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[FeFe] hydrogenases are enzymes that have acquired a unique capacity to synthesize or consume molecular hydrogen (H 2 ). This function relies on a complex catalytic mechanism involving the active site and two distinct electron and proton transfer networks working in concert. By an analysis based on terahertz vibrations of [FeFe] hydrogenase structure, we are able to predict and identify the existence of rate-promoting vibrations at the catalytic site and the coupling with functional residues involved in reported electron and proton transfer networks. Our findings suggest that the positioning of the cluster is influenced by the response of the scaffold to thermal fluctuations, which in turn drives the formation of networks for electron transfer through phonon-assisted mechanisms. Thus, we address the problem of linking the molecular structure to the catalytic function through picosecond dynamics, while raising the functional gain brought by the cofactors or clusters, using the concept of fold-encoded localized vibrations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57 Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe] H subcluster. A - 13 C 2 H 2 - ADT labeling in the synthetic diiron precursor of [2Fe] H produced isotope effects observed throughout the NRVS spectrum. The two precursor isotopologues were then used to reconstitute the H-cluster of [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1), and NRVS was measured on samples poised in the catalytically crucial H hyd state containing a terminal hydride at the distal Fe site. The 13 C 2 H isotope effects were observed also in the H hyd spectrum. DFT simulations of the spectra allowed identification of the 57 Fe normal modes coupled to the ADT ligand motions. Particularly, a variety of normal modes involve shortening of the distance between the distal Fe-H hydride and ADT N-H bridgehead hydrogen, which may be relevant to the formation of a transition state on the way to H 2 formation.