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Abstract

[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.