Vibrational Perturbation of the [FeFe] Hydrogenase H-Cluster Revealed by 13C2H-ADT Labeling

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, Fe-57 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 -(CH2)-C-13-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 (CrHydA1), and NRVS was measured on samples poised in the catalytically crucial H-hyd state containing a terminal hydride at the distal Fe site. The (CH)-C-13-H-2 isotope effects were observed also in the H-hyd spectrum. DFT simulations of the spectra allowed identification of the Fe-57 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.

Automated summary

This study used isotopic labeling, Fe-57 nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize vibrational modes involving motion of the ligand in [FeFe] hydrogenases. The findings provide insights into the reaction mechanism and dynamics of the hydrogenase enzyme.