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A conformational study of the GTPase domain of [FeFe]-hydrogenase maturation protein HydF by PELDOR spectroscopy

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Abstract

[FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydrogen (H2) at an active site called H-cluster. The maturation pathway of these enzymes is a complex process involving three proteins, HydE, HydF and HydG. The maturase protein HydF has been suggested to interact with HydE and HydG and to be the transferase that shuttles the complete H-cluster to the hydrogenase; however, the exact molecular mechanism driving this translocation remains unclear. HydF is constituted by three different domains: a N-terminal GTP-binding domain, a dimerization domain and a C-terminal [4Fe4S] cluster-binding domain. To investigate possible conformational changes induced by the GTP binding in the N-terminal domain, we have expressed, in Escherichia coli, a recombinant HydF protein from Thermotoga neapolitana including the GTP-binding domain only. Site-directed mutants were designed in which the native residues were substituted by cysteines and subsequently spin labeled with the nitroxide MTSSL. CW-EPR was used to study the local mobility of the nitroxides at each site, and double spin-labeled mutants have been investigated by PELDOR spectroscopy. We found that the binding of the nucleotide does not induce large conformational effects within the isolated GTP domain, at least at the level of the elements investigated in this work. However, small changes in the distance between spin labels were observed which might reflect diffuse structural rearrangements. We suggest that the variations following the GTP binding could affect the dimer form adopted by the whole HydF protein in solution and, as a consequence, the interactions with the other maturases.

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References

  1. P.M. Vignais, B. Billoud, Chem. Rev. 107, 4206–4272 (2007)

    Article  Google Scholar 

  2. J.W. Peters, W.N. Lanzilotta, B.J. Lemon, L.C. Seefeldt, Science 282, 1853–1858 (1998)

    Article  ADS  Google Scholar 

  3. Y. Nicolet, C. Piras, P. Legrand, C.E. Hatchikian, J.C. Fontecilla-Camps, Structure 7, 13–23 (1999)

    Article  Google Scholar 

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

    Article  Google Scholar 

  5. E.M. Shepard, F. Mus, J.N. Betz, A.S. Byer, B.R. Duffus, J.W. Peters, J.B. Broderick, Biochemistry 53, 4090–4104 (2014)

    Article  Google Scholar 

  6. J.W. Peters, J.B. Broderick, Annu. Rev. Biochem. 81, 429–450 (2012)

    Article  Google Scholar 

  7. M.C. Posewitz, P.W. King, S.L. Smolinski, L. Zhang, M. Seibert, M.L. Ghirardi, J. Biol. Chem. 279, 25711–25720 (2004)

    Article  Google Scholar 

  8. J.K. Ruback, X. Brazzolotto, J. Gaillard, M. Fontecave, FEBS Lett. 579, 5055–5060 (2005)

    Article  Google Scholar 

  9. X. Brazzolotto, J.K. Rubach, J. Gaillard, S. Gambarelli, M. Atta, M. Fontecave, J. Biol. Chem. 281, 769–774 (2006)

    Article  Google Scholar 

  10. S.E. McGlynn, E.M. Shepard, M.A. Winslow, A.V. Naumov, K.S. Duschene, M.C. Posewitz, W.E. Broderick, J.B. Broderick, J.W. Peters, FEBS Lett. 582, 2183–2187 (2008)

    Article  Google Scholar 

  11. D.W. Mulder, E.S. Boyd, R. Sarma, R.K. Lange, J.A. Endrizzi, J.B. Broderick, J.W. Peters, Nature 465, 248–251 (2010)

    Article  ADS  Google Scholar 

  12. I. Czech, S. Stripp, O. Sanganas, N. Leidel, T. Happe, M. Haumann, FEBS Lett. 585, 225–230 (2011)

    Article  Google Scholar 

  13. G. Berggren, A. Adamska, C. Lambertz, T.R. Simmons, J. Esselborn, M. Atta, S. Gambarelli, J.M. Mouesca, E. Reijerse, W. Lubitz, T. Happe, V. Artero, M. Fontecave, Nature 499, 66–69 (2013)

    Article  ADS  Google Scholar 

  14. J. Esselborn, C. Lambertz, A. Adamska-Venkatesh, T. Simmons, G. Berggren, J. Noth, J. Siebel, A. Hemschemeier, V. Artero, E. Reijerse, M. Fontecave, W. Lubitz, T. Happe, Nat. Chem. Biol. 9, 607–609 (2013)

    Article  Google Scholar 

  15. J.M. Kuchenreuther, W.K. Myers, D.L.M. Suess, T.A. Stich, V. Pelmenschikov, S.A. Shiigi, S.P. Cramer, J.R. Swartz, R.D. Britt, S.J. George, Science 343, 424–427 (2014)

