory. J. Am. Chem. Soc. 129, 13394–13395 (2007).

    CAS  PubMed  Google Scholar 

  42. Garcia-Serres, R. et al. Distinct reaction pathways followed upon reduction of oxy-heme oxygenase and oxy-myoglobin as characterized by Mössbauer spectroscopy. J. Am. Chem. Soc. 129, 1402–1412 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Spiro, T. G., Soldatova, A. V. & Balakrishnan, G. CO, NO and O2 as vibrational probes of heme protein interactions. Coord. Chem. Rev. 257, 511–527 (2013).

    CAS  PubMed  Google Scholar 

  44. Raven, E. L. & Mauk, A. G. Chemical reactivity of the active site of myoglobin. Adv. Inorg. Chem. 51, 1–49 (2001).

    CAS  Google Scholar 

  45. Noodleman, L., Han Du, W.-G., Fee, J. A., Gotz, A. W. & Walker, R. C. Linking chemical electron–proton transfer to proton pumping in cytochrome c oxidase: broken-symmetry DFT exploration of intermediates along the catalytic reaction pathway of the iron–copper dinuclear complex. Inorg. Chem. 53, 6458–6472 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Babcock, G. T., Varotsis, C. & Zhang, Y. Oxygen activation in cytochrome oxidase and in other heme proteins. Biochim. Biophys. Acta 1101, 192–194 (1992).

    CAS  PubMed  Google Scholar 

  47. Babcock, G. T. How oxygen is activated and reduced in respiration. Proc. Natl Acad. Sci. USA 96, 12971–12973 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Saraste, M. & Castresana, J. Cytochrome oxidase evolved by tinkering with denitrification enzymes. FEBS Lett. 341, 1–4 (1994).

    CAS  PubMed  Google Scholar 

  49. Liu, X. et al. Significant increase of oxidase activity through the genetic incorporation of a tyrosine–histidine cross-link in a myoglobin model of heme–copper oxidase. Angew. Chem. Int. Ed. 51, 4312–4316 (2012).

    CAS  Google Scholar 

  50. Marshall, N. M. et al. Rationally tuning the reduction potential of a single cupredoxin beyond the natural range. Nature 462, 113–117 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang, Y. & Oldfield, E. On the Mössbauer spectra of isopenicillin n synthase and a model {FeNO}7 (S = 3/2) system. J. Am. Chem. Soc. 126, 9494–9495 (2004).

    CAS  PubMed  Google Scholar 

  52. Zhang, Y. & Oldfield, E. NMR hyperfine shifts in blue copper proteins: a quantum chemical investigation. J. Am. Chem. Soc. 130, 3814–3823 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Ling, Y. & Zhang, Y. Mössbauer, NMR, geometric, and electronic properties in S = 3/2 iron porphyrins. J. Am. Chem. Soc. 131, 6386–6388 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Yang, L., Ling, Y. & Zhang, Y. HNO binding in a heme protein: structures, spectroscopic properties, and stabilities. J. Am. Chem. Soc. 133, 13814–13817 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank A. R. Damodaran and R. B. Gennis for helpful comments and suggestions on the manuscript. We also thank P. Hosseinzadeh, M. R. Sponholtz and S. Dwaraknath for help with various aspects of data collection and analysis. J. R. was supported by a predoctoral training grant 5T32-GM070421 from the US National Institute of Health. This material is based on work supported by the US National Institutes of Health (NIH) under Award NIH R01GM06211 (to Y.L.) and NIH R01GM074785 (to P.M.-L.) and by a US National Science Foundation (NSF) Award (NSF CHE-1300912 to Y.Z.). 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 under Contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research, and by the NIH National Institute of General Medical Sciences (NIGMS) (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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A.B.-D. and Y.L. designed the research. A.B.-D., J.R. and B.A.S. purified and characterized the proteins, performed the oxygen-reduction assays, and the electrochemical, EPR and stopped-flow experiments. E.M. performed the XANES study and analysed the data. S.C. helped with the initial assay design, M.A.M. and Y.Z. performed the DFT calculations and Q.Z. and P.M.-L. performed the RR experiments. A.B.D. Y.Z., P.M.-L. and Y.L. wrote the paper. All the authors contributed to developing the rationale of the manuscript and analysis of results.

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Correspondence to Pierre Moënne-Loccoz, Yong Zhang or Yi Lu.

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Bhagi-Damodaran, A., Michael, M., Zhu, Q. et al. Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases. Nature Chem 9, 257–263 (2017). https://doi.org/10.1038/nchem.2643

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