Terminal Hydride Species in [FeFe]-Hydrogenases are Vibrationally Coupled to the Active Site Environment

Abstract

A combination of NRVS and FT-IR spectroscopies and DFT calculations was used to observe and characterize Fe-H/D bending modes in CrHydA1 [FeFe]-hydrogenase Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a –SH to –OH, thus altering the proton transfer pathway. C169S has a significant reduction in catalytic activity compared to the native CrHydA1, presumably due to less efficient transfer of protons to the H-cluster. This mutation allowed effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We find a significant shift to higher frequency in a Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT) substituted CrHydA1. Rationalized by DFT calculations, we propose that this shift is caused by a stronger interaction between the –OH of C169S with the bridgehead –NH– of the active site, as compared to the –SH of C169 in the native enzyme.

Graphical Abstract

Combination of NRVS measurements, DFT calculations, and mutagenesis identified Fe_d-H bond vibrational modes and observed effects of amino acid environment that surrounds the [FeFe]-hydrogenase H-cluster.

Hydrogenases are enzymes that catalyze the reversible interconversion of molecular hydrogen with protons and electrons: H₂ ⇄ 2H⁺ + 2e⁻.^[1] Under optimal electron delivery hydrogenases require little or no driving force while utilizing base transition metals as catalytic cofactors. The [FeFe]-hydrogenases ([FeFe]-H₂ases) are known to possess exceptionally high turnover rates of >10⁴ s⁻¹.^{[1a, 2]} Resolving the mechanistic properties of [FeFe]-H₂ase requires identification of the catalytically relevant intermediates,^[3] and is of interest to advancing catalyst design for developing H₂ fuel-cells and renewable energy production technologies.^[4]

The active site for all known [FeFe]-H₂ases consists of an ‘H-cluster’ that is composed of [4Fe-4S]_H and [2Fe]_H subclusters, linked by a cysteine thiolate (Figure 1).^[6] The main site of catalytic H₂ bond activation is the Fe_d iron site of [2Fe]_H distal to [4Fe-4S]_H, where the azadithiolate (ADT) group has a fundamental role in proton transfer. The nitrogen N_ADT of the ADT bridge is involved in transporting solvent protons to and from Fe_d through a relay of conserved amino acids that lead to the surface of the protein.^[7] In one of the simplest structural forms of the enzyme, Chlamydomonas reinhardtii [FeFe]-H₂ase HydA1 (CrHydA1), the conserved relay is composed of residues C169, E141, S189, and E144 (Figure 1). The C169 side chain plays a crucial role in catalysis due to the –SH group positioned within H-bonding distance (≈2 Å) to the bridgehead amine of ADT.^[7b]

Figure 1.

Structural representation of the primary proton transport chain and H₂ bond activation at the H-cluster of [FeFe]-H₂ase. The ADT ligand and C169 residue link proton transfer events to terminal hydride formation at [2Fe]_H through an extended hydrogen-bonding network (dotted lines) composed of several conserved amino acids; crystal structure from PDB 3C8Y^[5] was used for this illustration, and the amino-acid numbering is from the CrHydA1 primary sequence. Protonation sites (−H) on [2Fe]_H of the H_hyd state are shown.

Alteration of the ADT bridge or critical amino acids along the proton transfer pathway can abolish or severely reduce the H₂ reduction activity. Examples include [FeFe]-H₂ase variants C169A and C169S of CrHydA1,^{[3b, 3e]} C299S of Clostridium pasteurianum (CpI), C298S of Clostridium acetobutylicum (CaHydA), and C178A of Desulfovibrio desulfuricans (DdHydAB).^{[7b, 8]} Furthermore, using these modified proteins, one can accumulate significant amounts of an intermediate known as H_hyd, which contains a trapped terminal hydride Fe_d-H (Figure 1), and features a [4Fe-4S]_H⁺-Fe_p(II)Fe_d(II) oxidation state of the H-cluster.^{[3b, 3e] [3c] [9]}

Nuclear Resonance Vibrational Spectroscopy (NRVS), also known as Nuclear Inelastic Scattering (NIS), is a synchrotron-based X-ray technique that probes vibrational modes using nuclear excitation in Mössbauer-active isotopes.^[10] When applied to ⁵⁷Fe-labeled proteins, it selectively probes ⁵⁷Fe nuclei vibrational motion. In previous works on CrHydA1 and DdHydAB [FeFe]-H₂ases,^{[3c] [9]} we have shown that ⁵⁷Fe_d-H bending modes can be observed by NRVS. The NRVS signature of the terminal Fe_d-H hydride in H_hyd contains two bands >670 cm⁻¹, rationalized by density functional theory (DFT) as H^– motions perpendicular to and within the approximate mirror symmetry plane of [2Fe]_H.

