Temperature Dependence of Structural Dynamics at the Catalytic Cofactor of [FeFe]-hydrogenase

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[FeFe]-hydrogenases are nature’s blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H 2 fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe] H ), cysteine-linked to a diiron site ([2Fe] H ) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (μCO) in the oxidized protein ( Hox ). Recently, the debate on the structure of reduced H-cluster states was intensified by the assignment of new species under cryogenic conditions. We investigated temperature effects (4–280 K) in infrared (IR) and X-ray absorption spectroscopy (XAS) data of [FeFe]-hydrogenases using fit analyses and quantum-chemical calculations. IR data from our laboratory and literature sources were evaluated. At ambient temperatures, reduced H-cluster states with a bridging hydride (μH –, in Hred and Hsred ) or with an additional proton at [4Fe] H ( Hred′ ) or at the distal iron of [2Fe] H ( Hhyd ) prevail. At cryogenic temperatures, these species are largely replaced by states that hold a μCO, lack [4Fe] H protonation, and bind an additional proton at the adt nitrogen ( HredH + and HsredH + ). XAS revealed the atomic coordinate dispersion (i.e., the Debye–Waller parameter, 2σ 2 ) of the iron–ligand bonds and Fe–Fe distances in the oxidized and reduced H-cluster. 2σ 2 showed a temperature dependence typical for the so-called protein–glass transition, with small changes below ∼200 K and a pronounced increase above this “breakpoint”. This behavior is attributed to the freezing-out of larger-scale anharmonic motions of amino acid side chains and water species. We propose that protonation at [4Fe] H as well as ligand rearrangement and μH – binding at [2Fe] H are impaired because of restricted molecular mobility at cryogenic temperatures so that protonation can be biased toward adt. We conclude that a H-cluster with a μCO, selective [4Fe] H or [2Fe] H protonation, and catalytic proton transfer via adt facilitates efficient H 2 conversion in [FeFe]-hydrogenase.