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 H2 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,
2σ2) of the iron–ligand bonds and Fe–Fe
distances in the oxidized and reduced H-cluster. 2σ2 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 H2 conversion in [FeFe]-hydrogenase.