Herein, we extend our methodology to identify the Fe_d-H/D modes in the C169S CrHydA1 variant. Specifically, we report the H_hyd ⁵⁷Fe-NRVS spectra of the biosynthesized [4⁵⁷Fe-4S]_H-[2⁵⁷Fe]_H C169S CrHydA1 (^†C169S) species. We compare this new data to previously reported H_hyd spectra for reconstituted [2⁵⁷Fe]_H native^[9] (^†RN) and [2⁵⁷Fe-ODT]_H bridgehead-altered^[3c] (^†ODT) enzymes. The NRVS data reveal surprising spectral shifts produced by the –SH (^†RN) to –OH (^†C169S) substitution in the side chain functional group. The results are interpreted using DFT modeling of the H-cluster and its environment.

NRVS spectra for the ^†C169S samples prepared under reducing conditions for hydride enrichment (documented by FT-IR in Figure S1) in either H₂O (^†C169S_H) or D₂O (^†C169S_D) buffer are illustrated in Figures 2 and S2. Features in the 200–400 cm⁻¹ range are primarily due to Fe-S motion, and hence they show a little change between either H or D isotopologues (Figure 2, top). A benefit from having an ⁵⁷Fe-enriched [4⁵⁷Fe-4S]_H subcluster is a presence of redox-sensitive [4Fe-4S]-specific bands in the NRVS data.^[12] As illustrated in Figure 2, the positions of the Fe-S stretch bands at 268 and 362 cm⁻¹ align nicely with a previously recorded spectrum of D14C variant of Pyrococcus furiosus (Pf) ferredoxin (Fd) in its reduced [4Fe-4S]⁺ form.^[11] This is consistent with the assignment of H_hyd to the [4Fe-4S]_H⁺-Fe_p(II)Fe_d(II) state having a reduced, paramagnetic Fe-S cubane.^{[3b, 3e]} The NRVS features in the 400–600 cm⁻¹ range, having weaker intensities and higher vibrational energies than the Fe-S bands, are mostly from modes with Fe-CN/CO character, as reported previously.^[13]

Figure 2.

Top: NRVS for H₂O and D₂O samples of [4⁵⁷Fe-4S]_H-[2⁵⁷Fe]_H C169S CrHydA1 (^†C169S) reduced with Na-dithionite (bottom), compared to the oxidized (top) and reduced (middle) [4⁵⁷Fe-4S] cluster from D14C Pf Fd.^[11] Bottom: Comparison of the Fe_d-H/D bending region recorded using the [2⁵⁷Fe-ODT]_H (^†ODT),^[3c] [2⁵⁷Fe]_H (^†RN),^[9] and ^†C169S samples. Bands A’, A” and B (see text) are marked.

At still higher energies are bands at 673 (A’) and 772 (A”) cm⁻¹, which have been assigned to the Fe_d-H bending modes in the H_hyd state.^{[3c, 9]} DFT modeling reveals that these modes contain only ≈8% ⁵⁷Fe and as much as ≈60–80% H^– contributions to their vibrational energies; see animations for the key normal modes in Supporting Information. The A’ and A” features red-shift under D₂O conditions, and an Fe_d-D band (B) appears at 630 cm⁻¹ (Figure 2, bottom) with its intensity enhanced due to coupling to the Fe-CO/CN motions. Band B is thus the most direct evidence for identifying an isotope effect on the Fe_d-H/D modes. The position of band B of the ^†C169S_D sample changes insignificantly compared with previously published NRVS data on bands B from samples ^†RN_D and ^†ODT_D. The relative positions of bands A’ and A” of ^†ODT_H, ^†RN_H, and ^†C169S_H show very different behavior (see additionally Table S1). Band A’ of ^†C169S_H, compared with ^†ODT_H and ^†RN_H, exhibits only small shifts within 5 cm⁻¹ (Figure 2, bottom), less than the 8 cm⁻¹ instrumental resolution of our spectra. However, band A” of ^†C169S_H shows a dramatic 28 cm⁻¹ upshift relative to ^†RN_H, and an even larger 45 cm⁻¹ upshift relative to ^†ODT_H.

