Abstract
[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 (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 (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.