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

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, 57Fe 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 −13C2H2– 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 Hhyd state containing a terminal hydride at the distal Fe site. The 13C2H isotope effects were observed also in the Hhyd spectrum. DFT simulations of the spectra allowed identification of the 57Fe 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 H2 formation.

Nuclear Resonance Vibrational Spectroscopy: A Modern Tool to Pinpoint Site-Specific Cooperative Processes

“…The further confirmation of this feature involved two more aspects: (3) a tripled incident X-ray intensity (with 0.8 meV energy resolution) at BL19LXU (vs. that at BL09XU), and (4) the H/D pair comparison, as shown in Figure 15d2. After the historical observation of this Ni-H-Fe peak, terminal X-Fe-H bending features inside several FeFe hydrogenses were also successfully observed, and their detailed structures were identified via DFT calculations [44,45,47,69]. In addition to the Fe-H bending features, Fe-D or Fe-H stretching features at highenergy regions stand out from all other features and can provide additional and better reference points for DFT calculation vs. proposed structures.…”

“…The further confirmation of this feature involved two more aspects: (3) a tripled incident X-ray intensity (with 0.8 meV energy resolution) at BL19LXU (vs. that at BL09XU), and (4) the H/D pair comparison, as shown in Figure 15d2. After the historical observation of this Ni-H-Fe peak, terminal X-Fe-H bending features inside several FeFe hydrogenses were also successfully observed, and their detailed structures were identified via DFT calculations [44,45,47,69].…”

“…People need 36-48 h to move in and set up the HRM and other instruments at the beginning of each beamtime [8]. Nevertheless, researchers are rewarded with amazing NRVS results [7,8,45,47,[65][66][67][68][69].…”