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.

Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase

Abstract

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Structure of D. fructosovorans NiFe hydrogenase depicting the “dry” hydrophobic cavities.
Figure 2: EPR characterization of the NiFe hydrogenase variants.
Figure 3: The inhibition by O2 of D. fructosovorans NiFe hydrogenase selected mutants and the reaction with CO and O2 of the FeFe hydrogenases from C. acetobutylicum and D. desulfuricans.
Figure 4: Kinetic properties of selected NiFe hydrogenase mutants.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Maynard, E.L. & Lindahl, P.A. Evidence of a molecular tunnel connecting the active sites for CO2 reduction and acetyl-CoA synthesis in acetyl-coa synthase from Clostridium thermoaceticum. J. Am. Chem. Soc. 121, 9221–9222 (1999).

    CAS  Article  Google Scholar 

  2. 2

    Fontecilla-Camps, J.C., Volbeda, A., Cavazza, C. & Nicolet, Y. Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. Chem. Rev. 107, 4273–4303 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Cohen, J., Kim, K., King, P., Seibert, M. & Schulten, K. Finding gas diffusion pathways in proteins: application to O2 and H2 transport in CpI [FeFe]-hydrogenase and the role of packing defects. Structure 13, 1321–1329 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Nienhaus, K., Deng, P., Olson, J.S., Warren, J.J. & Nienhaus, G.U. Structural dynamics of myoglobin: ligand migration and binding in valine 68 mutants. J. Biol. Chem. 278, 42532–42544 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Ruscio, J.Z. et al. Atomic level computational identification of ligand migration pathways between solvent and binding site in myoglobin. Proc. Natl. Acad. Sci. USA 105, 9204–9209 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Cohen, J. & Schulten, K. O2-migration pathways are not conserved across proteins of a similar fold. Biophys. J. 93, 3591–3600 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Salomonsson, L., Lee, A., Gennis, R.B. & Brzezinski, P. A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter. Proc. Natl. Acad. Sci. USA 101, 11617–11621 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Leroux, F. et al. Experimental approaches to kinetics of gas diffusion in hydrogenase. Proc. Natl. Acad. Sci. USA 105, 11188–11193 (2008).

    CAS  Article  Google Scholar 

  9. 9

    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 

  10. 10

    Vincent, K.A. et al. Enzymatic catalysis on conducting graphite particles. Nat. Chem. Biol. 3, 761–762 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Karyakin, A.A. et al. The limiting performance characteristics in bioelectrocatalysis of hydrogenase enzymes. Angew. Chem. Int. Ed. 46, 7244–7246 (2007).

    CAS  Article  Google Scholar 

  12. 12

    Hambourger, M. et al. [FeFe]-hydrogenase-catalyzed H2 production in a photo-electrochemical biofuel cell. J. Am. Chem. Soc. 130, 2015–2022 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Ghirardi, M.L., Dubini, A., Yu, J. & Maness, P.-C. Photobiological hydrogen-producing systems. Chem. Soc. Rev. 38, 52–61 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Volbeda, A., Montet, Y., Vernède, X., Hatchikian, E.C. & Fontecilla-Camps, J.C. High-resolution crystallographic analysis of Desulfovibrio fructosovorans NiFe hydrogenase. Int. J. Hydrogen Energy 27, 1449–1461 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Buhrke, T., Lenz, O., Krauss, N. & Friedrich, B. Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site. J. Biol. Chem. 280, 23791–23796 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Duché, O., Elsen, S., Cournac, L. & Colbeau, A. Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity. FEBS J. 272, 3899–3908 (2005).

    Article  Google Scholar 

  17. 17

    Ludwig, M., Cracknell, J.A., Vincent, K.A., Armstrong, F.A. & Lenz, O. Oxygen-tolerant H2 oxidation by membrane-bound [NiFe]-hydrogenases of Ralstonia species: coping with low-level H2 in air. J. Biol. Chem. 284, 465–477 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Dementin, S. et al. Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant. J. Am. Chem. Soc. 131, 10156–10164 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Baffert, C. et al. Hydrogen-activating enzymes: activity does not correlate with oxygen-sensitivity. Angew. Chem. Int. Ed. 47, 2052–2055 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Vincent, K.A., Parkin, A. & Armstrong, F.A. Investigating and exploiting the electrocatalytic properties of hydrogenases. Chem. Rev. 107, 4366–4413 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Léger, C., Dementin, S., Bertrand, P., Rousset, M. & Guigliarelli, B. Inhibition and aerobic inactivation kinetics of Desulfovibrio fructosovorans NiFe hydrogenases studied by protein film voltammetry. J. Am. Chem. Soc. 126, 12162–12172 (2004).

