© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. NATURE CHEMICAL BIOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology 1 ARTICLE PUBLISHED ONLINE: 20 MARCH 2017 | DOI: 10.1038/NCHEMBIO.2335 H ydrogenases catalyze the simplest of chemical reactions— the reversible conversion of protons and electrons to hydro- gen. These metalloenzymes have attracted immense interest because they are extremely active catalysts for these reactions and can be applied in fuel cells, electrocatalytic or photocatalytic devices and serve as models for synthetic catalysts 1–4 . Of particular interest are systems aiming at artificial photosynthesis for solar-based H 2 production from water splitting, forming the basis for a carbon-free, hydrogen-fueled economy 5,6 . In terms of enzyme-based systems, [FeFe] hydrogenases are an obvious choice for this application, as they have the highest H 2 production activities 7,8 , but these enzymes are irreversibly inactivated by even trace amounts of O 2 , which limits their use in H 2 evolution devices. In contrast, [NiFe] hydrogenases can be reductively reactivated after exposure to O 2 , but the standard enzymes form inactive Ni(III) species (Ni-A and Ni-B), of which Ni-A reactivates only very slowly 9 . A group of [NiFe] hydroge- nases are O 2 tolerant 10 , producing only the rapidly reactivated Ni-B species upon contact with O 2 , but these enzymes are not suited for H 2 production owing to a very strong bias toward H 2 oxidation and pronounced H 2 inhibition of H + reduction 3 . The subfamily of [NiFeSe] hydrogenases 11 , which have a seleno- cysteine as a direct ligand to the active site Ni (Fig. 1a,b), are the enzymes that display the most interesting properties for H 2 evolu- tion applications 12 . They have a fast rate and catalytic bias toward H 2 production, in contrast to standard [NiFe] hydrogenases 13–15 , and show much less product inhibition by H 2 (refs. 15–17). In addition, they do not form the inactive Ni(III) species characteristic of [NiFe] hydrogenases and are reactivated quickly at low potentials 16,18–21 , being capable of H 2 production in the presence of small amounts of O 2 (refs. 15,16). These properties have been exploited in biocata- lytic applications of [NiFeSe] hydrogenases for photo- and electro- chemical H 2 production 14,17,22–26 and also for electrochemical ATP synthesis 27 . Furthermore, the superiority of [NiFeSe] hydrogenases has also been revealed in vivo, as these enzymes are preferentially expressed when selenium is available 28,29 . For example, in D. vulgaris Hildenborough the [FeFe] and [NiFe] hydrogenases are down- regulated in the presence of selenium, indicating a physiological preference for the [NiFeSe] hydrogenase 29 . However, the incorporation of selenocysteine requires a com- plex dedicated machinery and has a very high energetic cost. Given also that sulfur is a much more abundant element than selenium, there must a strong biological advantage for using selenocysteine over cysteine 30,31 . Selenoproteins are mostly oxidoreductases in which selenocysteine is involved in the catalytic reaction. Despite numerous studies, there is still no consensus about why seleno- cysteine is used in selenoenzymes. The most studied group is that involved in thiol-disulfide exchange reactions, and possible factors discussed include selenocysteine’s lower pK a compared to cysteine, its increased nucleophilicity, increased electrophilicity, higher polarizability and hypervalency, better leaving group ability or a combination of all these, as selenocysteine performs multiple roles during the catalytic cycle 31,32 . However, several cysteine homologs of selenocysteine-containing enzymes can catalyze their enzymatic reactions with high catalytic efficiency, raising questions about the real necessity for selenium 32,33 . Another important argument for the superiority of selenocysteine is its ability to resist irreversible oxi- dative inactivation 31,33,34 . In fact, although selenium is more easily oxidized than sulfur, the resulting selenium oxides are much more electrophilic and unstable than their sulfur analogs and therefore easier to reduce back to the parent state. Thus, oxidation of the sele- nocysteine residue to the corresponding selenenic or seleninic acids is readily reversible, whereas reduction of a sulfenic acid is more dif- ficult, and that of a sulfinic acid virtually impossible 34,35 . This prop- erty apparently enables selenoenzymes to better resist irreversible oxidative inactivation compared to their cysteine counterparts 34 . Here we report the first recombinant expression system for a [NiFeSe] hydrogenase allowing the production of engineered forms of the enzyme. We generated a protein variant in which the selenocysteine residue was replaced by cysteine, converting the [NiFeSe] enzyme into a [NiFe] hydrogenase and thus enabling us to 1 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal. 2 Instituto de Catálisis y Petroleoquímica (CSIC), Madrid, Spain. 3 Biochemistry Department, University of Missouri, Columbia, Missouri, USA. 4 Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA), Berkeley, California, USA. 5 Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal. 6 Present address: Biology Department, William Woods University, Fulton, Missouri, USA. *e-mail: ipereira@itqb.unl.pt or matias@itqb.unl.pt The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis Marta C Marques 1 , Cristina Tapia 2 , Oscar Gutiérrez-Sanz 2 , Ana Raquel Ramos 1 , Kimberly L Keller 3,4,6 , Judy D Wall 3,4 , Antonio L De Lacey 2 , Pedro M Matias 1,5 * & Inês A C Pereira 1 * Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which seleno- cysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H 2 production. In contrast to [NiFe] hydroge- nases, they display reduced H 2 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 seleno- cysteine-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.