A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage

Subjects

Abstract

Hydrogenases are nature's efficient catalysts for both the generation of energy via oxidation of molecular hydrogen and the production of hydrogen via the reduction of protons. However, their O2 sensitivity and deactivation at high potential limit their applications in practical devices, such as fuel cells. Here, we show that the integration of an O2-sensitive hydrogenase into a specifically designed viologen-based redox polymer protects the enzyme from O2 damage and high-potential deactivation. Electron transfer between the polymer-bound viologen moieties controls the potential applied to the active site of the hydrogenase and thus insulates the enzyme from excessive oxidative stress. Under catalytic turnover, electrons provided from the hydrogen oxidation reaction induce viologen-catalysed O2 reduction at the polymer surface, thus providing self-activated protection from O2. The advantages of this tandem protection are demonstrated using a single-compartment biofuel cell based on an O2-sensitive hydrogenase and H2/O2 mixed feed under anode-limiting conditions.

Figure 1: Inactivation pathways of a [NiFe] hydrogenase and reaction layers inside a hydrogenase/viologen hydrogel.
Figure 2: Electrochemical and spectroelectrochemical experiments demonstrating protection of the hydrogenase from high potential and O2 by the redox hydrogel.
Figure 3: Biofuel cell performance test.

References

  1. 1

    Armstrong, F. A. Copying biology's ways with hydrogen. Science 339, 658–659 (2013).

    CAS  Article  Google Scholar 

  2. 2

    Lubitz, W., Ogata, H., Rüdiger, O. & Reijerse, E. Hydrogenases. Chem. Rev. 114, 4081–4148 (2014).

    CAS  Article  Google Scholar 

  3. 3

    Fourmond, V. et al. The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster. Nature Chem. 6, 336–342 (2014).

    CAS  Article  Google Scholar 

  4. 4

    Ogo, S. et al. A functional [NiFe]hydrogenase mimic that catalyzes electron and hydride transfer from H2 . Science 339, 682–684 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Helm, M. L., Stewart, M. P., Bullock, R. M., Rakowski DuBois, M. & DuBois, D. L. A synthetic nickel electrocatalyst with a turnover frequency above 100,000 s−1 for H2 production. Science 333, 863–866 (2011).

    CAS  Article  Google Scholar 

  6. 6

    Le Goff, A. et al. From hydrogenases to noble metal-free catalytic nanomaterials for H2 production and uptake. Science 326, 1384–1387 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Camara, J. M. & Rauchfuss, T. B. Combining acid–base, redox and substrate binding functionalities to give a complete model for the [FeFe]-hydrogenase. Nature Chem. 4, 26–30 (2011).

    Article  Google Scholar 

  8. 8

    Vincent, K. A. et al. Electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels. Proc. Natl Acad. Sci. USA 102, 16951–16954 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Vincent, K. A. et al. Electricity from low-level H2 in still air—an ultimate test for an oxygen tolerant hydrogenase. Chem. Commun. 5033–5035 (2006).

  10. 10

    Tsujimura, S., Fujita, M., Tatsumi, H., Kano, K. & Ikeda, T. Bioelectrocatalysis-based dihydrogen/dioxygen fuel cell operating at physiological pH. Phys. Chem. Chem. Phys. 3, 1331–1335 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Xu, L. & Armstrong, F. A. Optimizing the power of enzyme-based membrane-less hydrogen fuel cells for hydrogen-rich H2–air mixtures. Energy Environ. Sci. 6, 2166–2171 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Krishnan, S. & Armstrong, F. A. Order-of-magnitude enhancement of an enzymatic hydrogen–air fuel cell based on pyrenyl carbon nanostructures. Chem. Sci. 3, 1015–1023 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Cracknell, J. A., Vincent, K. A. & Armstrong, F. A. Enzymes as working or inspirational electrocatalysts for fuel cells and electrolysis. Chem. Rev. 108, 2439–2461 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Abou Hamdan, A. et al. O2-independent formation of the inactive states of NiFe hydrogenase. Nature Chem. Biol. 9, 15–17 (2013).

