Direct Detection of the Terminal Hydride Intermediate in [FeFe

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Direct Detection of the Terminal Hydride Intermediate in [FeFe] Hydrogenase by NMR Spectroscopy Sigrun Rumpel, Constanze Sommer, Edward Reijerse, Christophe Farès, and Wolfgang Lubitz J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b00459 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Journal of the American Chemical Society

Direct Detection of the Terminal Hydride Intermediate in [FeFe] Hydrogenase by NMR Spectroscopy Sigrun Rumpel,† Constanze Sommer,† Edward Reijerse,† Christophe Farès‡ and Wolfgang Lubitz† † ‡

Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm Platz 1, 45470 Mülheim an der Ruhr, Germany

Supporting Information Placeholder ABSTRACT: Hydride state intermediates are known to occur in various hydrogen conversion enzymes, including the highly efficient [FeFe] hydrogenases. The intermediate state involving a terminal iron-bound hydride has been recognized as crucial for the catalytic mechanism, but its occurrence has up to now eluded unequivocal proof under (near) physiological conditions. Here we show that the terminal hydride in the [FeFe] hydrogenase from Chlamydomonas reinhardtii can be directly detected using solution 1H NMR spectroscopy at room temperature, opening new avenues for detailed in-situ investigations under catalytic conditions.

[FeFe] hydrogenases are the most efficient catalysts for H2 production.1 Their active site, the H-cluster is comprised of a classical [4Fe-4S]H cluster which is linked by a bridging cysteine to a unique [2Fe]H site ([Fe2(adt)(CO)4(CN)2]2-, adt = azadithiolate, Figure 1). The iron atoms of [2Fe]H are located proximal (Fep) and distal (Fed) to the [4Fe-4S]H cluster and are bridged to one another via the adt ligand. This adt ligand plays a key role in shuttling protons between the H-cluster and the protein surface. Owing to its acid-base properties, the secondary amine exchanges protons with Fed and the protein surrounding. The amino acid residue closest to the amine (Cys 169) participates in a hydrogen bonding interaction with the amine nitrogen2 and has been shown to play a central role in proton transfer.3,4 The current working model of the catalytic cycle involves H2 binding to the active oxidized state and subsequent heterolytic H2 cleavage resulting in an intermediate state Hhyd with a terminal hydride bound at Fed (Figure 1).5-7 This state is characterized by a formal Fe(II)Fe(II) redox configuration for [2Fe]H (S=0) and a reduced [4Fe-4S]H sub-cluster (S=1/2). Most [2Fe]H model complexes of Hhyd contain the hydride in the thermodynamically more stable bridging position between Fep and Fed.8 However, a few diiron dithiolate model complexes mimicking the H-cluster have been reported with a bound terminal hydride. They have also revealed faster and more reversible H2 activation.8 Terminal hydrides are typically kinetic products which are frequently detected at temperatures below 253 K by 1H NMR spectroscopy at relatively low frequency between -2 and -7 ppm (Table S1).9-14 A dihydride diiron dithiolate model complex has also been reported to contain a bridging as well as a terminal hydride with 1H chemical shifts at -12.2 and -18.9 ppm, respectively.15 In general, 1H NMR chemical shifts of transition metal hydrides are strongly influenced by their coordination to

the metal since hydrogen uses almost exclusively its 1s orbital for bonding. Large negative 1H chemical shifts are most common for transition metal hydrides especially for d6 and d8 complexes but also positive values are possible in d10 and d0 hydride complexes.16,17 Bridging hydrides resonate at lower frequency than terminal hydrides due to the additional shielding effect from the two metals. For the dihydride diiron dithiolate complex the 1H NMR signals are reversed as the trend may apply only to terminal, apical hydrides.15

Figure 1. The active site H-cluster of the [FeFe] hydrogenase HydA1 in the hydride state based on pdb-entry 3C8Y with the surrounding protein framework shown in gray. The H-cluster and the surrounding cysteines are shown as sticks with the following color coding; iron: orange, sulfur: yellow, carbon: cyan, oxygen: red, nitrogen: blue, hydrogen: gray. In contrast to the H-cluster, in which Fed adopts a reactive rotated state, synthesized terminal hydride model compounds lack conformational rigidity. The protein residues surrounding of the Hcluster in [FeFe] hydrogenases sterically constrains dynamics and regiochemistry at Fed thereby favoring a terminal instead of a bridging hydride as an intermediate of the catalytic cycle. This Hhyd state of [FeFe] hydrogenases has recently been characterized at low temperatures by Mössbauer18 and nuclear resonance vibrational spectroscopy6,7 as well as at ambient temperature by Fourier transform infrared spectroscopy (FTIR).19,20

