## 1 Introduction  
The present work addresses the structure of the active site of recombinant [NiFeSe] hydrogenase isolated from _Desulfovibrio vulgaris_ Hildenborough [1]. The [NiFeSe] hydrogenase represents, in addition to the [NiFeS] hydrogenase, one of the two subclasses of [NiFe] hydrogenases. Hydrogenases are metalloenzymes with an active site that contains, under a reducing atmosphere, iron, nickel and sulfur as well as diatomic ligands like CN and CO, which catalyze the reversible oxidation of molecular hydrogen,  
\[\mathrm{H_{2}}\leftrightarrow 2\;\mathrm{H^{+}}+2\;\mathrm{e^{-}}.\]  
Unlike [FeFe] hydrogenases, which are irreversibly inactivated after exposure to O\({}_{2}\), [NiFe] hydrogenases can be reductively reactivated after exposure to O\({}_{2}\)[1]. Due to their potential of producing hydrogen, these enzymes represent a design template for molecular catalysts being applied in electrolyzers, which use electricity to split water into hydrogen and oxygen, as well as in fuel cells [2]. Nonetheless, as O\({}_{2}\) acts as an oxidant that competes with protons, the hydrogenase's protein scaffold protects the redox centers from high O\({}_{2}\) concentrations that would inhibit H\({}_{2}\) production at the active site [2]. Representing the predominant form, the [NiFeS] hydrogenase contains the catalytic Ni-Fe center, bound by two bridging cysteines and surrounded by two additional cysteine ligands on the Ni site and two CN- and one CO ligands coordinated to the Fe site. The [NiFeSe] hydrogenase is structured analogously, with the exception that a selenocysteine (Sec489) replaces a terminal cysteine (Figure 1A). Three iron-sulfur clusters (proximal, medial, and distal) form an electron transfer path connecting the catalytic center to the protein surface (Figure 1B-E). In general, the activity of selenoproteins is usually higher than that of sulfur-containing protein homologues due to the lower pKa and higher nucleophilicity of the selenol group compared to that of the thiol group [3,4]. Selenium-containing cysteine (Se-Cys) is more acidic than sulfur-containing cysteine (S-Cys), and the high nucleophilicity of selenium leads to a higher O\({}_{2}\) tolerance of [NiFeSe] hydrogenase. The insertion of selenocysteine requires intricate biosynthetic machinery and is energetically more costly than cysteine insertion, so [NiFeSe] hydrogenases have evolved in only a few microorganisms, such as in _Desulfovibrio vulgaris_ and _Methanococcus voltae_[2]. The enzymes belonging to the subfamily of [NiFeSe] hydrogenases are special because they have a fast H\({}_{2}\) production rate [5, 6, 7]. Compared to [NiFeS] hydrogenases, the Se enzyme records a higher H\({}_{2}\) production activity [8]. Indeed, if Se is available to the microorganism, a preferred expression of NiFeSe over NiFeS hydrogenase is observed [8]. Importantly, the recombinant expression system developed by [PERSON] et al. is a step toward engineering large quantities of these valuable enzymes [1]. An earlier, lower-resolution (2.04 A) structure of aerobically crystallized [NiFeSe] hydrogenase revealed the oxidized, \"as-isolated\" active site that lacks an oxide bridging ligand [9]. More recently, high-resolution (0.95-1.4 A) crystal structures of [NiFeSe] hydrogenase have revealed three conformers of the active site that differ not only in the arrangement, but also in the number of sulfur atoms (Figure 2). In the recent, high-resolution structures, conformers I and II contain a sulfur atom bound between the nickel atom and the selenocysteine unit (Figure 2A,B), while conformer III does not contain this additional sulfur atom (Figure 2C). In contrast, the nickel atom of conformer III is only bound to a selenocysteine. Conformer I and II, in turn, differ in their binding geometries of the Ni-S-Se-Cys unit. While in conformer II, the additional sulfur atom is directly inserted between the Ni-Se-Cys bond compared to conformer III, and in conformer I, the nickel atom forms a direct bond to the additional sulfur atom as well as to the selenocysteine unit.