Since the NRVS spectra for the H_hyd state accumulated in the three CrHydA1 samples exhibit prominent shifts in their Fe_d-H bands, a computational model explaining these differences has to include the sample-specific structural variations. Recently, we proposed three levels of DFT modeling for the H-cluster in its H_hyd state: S, L, and L’.^[9] S is a small model that contains the [2Fe]_H subcluster only, sufficient to rationalize the variation between the ^†ODT_H and ^†RN_H spectra. L and L’ are larger models, both incorporating as well the [2Fe]_H-surrounding amino acids including the one at position 169; L’ additionally includes the [4Fe-4S]_H cluster (see Figures S4–S7). Model L is superior in reproducing the NRVS spectra >400 cm⁻¹ containing the Fe_d-H/D bands, and model L’ is optimal for the spectra simulation <400 cm⁻¹ (see Figure S2). In our previous work on the ^†RN system, the DFT modeling predicted that the Fe_d-H band structure is indicatively sensitive to the redox level of the [2Fe]_H subcluster; Fe-H vibrations in both [FeFe]-H₂ase and its model system^[14] were found to be sensitive to the details of the environment. A model of H_hyd having the –NH– amino form with its H_ADT in an ‘Axial’ position pointing towards the H_h hydride (H_hyd-A) as illustrated in Figure 3, top was found to be the best fit to the observed NRVS spectra.^[9]

Figure 3.

Top: Representative DFT L-models of the H_hyd-A state in CrHydA1 variants C169 and C169S, overlaid with the X-ray structural reference PDB 5BYQ.^[6b] H_h, H_ADT, and H_C/S sites are shown in ball representation, and other hydrogen nuclei are omitted for clarity. Dashed lines indicate interatomic interactions H_h⋯H_ADT and N_ADT⋯H_C/S within 2.3 Å as detailed in Table 1. For the full-size structural view and model alternatives, see Figures S4–S7. Bottom: DFT ⁵⁷Fe-PVDOS spectra >600 cm⁻¹ for the ^†RN and ^†C169S species based on the best-fit L-H_hyd-A modeling. H and D nuclei relevant to the isotope labeling are specified in square brackets. Important bands (A’, A’’, and B) associated with the Fe_d-H_h/D_h bending motion are labeled. See Figures S2–S3 for the 0–900 cm⁻¹ spectra, as well as comparison to the NRVS data.

The DFT modeling further indicates that inclusion of the N_ADT⋯H_C/H_S hydrogen bond between the ADT bridgehead and C/S169 side chain effectively strengthens the H_h⋯H_ADT dihydrogen interaction (Table 1, S vs L values) in both the native and C169S variant. The H_h⋯H_ADT interaction, absent in the bridgehead-altered ODT (–O–) species, is a crucial step for H₂ formation from the H_hyd state in the proposed [FeFe]-H₂ase mechanism. Interestingly, the C169S variant is predicted to have a ≈0.1 Å shorter H_h⋯H_ADT distance than the native enzyme (Table 1, L values). Moreover, the optimized C169S L-model has noticeably shorter N_ADT⋯H_S and N_ADT⋯O_S distances, which characterize the hydrogen bonding between ADT and S169. Overall the delocalized interactions involving Fe_d–H_h⋯H_ADT–N_ADT⋯H_C/H_S–S_C/O_S have shorter non-covalent bonding (⋯) distances in C169S versus the native enzyme and a ≈0.8 Å contraction of Fe_d⋯O_S to 5.64 Å versus 6.24 Å for Fe_d⋯S_c.

Table 1.

Comparison of key bond lengths and distances in the H_hyd-A state of C169 / C169S variants of CrHydA1, optimized at the DFT modeling levels S and L.

