X‐ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases

[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H 2 ). However, structural determinants of efficient H 2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational‐spectroscopic insights into the unexplored structure of the H 2 ‐binding [NiFe] intermediate. Using an F 420 ‐reducing [NiFe]‐hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H 2 binding and conversion. The protein matrix also directs H 2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit‐bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio‐inspired H 2 ‐conversion catalysts.