Caserta, GiorgioLorent, ChristianPelmenschikov, VladimirSchoknecht, JannaYoda, YoshitakaHildebrandt, PeterCramer, Stephen P.Zebger, IngoLenz, Oliver2023-01-192023-01-192020-11-16https://depositonce.tu-berlin.de/handle/11303/18014https://doi.org/10.14279/depositonce-16806[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.en541 Physikalische Chemiemetalloenzymehydrogenasehydrogennickelironcatalytic cyclespectroscopyArticle2155-5435