Vibrational characterization of a diiron bridging hydride complex – a model for hydrogen catalysis
A diiron complex containing a bridging hydride and a protonated terminal thiolate of the form [(m,k 2 -bdtH)(m-PPh 2 )(m-H)Fe 2 (CO) 5 ] + has been investigated through 57 Fe nuclear resonance vibrational spectroscopy (NRVS) and interpreted using density functional theory (DFT) calculations. We report the Fe-mH-Fe wagging mode, and indications for Fe-mD stretching vibrations in the D-isotopologue, observed by 57 Fe-NRVS. Our combined approach demonstrates an asymmetric sharing of the hydride between the two iron sites that yields two nondegenerate Fe-mH/D stretching vibrations. The studied complex provides an important model relevant to biological hydrogen catalysis intermediates. The complex mimics proposals for the binuclear metal sites in [FeFe] and [NiFe] hydrogenases. It is also an appealing prototype for the 'Janus intermediate' of nitrogenase, which has been proposed to contain two bridging Fe-H-Fe hydrides and two protonated sulfurs at the FeMo-cofactor. The significance of observing indirect effects of the bridging hydride, as well as obstacles in its direct observation, is discussed in the context of biological hydrogen intermediates.
Introduction<!>NRVS spectra and qualitative assignments<!>DFT calculations and quantitative comparisons<!>Asymmetric hydride sharing by Fe p and Fe d<!>Conformational heterogeneity of the Fe thiol<!>Chemical heterogeneity of the Fe thiolate<!>Intermolecular couplingthe dimer model<!>Discussion<!>Summary and outlook<!>Conflicts of interest
<p>Biological hydrogen catalysis is driven by a ubiquitous and diverse set of enzymes called hydrogenases [1][2][3][4] and to a lesser extent by the nitrogenase family. 5 Hydrogenases reversibly convert molecular hydrogen into reducing power at binuclear [FeFe], binuclear [NiFe], 6 or mononuclear [Fe] active sites. 7 Similarly, hydrogen evolution in Mo-nitrogenase occurs at a unique Fe 7 MoS 9 C cluster called the FeMo-cofactor, and analogous chemistry likely occurs at the Fe(V,Fe)-cofactors. 8,9 The activities of all these enzymes are presumably related by the formation of metastable Fe-H À hydrides and protonation of nearby sulfurs during catalysis (Fig. 1). 6,10,11 Synthesis and characterization of model complexes that can simulate biochemical intermediates and their proton transfer capabilities by the stabilization of transition metal hydrides (M-H À ) and nearby thiols (-SH) are key to understanding hydrogenase and nitrogenase active site hydrogen catalysis. 10,[12][13][14][15][16]  These special -SH/M-H À interactions take various forms. In [FeFe] hydrogenases there is a conserved cysteine situated at the end of a proton transfer chain formed by hydrophilic amino acid residues and water molecules, with the -SH thiol group adjacent to an azadithiolate (ADT) bridge of the [2Fe] H subcluster (Fig. 1c). There is considerable evidence that the ADT bridge plays a critical role in proton acceptance and relay to the catalytic Fe d site. 17 Moreover, disruption of the proton supply chain in the enzymes from Chlamydomonas reinhardtii (CrHydA1) or Desulfovibrio desulfuricans (DdHydAB) has allowed the transient H hyd catalytic state to be trapped and unequivocally shown to involve a terminal Fe d -H À hydride (Fig. 1c). 14,[18][19][20][21] For [NiFe] hydrogenases, the catalytic Ni-R state has been shown to feature a bridging hydride, with a Ni-bound protonated cysteine sulfur (Fig. 1b). 6 Finally, in nitrogenases the E 2 state is presumed to have one bridging Fe-H À -Fe hydride with a nearby protonated sulfur capable of H 2 release, while two hydrides capable of reductive elimination have been proposed for the E 4 or 'Janus intermediate' of the Fe(Mo,V,Fe)-cofactor (Fig. 1d). 