The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center

Background: [NiFeSe] hydrogenases are metalloenzymes that catalyze the reac tion H-2<----> 2H(+)+2e(-). They are generally heterodimeric, contain three iron-sulfur clusters in their small subunit and a nickel-iron-containing a ctive site in their large subunit that includes a selenocysteine (SeCys) li gand. Results: We report here the X-ray structure at 2.15 Angstrom resolution of the periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-pre pared, Desulfovibrio gigas and the reduced D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 Angstrom shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detecte d in the as prepared D. gigas enzyme, is absent from the D. baculatum hydro genase. We also observe higher-than-average temperature factors for both th e active site nickel-selenocystein ligand and the neighboring Glu18 residue , suggesting that both these moieties are involved in proton transfer betwe en the active site and the molecular surface. Other differences between [Ni FeSe] and [NiFe] hydrogenases are the presence of a third [4Fe4S] cluster r eplacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative i ron center that substitutes the magnesium ion that has already been describ ed at the C terminus of the large subunit of two [Nife] hydrogenases. Conclusions: The heterolytic cleavage of molecular hydrogen seems to be med iated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive 'unready' [NiFe] hydrogenases.