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

[FeFe]-hydrogenases are nature's blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe]), cysteine-linked to a diiron site ([2Fe]) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (μCO) in the oxidized protein (). 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 and ) or with an additional proton at [4Fe] () or at the distal iron of [2Fe] () prevail. At cryogenic temperatures, these species are largely replaced by states that hold a μCO, lack [4Fe] protonation, and bind an additional proton at the adt nitrogen ( and ). XAS revealed the atomic coordinate dispersion (i.e., the Debye-Waller parameter, 2σ) of the iron-ligand bonds and Fe-Fe distances in the oxidized and reduced H-cluster. 2σ 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] as well as ligand rearrangement and μH binding at [2Fe] 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] or [2Fe] protonation, and catalytic proton transfer via adt facilitates efficient H conversion in [FeFe]-hydrogenase.