9 To model the aforementioned biological hydrides, previous efforts produced and characterized synthetic protonated thiols coordinated to diiron centers. [22][23][24][25] Here we extend this work by exploring 57 Fe-specic vibrational dynamics for a diiron center with a bridging hydride and a protonated thiol ligand. Specically, we have characterized the [(m,k 2 -bdtH)(m-PPh 2 )(m-H) Fe 2 (CO) 5 ] + (bdt ¼ 1,2-benzenedithiolate) model compound, referred hereaer as 'mHSH' (Fig. 1a), and as well its deuterated isotopologue [(m,k 2 -bdtD)(m-PPh 2 )(m-D)Fe 2 (CO) 5 ] + , mDSD, using nuclear resonance vibrational spectroscopy (NRVS). We also recorded the spectrum of the S-methylated complex: [(m,k 2 -bdtCH 3 )(m-PPh 2 )(m-H)Fe 2 (CO) 5 ] + , here referred to as 'mHSMe'.</p><p>57 Fe-NRVS is a synchrotron-based technique that observes excitation of an 57 Fe nucleus together with the excitation/ deexcitation of vibrational modes. [26][27][28] NRVS is essentially the recoil fraction that steals intensity from the recoil-free Mössbauer effect, with the measured intensity for a given normal mode proportional to the 57 Fe kinetic energy in that mode, ultimately yielding an 57 Fe partial vibrational density of states (PVDOS). The experimental frequencies and intensities can be directly compared with normal mode calculations from density functional theory (DFT) or even empirical force elds.</p><p>The asymmetric diferrous compound mHSH is relevant to the protonated and bridging hydride-bound states proposed for H 2 production in [NiFe] hydrogenases and to the nitrogenase E 2 and E 4 intermediates. It is also a good benchmark for comparison with the NRVS for intermediates in [FeFe] hydrogenase proposed to contain a m-hydride ligand bridging the Fe p (II) and Fe d (II) sites (Fig. S1 †). 29 In line with a recent interpretation that [FeFe] hydrogenase operates exclusively with bridging CO intermediates, 30 the m-hydride intermediates were attributed to the enzyme's 'slow cycle'.</p><!><p>The overall NRVS-derived 57 Fe-PVDOS for the mHSH, mDSD and mHSMe samples are compared in Fig. 2. Qualitative assignments can be made based on comparison with previous spectra for Fe-S proteins and model compounds. The low energy <200 cm À1 portion of the spectrum is related to acoustic and torsional modes largely involving the bulky Ph and bdt subunits, as well as bending modes at the Fe sites. The next region up to 400 cm À1 comprises predominantly Fe-S and Fe-P stretching modes. The 400-650 cm À1 region has prominent features from modes that are a mix of nFe-CO stretching and dFe-CO bending.</p><p>At higher energies, a band at 745 cm À1 is particularly signicantit represents an Fe-H-Fe wagging mode. This assignment is based on comparison with results for similar Fe-mH À -Fe synthetic complexes and their computational models, 31,32 as well as Ni-mH À -Fe models and the Ni-R state of [NiFe] hydrogenase. 11 The signicant weakening at this position in the mDSD spectrum supports this assignment; residual Fig. 2 Top: the NRVS-derived 57 Fe-PVDOS spectra for mHSH ( ) and its deuterium isotopologue mDSD ( ), vertically offset by 1 Â 10 À3 cm from each other in the high-energy region. Bottom: the 57 Fe-PVDOS spectra for mHSMe ( ). In all cases the high-energy region intensity above 650 cm À1 is multiplied by 5 for visibility. The bands are labelled with their top positions, with those above 800 cm À1 assigned tentatively.</p><p>intensity likely results from an incomplete H-to-D exchange. Below the 745 cm À1 band is a second candidate wagging mode feature at $701 cm À1 . Although this energy region coincides with benzene-dithiol modes, their Fe motion is predicted to be low. The energy is also consistent with the predicted hydride wagging motion of a deprotonated species, mHS À , based on DFT calculations as described below. For the mHSMe complex, a slight downshi to 733 cm À1 is observed for the main wagging mode feature.