Figure 3. Ni–H–Fe-hydride wag exposed in the reduced state Ni-R of [NiFe]-hydrogenase.

(a–c) High-frequency NRVS for [NiFe]-hydrogenase reduced in H₂O (blue trace) and D₂O (red trace; a) and the corresponding ⁵⁷Fe PVDOS simulations given for the representative DFT models VI^S (b) and V^S (c). The higher regions of spectra containing the Ni–H–Fe wag band (in H₂O samples) are repeated with their intensities × 4 amplified. The low-energy region of the Ni-R spectrum in H₂O reveals a triplet of bands (454, 475 and 502 cm⁻¹) that correspond to those located at 440, 461 and 504 cm⁻¹ in the calculated spectrum of the model V^S. Further, two intense bands seen at 549 and 609 cm⁻¹ in Ni-R map on calculated bands at 543 and 613 cm⁻¹, with an additional weak band observed at 590 cm⁻¹ that can be correlated with the calculated band appearing at 588 cm⁻¹. (d,e) Representative DFT-optimized models VI^S (d) and V^S (e) for the Ni-R active site. Arrows indicate the position of CysSH. Non-substrate H atoms have been omitted for clarity (excluding HNɛ of His88).