Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations

Abstract

The catalytic mechanism of [NiFe]-hydrogenases is a subject of extensive research. Apart from at least four reaction intermediates of H₂/H⁺ cycling, there are also a number of resting states, which are formed under oxidizing conditions. Although not directly involved in the catalytic cycle, the knowledge of their molecular structures and reactivity is important, because these states usually accumulate in the course of hydrogenase purification and may also play a role in vivo during hydrogenase maturation. Here, we applied low-temperature infrared (cryo-IR) and nuclear resonance vibrational spectroscopy (NRVS) to the isolated catalytic subunit (HoxC) of the heterodimeric regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha. Cryo-IR spectroscopy revealed that the HoxC protein can be enriched in almost pure resting redox states suitable for NRVS investigation. NRVS analysis of the hydrogenase catalytic center is usually hampered by strong spectral contributions of the FeS clusters of the small, electron-transferring subunit. Therefore, our approach to investigate the FeS cluster-free, ⁵⁷Fe-labeled HoxC provided an unprecedented insight into the [NiFe] site modes, revealing their contributions in a spectral range otherwise superimposed by FeS cluster-derived bands. Rationalized by density functional theory (DFT) calculations, our data provide structural descriptions of the previously uncharacterized hydroxy- and water-containing resting states. Our work highlights the relevance of cryogenic vibrational spectroscopy and DFT to elucidate the structure of barely defined redox states of the [NiFe]-hydrogenase active site.

Fig. 1. (Left) Schematic representation of the subunit composition of the RH and the proposed catalytic cycle of [NiFe]-hydrogenases. The states involved in the H₂ transformation include the Ni_a–S intermediate that presumably binds H₂ in the bridging position between the nickel and the iron ions. H₂ splitting results in a two-electron reduced Ni_a–SR state characterized by a bridging hydride. Release of one electron and one proton results in a hydride-carrying Ni_a–C species, which is a tautomeric form of the Ni_a–L state. Release of another electron and a further proton restores the Ni_a–S intermediate. Protonation of a terminal cysteine as well as the bridging hydride are highlighted in red. The Ni and its oxidation state are depicted in green. (Right) Schematic representation of the [NiFe]-hydrogenase large subunit HoxC and proposed structures of its resting states. The as-isolated HoxC protein occurs in a mixture of the Ni_r–S_I and Ni_r–S_II species. One-electron oxidation results in the formation of the Ni_r–B′′ species. Protonation of the terminal cysteine, as found for the Ni_a–SR and the light-induced Ni_a–L intermediates (left panel), and the OH⁻/H₂O active site ligands assigned in this work are depicted in bold case letters.

Fig. 2. IR spectra of HoxC_ai, HoxC_ox and RH (top to bottom) taken at 283 K and 85 K. (a) IR spectral region characteristic of CO and CN stretching modes recorded at 283 K. The spectrum of HoxC_ai exhibits spectral contributions of the Ni_r–S_I (red) and Ni_r–S_II (dark blue) resting states, respectively. The spectrum of RH is dominated by signals attributed to the Ni_a–S state (labeled in black). The IR data of HoxC_ox comprise contributions from two paramagnetic oxidized Ni_r–B′ (minor species) and Ni_r–B′′ states labeled in purple and blue, respectively. (b) IR spectral region characteristic of CO and CN stretching modes recorded at 85 K for the same samples. Contributions from the corresponding CN absorptions of Fe(CN)₆³⁻ and Fe(CN)₆²⁻ present in the chemically oxidized HoxC_ox were subtracted for the sake of clarity.

Fig. 3. DFT models: (a) Ni_r–S^SH_μOH of the reduced Ni_r–S_I state, (b) of the reduced Ni_r–S_II state, and (c) Ni_r–B^SH_μOH of the oxidized Ni_r–B′′ state, displaying the [NiFe] cofactor metal ligands and their contacts with the nearby side chains in HoxC. For alternative models and the entire HoxC homology model employed, see Fig. S4–S10.

Fig. 4. NRVS partial vibrational density of states (PVDOS) of ⁵⁷Fe-labeled RH (bottom) and its large subunit HoxC (top), in their isolated forms, normalized to an integrated PVDOS of 3. Different spectral regions are indicated with arrows using the following color code: red, bands related to Fe–CO/CN of the [NiFe] active site; orange, bands related to [NiFe] site/protein modes; blue, bands related to Fe–μS modes involving bridging cysteines; olive, bands related to the Fe–S modes of the [4Fe4S]-clusters. Representative bands in the RH and HoxC spectra are labeled with numbers in black. The corresponding NRVS data including the error bars are presented in Fig. S20.

Fig. 5. Comparison of the experimental NRV spectrum of the HoxC_ai sample (Ni_r–S_I, grey trace reiterated in (a–e)) overlaid with the corresponding DFT-calculated ⁵⁷Fe-PVDOS bands using alternative [Ni^IIFe^II] models, schematically shown on the right side of the spectra. The DFT spectra derived using either protonated (solid lines) or deprotonated (broken lines) Cys479 are shown for the following models: (a) best-fit Ni_r–S^SH_μOH and Ni_r–S^S−_μOH (Fig. S6†); (c) and (Fig. S8†); (d) and (Fig. S9†); (e) and (Fig. S10†). The DFT spectrum derived from the alternative hydroxy model with protonated Cys60, Ni_r–S^Cys60-SH_μOH, is highlighted in (b). Both μOH⁻ models in (a) reproduce the experimental data in the Fe–CO/CN region above 400 cm⁻¹. Minor differences in the low-frequency spectral region around 180 cm⁻¹ (Fig. S16†) strengthen the Ni_r–S^SH_μOH model, in line with the better prediction of the experimental IR absorptions for the CO and CN stretching modes (Fig. S11a†).

Fig. 6. Comparison of the experimentally observed and DFT-calculated ⁵⁷Fe-PVDOS bands in the 400–650 cm⁻¹ spectral region for HoxC_ai and HoxC_ox. Experimental spectra (top: red trace, HoxC_ai; blue trace, HoxC_ox) are compared to their DFT-calculated counterparts based on the corresponding models that contain either protonated (middle: Ni_r–S^SH_μOH and Ni_r–B^SH_μOH states) or deprotonated Ni-bound terminal cysteine (bottom: Ni_r–S^S−_μOH and Ni_r–B^S−_μOH states). Bands calculated using Ni_r–S^SH_μOH and Ni_r–B^SH_μOH models are in better agreement with the NRVS data, including their shift magnitudes marked in the figure. The corresponding NRVS data including the error bars are presented in Fig. S20.

Fig. 7. Arrow-style representation of important Fe/Ni–μOH vibrational modes from representative DFT models for the Ni_r–S_I (model Ni_r–S^SH_μOH, top) and Ni_r–B′′ (model Ni_r–B^SH_μOH, bottom) states. The corresponding vibrational energies (cm⁻¹) are indicated in the top left of each panel. Left to right: Ni–μOH–Fe wagging, Fe–μOH stretching, and Ni–μOH stretching modes. The stick-style mode intensities relevant to the interpretation of the ⁵⁷Fe-PVDOS spectra are marked in Fig. S18b and S19b–d. Animations of these and other normal modes are available as part of the ESI.