</p><p>From previous NRVS work on Fe-H/D À complexes, 11,31,32 as well as guidance from the conventional vibrational spectroscopy literature, 33 we expect observation of the Fe-H stretching modes at vibrational energies above 1100 cm À1 . There are hints of such features at 1165 cm À1 and 1403 cm À1 , but the signal-tonoise is not convincing. Even in the best of cases, these modes are extremely weak in NRVS because they primarily involve the hydride nucleus motion with an almost static Fe partner. Below, we will use DFT calculations to investigate additional factors such as sample heterogeneity and intermolecular coupling that conspire to split the already weak Fe-H stretches into a multitude of even weaker modes that are below the current detection limit.</p><!><p>The 57 Fe-PVDOS predictions from the DFT models for mHSH, mDSD, and mHSMe (Fig. S3a and S4 †) are compared in Fig. 3, S8 and S9. † The key experimental feature at 745 cm À1 , attributed to the Fe-H-Fe wagging mode, is reproduced very well in terms of energy and intensity by the calculated band at 746 cm À1 . In this mode, the calculated mH À hydride motion is normal to the 'Fe-H-Fe' plane, as shown in Fig. S5 (see additionally ESI for animated vibrational modes, and Fig. S8 for an overlay of the NRVS-observed and DFT-predicted spectra). † A corresponding Fe-D-Fe wagging is not specically resolved in the NRVS of the deuterated sample.</p><p>To better understand the differences in these two spectra, we calculated the PVDOS for the bridging H and D nuclei, as shown in Fig. 4 and S7d. † Inspection of these curves reveals three distinct hydride bands, whereas there are only two distinct deuteride bands above 650 cm À1 . The intensity for the third deuteride band (corresponding to the wagging motion) is redistributed throughout the 400-600 cm À1 region, indicating signicant coupling in the motions of the D nucleus with the ve CO ligands. A comparison of the single wagging mode in mHSH with three prominent mDSD modes involving coupled motions is also depicted in Fig. S5. †</p><p>The computed spectrum of mDSD sees shis of the Fe-H stretching modes at 1424/1196 cm À1 to respectively 1015/ 855 cm À1 . The 57 Fe-PVDOS intensities of the Fe-D (vs. Fe-H) bands are enhanced due to the amplied 57 Fe displacements when the heavier deuteride is the bridging ligand, which allows the Fe-D stretching signals to raise above the NRVS noise level (Fig. S11 and S12 †). For future NRVS observation of such stretching modes, Fe-D complexes are clearly favored over Fe-H isotopologues.</p><!><p>We also calculated separate PVDOS for the 57</p><!><p>We also considered the possibility of alternate structural conformations having an effect on the NRVS proles. We found from DFT calculations that a local minimum was produced by reorientation of the protonated S p -H S thiolate ligand of Fe p towards the bridging hydride, yielding a 'mHSH in ' (or 'mDSD in ') structure (Fig. S3a †). The calculated energy difference was a trivial +0.3 kcal mol À1 compared to the above described best-t model having the S p -H S moiety oriented away from the hydride, referred to simply as mHSH (or mDSD). We thus expect mHSH (¼ mHSH out ) and mHSH in species to reach an equilibrium and co-exist in the sample crystals. The calculated 57 Fe-PVDOS for mHSH in produced an Fe-H-Fe wag at 752 cm À1 , upshied by 6 cm À1 from the spectrum for mHSH (Fig. 3 and S8 †). Slightly larger upshis of 7 cm À1 and 10 cm À1 were predicted for the Fe-D and Fe-H stretching modes, respectively.</p><!><p>From previous work with this compound, we know that deprotonation of the S p -H S moiety occurs readily (for example, upon solvation of mHSH in THF), and we refer to the resultant thiolate species as 'mHS À ' (Fig. S3a †). DFT calculations on this candidate predict a 35 cm À1 red-shi in the Fe-H-Fe wagging mode to 711 cm À1 (Fig. S8b †). The mHS À model is thus a possible explanation for the weak feature observed at $701 cm À1 in the experimental spectrum of mHSHalthough a similar, but weaker, feature was observed in mHSMe, implying the source of this intensity need not be limited to the mHS À species in the spectrum of mHSH (Fig. 2, S7a, and S9 †).