Abstract
Abstract: A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize FeÀH/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. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe À H/D normal modes. We observed a significant shift to higher frequency in an Fe À H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH-moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.

Abstract: A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize FeÀH/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. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe À H/D normal modes. We observed a significant shift to higher frequency in an Fe À H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH-moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.

Hydrogenases (H 2 ases) are enzymes that catalyze the reversible interconversion of molecular hydrogen into protons and electrons: H 2 Q2 H + + 2 e À . [1] Under optimal electron delivery, H 2 ases require little or no driving force while utilizing base transition metals as catalytic cofactors. The [FeFe]-H 2 ases are known to display exceptionally high turnover rates of > 10 4 s À1 . [1a, 2] Resolving the mechanistic properties of [FeFe]-H 2 ases requires identification of the catalytically relevant intermediates, [3] and is of interest to advancing catalyst design for developing H 2 fuel cells and renewable energy production technologies. [4] The active site for all known [FeFe]-H 2 ases 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 group plays 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]-H 2 ase 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 as the -SH group is positioned within hydrogen-bonding distance (ca. 2 ) to the bridgehead amine of ADT.

[7b]

Alteration of the ADT bridge or critical amino acids along the proton transfer pathway can abolish or severely reduce the H 2 H through an extended hydrogen-bonding network (dotted lines) composed of several conserved amino acids; the crystal structure from PDB 3C8Y [5] was used for this illustration, and the amino acid numbering is from the CrHydA1 primary sequence. Nuclear resonance vibrational spectroscopy (NRVS), also known as nuclear inelastic scattering (NIS), is a synchrotronbased X-ray technique that probes vibrational modes using nuclear excitation in Mçssbauer-active isotopes.

[ [12] As illustrated in Figure 2 , the positions of the FeÀS stretching bands at 268 and 362 cm À1 align nicely with a previously recorded spectrum of the D14C variant of Pyrococcus furiosus (Pf) ferredoxin (Fd) in its reduced [4Fe-4S] + form. [11] [3b,e] The NRVS features in the 400-600 cm À1 range, with weaker intensities and higher vibrational energies than the Fe À S bands, are mostly from modes with Fe À CN/CO character, as reported previously. [13] At still higher energies, there are bands at 673 (A') and 772 cm À1 (A''), which have been assigned to the Fe d ÀH bending modes in the H hyd state (Figure 2, bottom) .

[3c, 9] DFT modeling reveals that these modes contain only about 8 % Fe] H ( † RN), [9] and † C169S samples. Bands A', A'' and B (see text) are marked.

( Figures S4-S7) . Model L is superior in reproducing the NRVS spectra above 400 cm À1 containing the Fe d À H/D bands, and model L' is optimal for the spectral simulation below 400 cm À1 ( Figure S2 ). In our previous work on the † RN system, the DFT modeling predicted that the Fe d ÀH band structure was indicatively sensitive to the redox level of the [2Fe] H subcluster; the FeÀH vibrations in both [FeFe]-H 2 ase and its model system [14] were found to be sensitive to the details of the environment. A model of H hyd having the -NHamino 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] 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 ( 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 in terms of the secondary coordination sphere substitution of the more polar -SH thiol group in cysteine (pK a % 8) for the less polar -OH hydroxy group in serine (pK a % 13), which sets off a cascade of hydrogen-bonding effects linked to the active site. Owing 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 ÀH h hydride that enables the fast kinetics of the reversible H 2 activation observed in [FeFe]-H 2 ases. Analogous model compound studies indeed show that the geometry and precise orientation of the Fe À H Lewis acid-base pair is required to achieve heterolytic H 2 bond cleavage. [15] Through the combined application of protein mutagenesis, NRVS, FTIR spectroscopy, and DFT calculations, we have elucidated the effects of the modification of the proton transport chain of [FeFe]-H 2 ase on its hydride-bound state H hyd . The results reveal that the Cys-to-Ser change modifies structural interactions near the H 2 binding site and the Hcluster 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 H 2 . The observation of these effects highlights the potential of NRVS in providing detailed structural information on the [FeFe]-H 2 ase 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 À 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 that is > 5 away, such as C/S169, modulates the Fe d ÀH/D bond in CrHydA1.

{'paper_id': '49309776', 'title': 'Terminal Hydride Species in [FeFe]‐Hydrogenases Are Vibrationally Coupled to the Active Site Environment', 's2_url': 'https://api.semanticscholar.org/CorpusID:49309776', 'source': 'S2ORC'}