    Article  Google Scholar 

  15. 15

    Evans, R. M. et al. Principles of sustained enzymatic hydrogen oxidation in the presence of oxygen—the crucial influence of high potential Fe–S clusters in the electron relay of [NiFe]-hydrogenases. J. Am. Chem. Soc. 135, 2694–2707 (2013).

    CAS  Article  Google Scholar 

  16. 16

    Wait, A. F., Parkin, A., Morley, G. M., dos Santos, L. & Armstrong, F. A. Characteristics of enzyme-based hydrogen fuel cells using an oxygen-tolerant hydrogenase as the anodic catalyst. J. Phys. Chem. C 114, 12003–12009 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Tatsumi, H., Takagi, K., Fujita, M., Kano, K. & Ikeda, T. Electrochemical study of reversible hydrogenase reaction of Desulfovibrio vulgaris cells with methyl viologen as an electron carrier. Anal. Chem. 71, 1753–1759 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Eng, L. H. et al. Viologen-based redox polymer for contacting the low-potential redox enzyme hydrogenase at an electrode surface. J. Phys. Chem. 98, 7068–7072 (1994).

    CAS  Article  Google Scholar 

  19. 19

    Karyakin, A. A., Vinogradova, D. V., Morozov, S. V. & Karyakina, E. E. Improvement of enzyme electrocatalysis using substrate containing electroactive polymers. Towards limiting efficiencies of bioelectrocatalysis. Electrochim. Acta 55, 7696–7700 (2010).

    CAS  Article  Google Scholar 

  20. 20

    De Lacey, A. L., Detcheverry, M., Moiroux, J. & Bourdillon, C. Construction of multicomponent catalytic films based on avidin–biotin technology for the electroenzymatic oxidation of molecular hydrogen. Biotechnol. Bioeng. 68, 1–10 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Thorneley, R. N. F. A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus. Biochim. Biophys. Acta 333, 487–496 (1974).

    CAS  Article  Google Scholar 

  22. 22

    Lin, Q., Li, Q., Batchelor-McAuley, C. & Compton, R. G. Use of ‘split waves’ for the measurement of electrocatalytic kinetics: methyl viologen mediated oxygen reduction on a boron-doped diamond electrode. Phys. Chem. Chem. Phys. 15, 7760–7767 (2013).

    CAS  Article  Google Scholar 

  23. 23

    Farrington, J. A., Ebert, M., Land, E. J. & Fletcher, K. Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides. Biochim. Biophys. Acta Bioenerg. 314, 372–381 (1973).

    CAS  Article  Google Scholar 

  24. 24

    Cracknell, J. A., Wait, A. F., Lenz, O., Friedrich, B. & Armstrong, F. A. A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases. Proc. Natl Acad. Sci. USA 106, 20681–20686 (2009).

    Article  Google Scholar 

  25. 25

    Lamle, S. E., Albracht, S. P. J. & Armstrong, F. A. Electrochemical potential-step investigations of the aerobic interconversions of [NiFe]-hydrogenase from Allochromatium vinosum. Insights into the puzzling difference between unready and ready oxidized inactive states. J. Am. Chem. Soc. 126, 14899–14909 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Vincent, K. A., Belsey, N. A., Lubitz, W. & Armstrong, F. A. Rapid and reversible reactions of [NiFe]-hydrogenases with sulfide. J. Am. Chem. Soc. 128, 7448–7449 (2006).

    CAS  Article  Google Scholar 

  27. 27

    Shafaat, H. S., Rüdiger, O., Ogata, H. & Lubitz, W. [NiFe] hydrogenases: a common active site for hydrogen metabolism under diverse conditions. Biochim. Biophys. Acta Bioenerg. 1827, 986–1002 (2013).