</p><p>An even higher degree of hydride sharing asymmetry is calculated for the mHS À /mDS À model (Table S1 †) and, consequently, larger energy splittings between the predicted bands for the Fe p/d -mH/D stretches (Fig. 3b and S8b †). Specically, the Fe p -mH/D stretches calculated respectively at 818/1138 cm À1 are signicantly red-shied when compared to the best-t mHSH/ mDSD model, due to a stronger Fe p -S p bond strength which in turn leads to a weaker adjacent Fe p -mH À bonding. Notably, the deprotonation of mHSH to mHS À leads to a predicted upshi by 15 cm À1 (332 to 347 cm À1 , Fig. S8b †) for the band observed at 330 cm À1 and having the strongest Fe p -S p stretching character (see ESI † for these modes animated). Finally, to produce a more realistic simulation of the observed NRVS spectrum, in Fig. 6 and S12 † we provide an average of the three DFT models considered above.</p><!><p>A common weakness of DFT simulations of the NRVS spectra of solids is the lack of predicted intensity in the low-energy regions (<100 cm À1 ). In solid model complexes, the experimental modes in this region are due to solid matrix motions, and lattice or acoustic modes. In larger biological samples, this energy region corresponds primarily to torsional/dihedral modes and larger scale motions of the protein, and it provides an indicator of coupling between the motion of the 57 Fe-enriched bioinorganic core and its environment. 18,20,35,36 To partially account for these modes in our calculations, we have used a DFT model based on a dimeric form [mHSH] 2 which is directly available from the crystallographic unit cell in the X-ray structure (Fig. S3b †); 34 this approach is justied as the NRVS experiment employed a polycrystalline powder sample.</p><p>The vibrational calculation on [mHSH] 2 shows an increased density of normal modes and signicant improvement in the low-energy <200 cm À1 region of the predicted NRVS spectrum over that of the mHSH monomer only (Fig. 5 and S10 †). Of note is reproduction of the broad experimental feature centered at 35 cm À1 produced by highly-delocalized expansions of the dimer, see Fig. 5 and ESI † for the representative normal mode at 38 cm À1 animated. The intermolecular dispersion interaction plays an important role here, included as a correction to our DFT setup on [mHSH] 2 (see ESI Methods †). The dimer computational model does not capture all of the features in the lowenergy region -specically the prominence at $70 cm À1which may be reproduced by models accounting for more molecules and/or their structural periodicity, however such a computational effort is beyond the scope of this work.</p><p>Notably, the dimer is composed of mHSH and its mirrorimage isomer mHSH m , or the enantiomer, yet the [mHSH] 2 structure itself does not exhibit a symmetry (higher than C 1 ). Deviation from the exact mirror-image symmetry between mHSH and mHSH m effectively splits the Fe-mH/D stretching normal modes >800 cm À1 , which are particularly sensitive to even minimal Fe-mH bond length changes of $0.01 A (Table S1 †), in line with the hydride mode variations indicated above for the monomer models. This results in a more faithful simulation of their low PVDOS intensitiessee the calculated [mHSH] 2 /[mDSD] 2 vs. mHSH/mDSD spectra compared to the observed H/D sample NRVS data in Fig. 6, S10, and S12. †</p><!><p>Our results on the bridging hydride samples show both the strengths and limitations of NRVS for observation of Fe-H normal modes. The experimental and calculated observations demonstrate that the Fe-H-Fe wag mode is sensitive to the chemistry at the Fe-S ligand, reected by the species-dependent redistribution of intensity in the 690-760 cm À1 region. The DFT calculations suggest the wag mode is even sensitive to the conformation of the SH proton, shiing 6 cm À1 between the 'in' and 'out' conformers. In the range of the models considered, $40 cm À1 upshi of the predicted wag band position is approximately correlated with  S1 †). This sensitivity makes NRVS a useful structural probe, but it also means that intensity can be lost by division into multiple channels in cases of sample heterogeneity.