    Article  ADS  Google Scholar 

  16. J.D. Lawrence, H.X. Li, T.B. Rauchfuss, M. Benard, M.M. Rohmer, Angew. Chem. Int. Ed. 40, 1768–1771 (2001)

    Article  Google Scholar 

  17. P.W. King, M.C. Posewitz, M.L. Ghirardi, M. Seibert, J. Bacteriol. 188, 2163–2172 (2006)

    Article  Google Scholar 

  18. L. Cendron, P. Berto, S. D’Adamo, F. Vallese, C. Govoni, M.C. Posewitz, G.M. Giacometti, P. Costantini, G. Zanotti, J. Biol. Chem. 286, 43944–43950 (2011)

    Article  Google Scholar 

  19. P. Berto, M. Di Valentin, L. Cendron, F. Vallese, M. Albertini, E. Salvadori, G.M. Giacometti, D. Carbonera, P. Costantini, BBA-Bioenergetics 1817, 2149–2157 (2012)

    Article  Google Scholar 

  20. E.M. Shepard, S.E. McGlynn, A.L. Bueling, C.S. Grady-Smith, S.J. George, M.A. Winslow, S.P. Cramer, J.W. Peters, J.B. Broderick, Proc. Natl. Acad. Sci. USA 107, 10448–10453 (2010)

    Article  ADS  Google Scholar 

  21. F. Vallese, P. Berto, M. Ruzzene, L. Cendron, S. Sarno, E. De Rosa, G.M. Giacometti, P. Costantini, J. Biol. Chem. 287, 36544–36555 (2012)

    Article  Google Scholar 

  22. A. Scrima, A. Wittinghofer, EMBO J. 25, 2940–2951 (2006)

    Article  Google Scholar 

  23. M.J. Ryle, W.N. Lanzilotta, L.C. Seefeldt, Biochemistry 35, 9424–9434 (1996)

    Article  Google Scholar 

  24. S.B. Jang, M.S. Jeong, L.C. Seefeldt, J.W. Peters, J. Biol. Inorg. Chem. 9, 1028–1033 (2004)

    Article  Google Scholar 

  25. H.J. Chiu, J.W. Peters, W.N. Lanzilotta, M.J. Ryle, L.C. Seefeldt, J.B. Howard, D.C. Rees, Biochemistry 40, 641–650 (2001)

    Article  Google Scholar 

  26. G. Jeschke, M. Pannier, H.W. Spiess, in Distance Measurements in Biological Systems, vol. 19, ed. by L.J. Berliner, S.S. Eaton, G.R. Eaton (Kluwer Academic, New York, 2000), pp. 493–512

  27. O. Schiemann, T.F. Prisner, Quart. Rev. Biophys. 40, 1 (2007)

    Article  Google Scholar 

  28. Y.D. Tsvetkov, A.D. Milov, A.G. Maryasov, Russ. Chem. Rev. 77, 487 (2008)

    Article  ADS  Google Scholar 

  29. G. Jeschke, Annu. Rev. Phys. Chem. 63, 419 (2012)

    Article  ADS  Google Scholar 

  30. S. Stoll, A. Schweiger, J. Magn. Reson. 178, 42–55 (2006)

    Article  ADS  Google Scholar 

  31. Y. Polyhach, E. Bordignon, G. Jeschke, Phys. Chem. Chem. Phys. 13, 2356–2366 (2010)

    Article  Google Scholar 

  32. G. Jeschke, V. Chechik, P. Ionita, A. Godt, H. Zimmermann, J. Banham, C.R. Timmel, D. Hilger, H. Jung, Appl. Magn. Reson. 30, 473–498 (2006)

    Article  Google Scholar 

Download references

Acknowledgments

This work has been supported by the CARIPARO Foundation (M3PC project) by the MIUR (PRIN2010-2011 prot. 2010FM38P_004). A special acknowledgement is due to Giovanni Giacometti to whom this issue is dedicated, for stimulating with his personal interest and involvement, the research of the authors in the field of photosynthesis and bio-hydrogen production.

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Authors and Affiliations

  1. Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy

    Lorenzo Maso, Edith De Rosa, Giorgio M. Giacometti & Paola Costantini

  2. Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy

    Laura Galazzo, Marilena Di Valentin, Marco Albertini & Donatella Carbonera

  3. Department of Biomedical Sciences, University of Padova, Viale G. G. Colombo 3, 35131, Padua, Italy

    Francesca Vallese

Authors
  1. Lorenzo Maso
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  2. Laura Galazzo
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  4. Marilena Di Valentin
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  7. Giorgio M. Giacometti
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  9. Donatella Carbonera
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Correspondence to Paola Costantini or Donatella Carbonera.

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Maso, L., Galazzo, L., Vallese, F. et al. A conformational study of the GTPase domain of [FeFe]-hydrogenase maturation protein HydF by PELDOR spectroscopy. Appl Magn Reson 46, 465–479 (2015). https://doi.org/10.1007/s00723-015-0641-z

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  • DOI: https://doi.org/10.1007/s00723-015-0641-z

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