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 H_hyd state containing a terminal hydride at the distal Fe site. The ¹³C²H isotope effects were observed also in the H_hyd 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.

Figure 1.

(a) [FeFe] hydrogenase active site including key amino acids in the proton transfer pathway (based on the PDB 4XDC structure of the CpI enzyme from Clostridium pasteurianum, but using CrHydA1 sequence numbering). (b) Schematic structure of the [2Fe]_H subcluster in the H_hyd state, showing the isotopically labeled nuclei ⁵⁷Fe, ¹³C, and D (i.e. ²H). The important hydrogens, H_h (catalytic hydride at the distal Fe_d iron), H_ADT (at the ADT N_ADT nitrogen), and H_C (at the S_C C169 sulfur), are shown.

Figure 2.

⁵⁷Fe-PVDOS for the [2Fe]_H precursor isotopologues 1 (blue) vs ¹³CD-1 (red) from (a) NRVS experiments and (b) DFT calculations. Sticks correspond to individual DFT normal mode energies and intensities before lineshape convolution. For ¹³CD-1, important band positions are labeled, and atomic motions in selected normal modes are shown.

Figure 3.

⁵⁷Fe-PVDOS for the H_hyd state isotopologues 1-CrHydA1 (blue) vs ¹³CD-1-CrHydA1 (red) from (a) an NRVS experiment and (b) DFT calculations. Sticks correspond to individual DFT normal mode energies and intensities before broadening. For ¹³CD-1-CrHydA1, important band positions are labeled, and atomic motions in selected normal modes are shown. Only the [2Fe]_H and C169 fragments of the DFT model are shown with the methylene, H_h, and H_ADT hydrogen nuclei displacements indicated by red arrows.