[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 (mu CO) in the oxidized protein (Hox). Recently, the debate on the structure of reduced Hcluster 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 (mu H-, 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 mu CO, 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, 2 sigma(2)) of the iron-ligand bonds and Fe-Fe distances in the oxidized and reduced Hcluster. 2 sigma(2) showed a temperature dependence typical for the so-called protein-glass transition, with small changes below similar to 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 mu H-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 mu CO, selective [4Fe](H) or [2Fe](H) protonation, and catalytic proton transfer via adt facilitates efficient H-2 conversion in [FeFe]-hydrogenase.