</p><p>The DFT calculations reveal that Fe-H stretching modes are even more sensitive to variability at the Fe-S ligand than the Fe-H-Fe wag modes. In contrast to the well-dened wag mode, current S/N limitations unfortunately prevented unambiguous experimental assignments of the stretching modes. The predicted position of the lower frequency Fe p -H stretch mode is especially sensitive to the chemistry at the sulfur ligand adjacent to Fe p , varying by almost 70 cm À1 in the range of the considered alternatives (S À : 1138 cm À1 , SH out : 1195 cm À1 , SMe in : 1204 cm À1 , SH in : 1206 cm À1 ).</p><p>Another result from the DFT calculations is the inequitable sharing of the bridging hydride. This is most directly seen in the optimized $0.1 A Fe-H distance differences. Similar differences have been predicted by DFT previously in the bimetallic cofactors of [FeFe] and [NiFe] hydrogenases and their models. 11,20,32 The difference in Fe p/d -mH bonding is also reected in the prediction of two distinct Fe-H stretching modes split by as much as 322 cm À1 . Even greater asymmetry was observed in a Ni-mH-Fe model complex, where Ni-H and Fe-H stretches were seen respectively at 954 cm À1 and 1468/1532 cm À1 , the latter split by 64 cm À1 due to a conformational heterogeneity in the m-pdt ligand. 11 It is noteworthy that the asymmetry is modulated by the status of the iron-bound sulfura larger asymmetry occurs when the sulfur is deprotonated.</p><p>The model studies have relevance to proposed [FeFe] hydrogenase intermediates. A terminal hydride state H hyd has been convincingly assigned using NRVS. [18][19][20]29 However, bridging hydride species have also been proposed for intermediates named H(s)red using other methods, 29,37 and a recent report has investigated the Hred state using a combination of 57 Fe-NRVS and other spectroscopies, supported by QM/MM calculations. 29 It was proposed that the Hred state contains a bridging Fe p -mH À -Fe d hydride with its wag distributed among the $600-650 cm À1 (Fe-)CO/CN À /ADT motions, thus prohibiting its direct observation.</p><p>Our experimental results on model compounds found wagging modes at higher frequencies, ranging from 694-702 cm À1 in a sterically congested diiron hydride 32 to 733-745 cm À1 for the current samples to 758 cm À1 in a Ni-mH-Fe model complex. 11 Of course, structural differences can conspire to move wagging modes to lower frequenciesin a recent study of a complex with an Fe(m-H) 2 Fe core the wagging modes were identied at 455 and 587 cm À1 , mostly screened by other NRVS bands. 38 For future work with improved S/N, observation of Fe-D and Fe-H stretching modes would be a more rigorous way to test various structural hypotheses.</p><!><p>In this work we have characterized the mHSH and mHSMe (and likely mHS À ) synthetic models with NRVS experiments and DFT calculations. Foremost, we have directly identied the wellseparated Fe-H-Fe wagging modes both in the experimental and DFT-simulated spectra. The related Fe-D-Fe wagging modes were found to be heavily mixed with Fe-CO vibrations. The wagging mode displays signicant variability that makes it a sensitive test of molecular structure.</p><p>Fe-H and Fe-D stretching modes were also scrutinized by DFT modeling for the isomeric mHSH in/out and deprotonated mHS À forms of mHSH, as well as a [mHSH] 2 dimer relevant to the polycrystalline NRVS sample. These modes exhibit larger frequency and intensity shis, making them an even more delicate probe of iron-hydride bonding. Their routine application awaits improvements in sample preparation, beamline ux, monochromators, and detectors.</p><!><p>There are no conicts to declare.</p>