Abstract

Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 production. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

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Acknowledgements

We thank F. Grein and M. Martins for advice and helpful discussions, R. Coelho and S. Silva for help with crystallization procedures and S. Zacarias for experimental assistance; ESRF, DLS and SOLEIL light sources for X-ray data collection; V. Olieric (Swiss Light Source) for the 0.95-Å data collection of the anaerobically purified and crystallized r[NiFeSe] hydrogenase. This work was supported by grants PTDC/BBB-BEP/0934/2012 and PTDC/BBB-BEP/2885/2014 (to I.A.C.P. and P.M.M.) from the Fundação para a Ciência e Tecnologia (FCT/MCTES), by research units GREEN-IT (UID/Multi/04551/2013) funded by FCT/MCTES, and MOSTMICRO (project LISBOA-01-0145-FEDER-007660) co-funded by FCT/MCTES and FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI); and by Spanish MINECO/FEDER project CTQ2015-71290-R (to A.L.D.L.). M.C.M. was a recipient of fellowship SFRH/BD/60879/2009 and C.T. was a recipient of predoctoral contract BES-2013-064099 from MINECO. This work was also supported by the European Community's Seventh Framework Program (FP7/2007–2013) under grant agreement 283570 (BioStruct-X).

Author information

Author notes

    • Kimberly L Keller

    Present address: Biology Department, William Woods University, Fulton, Missouri, USA.

Affiliations

  1. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

    • Marta C Marques
    • , Ana Raquel Ramos
    • , Pedro M Matias
    •  & Inês A C Pereira
  2. Instituto de Catálisis y Petroleoquímica (CSIC), Madrid, Spain.

    • Cristina Tapia
    • , Oscar Gutiérrez-Sanz
    •  & Antonio L De Lacey
  3. Biochemistry Department, University of Missouri, Columbia, Missouri, USA.

    • Kimberly L Keller
    •  & Judy D Wall
  4. Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA), Berkeley, California, USA.

    • Kimberly L Keller
    •  & Judy D Wall
  5. Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.

    • Pedro M Matias

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Contributions

I.A.C.P., P.M.M. and M.C.M. conceived the study. A.R.R., K.L.K., J.D.W. and M.C.M. carried out the molecular biology work. M.C.M. and I.A.C.P. produced and characterized hydrogenase variants. M.C.M. and P.M.M. were involved in crystallization and structure determination. O.G.-S., C.T. and A.L.D.L. carried out FTIR and MS experiments. I.A.C.P., P.M.M. and M.C.M. wrote the manuscript with input from other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Pedro M Matias or Inês A C Pereira.

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DOI

https://doi.org/10.1038/nchembio.2335

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