Vibrational Perturbation of the [FeFe] Hydrogenase H-Cluster Revealed by ~(13)C~2H-ADT Labeling

摘要

[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-l,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe]_H subduster. A -~(13)C~2H_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 (CrHydAl), 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~2H 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.