DFT Modeling Level	Internuclear Distance (Å), C169 / C169S Variant[a]
	Fed–Hh	Hh⋯HADT	NADT–HADT	NADT⋯HC / HS	SC / OS–HC / HS	NADT⋯SC / OS	Fed⋯SC / OS
S[b]	1.51	2.14	1.03	NA	NA	NA	NA
L	1.52 / 1.52	2.01 / 1.92	1.03 / 1.03	2.26 / 2.15	1.37 / 0.98	3.59 / 3.04	6.24 / 5.64

^[a]Atomic labels follow those used in Figures 3 top and S4–S7. Bonding and non-bonding interactions are marked in column headers as ‘-’ and ‘⋯’, correspondingly.

^[b]At the modeling level S, the C/S169 residue is not included. The internuclear distances within the [2Fe]_H subcluster therefore take their single values, and ‘NA’ implies distances not applicable.

Even though the Fe_d⋯S_C/O_S distance is >5 Å, DFT was able to predict the effects of the change in functional groups on the Fe_d-H_h vibrational modes. Specifically, there is a ≈20 cm⁻¹ calculated upshift of the ⁵⁷Fe-PVDOS band A”, produced by the delocalized ‘in-plane’ normal mode (involving hydrogen nuclei H_h of hydride, H_ADT of ADT, and H_C/S of C/S169) from 749 (C169) to 768 cm⁻¹ (C169S), see Figures 3 bottom and S3. In contrast, the lower energy band A’, produced by the localized ‘out-of-plane’ normal mode (which is an essentially a pure Fe_d-H_h motion decoupled from other nuclei) is predicted to be two-fold less affected by ≈10 cm⁻¹, from 665 (C169) to 675 (C169S) cm⁻¹. The calculations therefore match sufficiently well with shifts observed by NRVS. Notably, the optimized Fe_d–H_h–H_ADT angle is ≈110° (Figure 3, top); the H_h displacement vector is therefore approximately inline to the H_h⋯H_ADT bonding for vibrational mode A”, and perpendicular to it for mode A’. Notably, the band B position is essentially invariant to the ^†ODT_D/^†RN_D/^†C169S_D system selection, from both the experiment and theory; the Fe_d-D_h normal mode responsible for B is decoupled from the cofactor bridgehead and C/S169.

Implicated in the catalysis, the H_h⋯H_ADT dihydrogen interaction of the H_hyd state is therefore stronger in the C169S variant than in the native enzyme. This can be rationalized due to the secondary coordination sphere substitution of the more polar –SH thiol group in cysteine (pK_a ≈ 8) for the less polar –OH hydroxyl group in serine (pK_a ≈ 13), which sets off a cascade of H-bonding effects linked to the active site. Due to the large difference in pK_a between the two groups, this structural difference has also been proposed to alter essential proton-transfer steps required for the catalytic mechanism.^[8c]

The above results indicate that the architecture of the amino acid environment around the H-cluster is critical for attaining an optimal form of the Fe_d-H_h hydride that enables the fast kinetics of reversible H₂ activation observed in [FeFe]-H₂ases. Analogous model compound studies indeed show that the geometry and precise orientation of the Fe-H Lewis acid-base pair is required to achieve heterolytic H₂ bond cleavage.^[15]

Through the combined application of protein mutagenesis, NRVS, FT-IR, and DFT calculations we have elucidated the effects of the modification of the proton transport chain of [FeFe]-H₂ase on hydride formation. The results reveal that the Cys-to-Ser change modifies structural interactions near the H₂ binding site and H-cluster ADT ligand. This suggests a broader role for the C169 residue through vibrational coupling in fine-tuning the hydrogen bonding interactions around the H-cluster required for rapid binding and turnover of H₂. The observation of these effects highlights the potential of NRVS in providing detailed structural information on the [FeFe]-H₂ase active site environment and, potentially, on other iron-hydride systems. In combination with DFT calculations and site-directed mutagenesis, we have found a synergistic approach to probing the Fe-H vibrational modes and uncovering how they are coupled to the secondary coordination sphere around the active site. We have shown that even an amino acid >5 Å away, such as C/S169, modulates the Fe_d-H/D bond in CrHydA1.