    Article  Google Scholar 

  22. 22

    Léger, C. & Bertrand, P. Direct electrochemistry of redox enzymes as a tool for mechanistic studies. Chem. Rev. 108, 2379–2438 (2008).

    Article  Google Scholar 

  23. 23

    Guigliarelli, B. et al. Structural organization of the Ni and [4Fe-4S] centers in the active form of Desulfovibrio gigas hydrogenase. Analysis of the magnetic interactions by electron paramagnetic resonance spectroscopy. Biochemistry 34, 4781–4790 (1995).

    CAS  Article  Google Scholar 

  24. 24

    Almeida, M.G. et al. A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase. FEBS Lett. 581, 284–288 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Rohlfs, R.J., Olson, J.S. & Gibson, Q.H. A comparison of the geminate recombination kinetics of several monomeric heme proteins. J. Biol. Chem. 263, 1803–1813 (1988).

    CAS  PubMed  Google Scholar 

  26. 26

    Riistama, S., Puustinen, A., Verkhovsky, M.I., Morgan, J.E. & Wikstrom, M. Binding of O2 and its reduction are both retarded by replacement of valine 279 by isoleucine in cytochrome c oxidase from Paracoccus denitrificans. Biochemistry 39, 6365–6372 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Chen, L., Lyubimov, A.Y., Brammer, L., Vrielink, A. & Sampson, N.S. The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase. Biochemistry 47, 5368–5377 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Barney, B.M., Yurth, M.G., Dos Santos, P.C., Dean, D.R. & Seefeldt, L.C. A substrate channel in the nitrogenase MoFe protein. J. Biol. Inorg. Chem. 14, 1015–1022 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Dementin, S. et al. A glutamate is the essential proton transfer gate during the catalytic cycle of the NiFe hydrogenase. J. Biol. Chem. 279, 10508–10513 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Dementin, S. et al. Changing the ligation of the distal [4Fe4S] cluster in NiFe hydrogenase impairs inter- and intramolecular electron transfers. J. Am. Chem. Soc. 128, 5209–5218 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Rousset, M. New shuttle vectors for the introduction of cloned DNA in Desulfovibrio. Plasmid 39, 114–122 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Hatchikian, E.C., Forget, N., Fernandez, V.M., Williams, R. & Cammack, R. Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757. Eur. J. Biochem. 209, 357–365 (1992).

    CAS  Article  Google Scholar 

  33. 33

    Fourmond, V. et al. Correcting for electrocatalyst desorption and inactivation in chronoamperometry experiments. Anal. Chem. 81, 2962–2968 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Rousset, M. et al. [3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 95, 11625–11630 (1998).

    CAS  Article  Google Scholar 

  35. 35

    Fourmond, V. et al. SOAS: a free software to analyse electrochemical data and other one-dimensional signals. Bioelectrochem. 76, 141–147 (2009).

    CAS  Article  Google Scholar 

  36. 36

    Demuez, M. et al. Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners. FEMS Microbiol. Lett. 275, 113–121 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the Centre National de la Recherche Scientifique, Commissariat à l'Energie Atomique, Agence Nationale de la Recherche, the University of Provence and the City of Marseilles, and supported by the Pôle de Compétitivité Capénergies. The Groupe de Recherche 2977 (“Bio-hydrogène”) paid the publication fees for this article.

Author information

Affiliations

Authors

Contributions

P.-P.L. designed mutants of the NiFe enzyme, performed mutagenesis, carried out protein purification, solution assays and electrochemical measurements, and analyzed data, with the support of M.R. and C.L. F.L. performed electrochemical measurements on several forms of the NiFe hydrogenase (WT, L122M V74M, V74M, L122F V74I). B.B. characterized by EPR the NiFe hydrogenase mutants, with the support of B.G. S.D. designed mutants of the NiFe enzyme, performed mutagenesis, carried out protein purification and solution assays, and interpreted studies, with the support of M.R. C.B. performed the electrochemical characterization of the two FeFe hydrogenases and analyzed the data. T.L. purified the FeFe hydrogenase from C. acetobutylicum and assayed its activity, with the support and advice of I.M.-S. and P.S. V.F. contributed to modeling. P.C. characterized the V74W mutant, and analyzed the data. C.C. purified the FeFe hydrogenase from D. desulfuricans, with the support of J.C.F.-C. P.-P.L., S.D., B.B., C.B., M.R., B.G., P.B. and C.L. co-designed research. S.D., P.B. and C.L. conceptualized, analyzed and interpreted all studies and co-wrote the manuscript.

Corresponding author

Correspondence to Christophe Léger.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 183 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liebgott, PP., Leroux, F., Burlat, B. et al. Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase. Nat Chem Biol 6, 63–70 (2010). https://doi.org/10.1038/nchembio.276

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