Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site (“H-cluster”) consists of a [4Fe–4S]_H cluster linked through a bridging cysteine to a [2Fe]_H subsite coordinated by CN^– and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fe_d). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe–H species fully consistent with widely accepted models of the catalytic cycle

Reaction Coordinate Leading to H₂ Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (H_hyd) in the [FeFe]-hydrogenases from both <i>Chlamydomonas reinhardtii</i> and <i>Desulfovibrio desulfuricans</i> using ⁵⁷Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe–H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe–H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that H_hyd is the catalytic state one step prior to H₂ formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H₂ bond formation by [FeFe]-hydrogenases

Synchrotron-based Nickel Mössbauer Spectroscopy

We used a novel experimental setup to conduct the first synchrotron-based ⁶¹Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. ⁶¹NiCr₂O₄ was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional ⁶¹Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, ⁶¹Ni­{N­(Si­Me₃)­Dipp}₂, where Dipp = C₆H₃-2,6-ⁱPr₂, was chosen as a sample with an “extreme” geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based ⁶¹Ni Mössbauer spectroscopy, we examined the spectra of ⁶¹Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et₄N]₂[⁶¹Ni­(SPh)₄] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based ⁶¹Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources