In Vitro Assembly as a Tool to Investigate Catalytic Intermediates of [NiFe]-Hydrogenase.

[NiFe]-hydrogenases catalyze the reversible reaction H2 ⇄ 2H+ + 2e-. Their basic module consists of a large subunit, coordinating the NiFe(CO)(CN)2 center, and a small subunit that carries electron-transferring iron-sulfur clusters. Here, we report the in vitro assembly of fully functional [NiFe]-hydrogenase starting from the isolated large and small subunits. Activity assays complemented by spectroscopic measurements revealed a native-like hydrogenase. This approach was used to label exclusively the NiFe(CO)(CN)2 center with 57Fe, enabling a clear view of the catalytic site by means of nuclear resonance vibrational spectroscopy. This strategy paves the way for in-depth studies of [NiFe]-hydrogenase catalytic intermediates.