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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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).
The authors declare no competing financial interests.
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Marques, M., Tapia, C., Gutiérrez-Sanz, O. et al. The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis. Nat Chem Biol 13, 544–550 (2017). https://doi.org/10.1038/nchembio.2335
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