[NiFe]-hydrogenases are among the few enzymes known to contain nickel in their active site.¹  Because of their ability to oxidize molecular hydrogen or reduce protons, according to the reaction H₂ ⇔ 2H⁺ + 2e⁻, they are potentially relevant to biotechnological applications such as the generation of H₂ from solar energy or the oxidation of H₂ in bio-fuel cells. The simplest [NiFe]-hydrogenases are heterodimeric consisting of a large (L) subunit where the active Ni–Fe site is found and a small (S) subunit that binds a proximal [Fe₄S₄], a medial [Fe₃S₄], and a distal [Fe₄S₄] cluster. These clusters can transfer electrons back and forth between the molecular surface and the active site. The [NiFe]-hydrogenase from Desulfovibrio gigas, which was the first hydrogenase for which the structure was determined by X-ray crystallography,²  is a representative of this class of enzymes (Figure 6.1). Variations from this archetypical structure include [NiFeSe]-hydrogenases, which contain a selenocysteine (Sec) as a Ni ligand and have a [Fe₄S₄] medial cluster, and O₂-tolerant enzymes, which have a modified proximal cluster corresponding to a [Fe₄S₃] species. Differences are also found in the nature of the protein ligands to the [Fe–S] clusters. A recently reported example is the replacement of a cysteine thiolate by an aspartate carboxylate at the proximal [Fe₄S₄] cluster found in the crystal structure of the O₂-tolerant actinobacterial-type [NiFe]-hydrogenase from Ralstonia eutropha.³