    CAS  Article  Google Scholar 

  28. 28

    Lauterbach, L. & Lenz, O. Catalytic production of hydrogen peroxide and water by oxygen-tolerant [NiFe]-hydrogenase during H2 cycling in the presence of O2 . J. Am. Chem. Soc. 135, 17897–17905 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Léger, C., Lederer, F., Guigliarelli, B. & Bertrand, P. Electron flow in multicenter enzymes: theory, applications, and consequences on the natural design of redox chains. J. Am. Chem. Soc. 128, 180–187 (2006).

    Article  Google Scholar 

  30. 30

    Fourmond, V., Infossi, P., Giudici-Orticoni, M.-T., Bertrand, P. & Léger, C. ‘Two-step’ chronoamperometric method for studying the anaerobic inactivation of an oxygen tolerant NiFe hydrogenase. J. Am. Chem. Soc. 132, 4848–4857 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Jones, A. K. et al. Enzyme electrokinetics: electrochemical studies of the anaerobic interconversions between active and inactive states of Allochromatium vinosum [NiFe]-hydrogenase. J. Am. Chem. Soc. 125, 8505–8514 (2003).

    CAS  Article  Google Scholar 

  32. 32

    De Lacey, A. L., Fernández, V. M., Rousset, M. & Cammack, R. Activation and inactivation of hydrogenase function and the catalytic cycle: spectroelectrochemical studies. Chem. Rev. 107, 4304–4330 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Millo, D. et al. Spectroelectrochemical study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F in solution and immobilized on biocompatible gold surfaces. J. Phys. Chem. B 113, 15344–15351 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Fichtner, C., Laurich, C., Bothe, E. & Lubitz, W. Spectroelectrochemical characterization of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F. Biochemistry 45, 9706–9716 (2006).

    CAS  Article  Google Scholar 

  35. 35

    Ciaccafava, A. et al. Electrochemistry, AFM, and PM-IRRA spectroscopy of immobilized hydrogenase: role of a hydrophobic helix in enzyme orientation for efficient H2 oxidation. Angew. Chem. Int. Ed. 51, 953–956 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Ciaccafava, A. et al. An innovative powerful and mediatorless H2/O2 biofuel cell based on an outstanding bioanode. Electrochem. Commun. 23, 25–28 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Ramasamy, R. P., Luckarift, H. R., Ivnitski, D. M., Atanassov, P. B. & Johnson, G. R. High electrocatalytic activity of tethered multicopper oxidase–carbon nanotube conjugates. Chem. Commun. 46, 6045–6047 (2010).

    CAS  Article  Google Scholar 

  38. 38

    Block, E. et al. Synthesis, structure, and chemistry of new, mixed group 14 and 16 heterocycles: nucleophile-induced ring contraction of mesocyclic dications. J. Am. Chem. Soc. 128, 14949–14961 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Yagi, T. et al. Properties of purified hydrogenase from the particulate fraction of Desulfovibrio vulgaris Miyazaki. J. Biochem. 79, 661–671 (1976).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank J. Henig and S. Stapf for help with synthetic aspects, P. Malkowski for purifying the [NiFe] hydrogenase from DvMF, C. Léger for discussions concerning reaction-diffusion layers, as well as the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft for financial support.

Author information

Affiliations

Authors

Contributions

N.P., O.R., W.S. and W.L. conceived the study and co-wrote the paper. N.P. designed and supervised the monomer and polymer synthesis. O.R. designed, executed and interpreted all spectroelectrochemical and direct electrochemistry experiments as well as some mediated electrochemistry experiments. A.A.O. performed polymer synthesis, hydrogel film formation, mediated electrochemistry and biofuel cell experiments. N.P., O.R., A.A.O. and W.S. designed and interpreted mediated electrochemistry and biofuel cell experiments. R.W. executed monomer synthesis. S.P. contributed to polymer synthesis and characterization. J.V. prepared the biocathode for the biofuel cell.

Corresponding authors

Correspondence to Wolfgang Schuhmann or Wolfgang Lubitz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 2737 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Plumeré, N., Rüdiger, O., Oughli, A. et al. A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage. Nature Chem 6, 822–827 (2014). https://doi.org/10.1038/nchem.2022

Download citation

Further reading

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