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Figure 2. High and low frequency regions of the 1H 1D NMR spectra (600 MHz) at 298 K of HydA1-C169A A: maturated with [Fe2(adt)(CO)4(CN)2]2- (black line), B: maturated with [Fe2(adt)(CO)4(CN)2]2- and in 100 % D2O (red line), C: maturated with [Fe2(2Hadt)(CO)4(CN)2]2- (blue line) and D: maturated with [Fe2(2H-adt)(CO)4(CN)2]2- and in 100 % D2O (green line). The arrow in A and C points at the 1H signal ‘j’ which is assigned to the terminal hydride. In addition, computational data indicate a terminal hydride bound to Fed as favorable for the catalytic mechanism of [FeFe] hydrogenases.21 In order to understand the enzyme’s unmatched H2 production properties and to obtain clues that could help in designing a more efficient synthetic catalyst, it is essential to obtain information about the pivotal hydride state at near physiological conditions; arguably solution NMR spectroscopy is the method of choice since it allows direct detection of the hydrogenic species (H2, H+, H-).8 However, direct detection of a hydride intermediate of [FeFe] hydrogenases or any other enzyme at room temperature is very challenging and has so far never been successful. Recently, we have reported the first solution NMR spectroscopic investigation of different states of HydA1.22 Here, we present the first successful detection of the hydride 1H NMR signal corresponding to the intermediate hydride state of an enzyme in solution at room temperature. In order to enrich Hhyd, the variant C169A of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii has been prepared (see Supporting Information 1.1 and 1.2). The exchange of Cys at position 169 by Ala (or Ser) has been shown to impair proton transfer.19,20 Thereby, Hhyd is stabilized and accumulates. To confirm the presence of Hhyd and to analyze its purity, FTIR spectra were recorded and revealed νCO bands at 1979, 1963 and 1863 cm-1 (see Supporting Information Figure S1) which have recently been reported for Hhyd of HydA1 variant C169A.19 For the bridging CO, νCO bands occur between 1790 and 1850 cm-1 for the different H-cluster states of the catalytic cycle.23 Presence of a terminal hydride has been shown to have a strong influence on its trans ligand,24 the bridging CO, whose νCO band is shifted to 1863 cm-1. Furthermore, substitution of hydrogen by deuterium results in a substantial νCO red shift of the bridging CO whereas

no other CO ligands shift to lower energies.11,20 This effect has been used to indirectly prove the presence of a terminal hydride bound to Fed.7,19,20 Here, the expected red shift by 5-6 cm-1 upon deuterium substitution is identified by the peak at about 1858 cm-1 (Figures S1 and S2). The FTIR spectra confirm Hhyd to constitute at least 90 % of each HydA1-C169A sample directly after preparation (Figures S1 and S2). 1 H NMR experiments at room temperature were measured in parallel to the FTIR experiments for unlabeled Hhyd with 10% and 100 % D2O (Figure 2A,B) as well as Hhyd prepared using deuterated [2Fe]H (2H-adt) with 10% and 100 % D2O (Figure 2C,D). In the high frequency region between 14 and 70 ppm six broad peaks of different intensities labeled ‘a’ to ‘f’ are observed (Figure 2 and Table 1). The different intensities are indicative of different relaxation times caused by the “through-bond” interaction of the unpaired electron(s) with the 1H nuclei. Importantly, six signals appear in the low frequency region between -1 and -20 ppm (Figure 2A). Using [Fe2(2H-adt)(CO)4(CN)2]2- for maturation, 1H signals of peaks d, e, l and m (Figure 2C and D) can be assigned to the four adt methylene protons with the chemical shifts of peaks l and m observed at the lowest frequency (-15.2 and -17.6 ppm, respectively). In analogy to the 1H NMR spectra of the Hox and Hox-CO state22 of HydA1, signals l and m are assigned to the equatorial and signals d and e to the axial protons of [2Fe]H (Figure 2). Importantly, the 1H signal j observed at -9.6 ppm (Figure 2A and C) disappears in presence of 100 % D2O (Figure 2B and D) and hence belongs to a solvent exchangeable 1H. Considering the already demonstrated H/D exchange properties of the hydride of Hhyd7,19 in combination with the significant negative chemical shift, the peak j can be assigned with high confidence to the terminal hydride bound to Fed. Two main effects result in the termi-

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Journal of the American Chemical Society nal hydride being observed at -9.6 ppm. First, very strong spinorbit coupling due to the interaction of the approximately πsymmetric Fe orbitals with the hydrogen 1s orbitals result in extreme shielding17. Second, a fundamental dependence has been reported very recently for the trans ligand on the sign and magnitude of spin-orbit effects and 1H shielding.16 In case of Hhyd, the bridging CO as a strong trans ligand causes deshielding. This deshielding effect diminishes the extreme shielding caused by the iron Fed and leads to the observation of the 1H signal of the terminal hydride at a moderately negative chemical shift of -9.6 ppm (Figure 2). Nevertheless, this shift is still at least 3.5 ppm more negative than the reported hydride chemical shifts of monohydride diiron dithiolate complexes (Table S2). Possibly, the increased shift is related to the delocalization of spin density from the reduced [4Fe-4S]H subcluster on the [2Fe]H subsite. The observed pseudo Curie temperature dependence (Table 1, Figure S3) would be consistent with such a mechanism. In addition to the signal at -9.6 ppm, a water exchangeable 1H signal is observed at -1.6 ppm (Figure 2). This signal ‘g’ could originate from the amine proton of the adt ligand or a water exchangeable -NH, -NH2, -OH, -SH or –COOH site of a protein residue. The temperature independence of its shift suggests a negligible contribution from paramagnetic spin density. Considering the unusual 1H shift of -1.6 ppm, the only possible residuebased candidates are the water exchangeable hydrogens of the NH2 group of Lys228 or the backbone amide 1H of Ala94 or Gln195 in hydrogen bond distance to one of the CN- ligands of the H-cluster.2 However, an assignment of the 1H signal ‘g’ ppm to one of the surrounding residues can likely be dismissed by the comparison with the 1H NMR spectra of the Hox-CO state22 in 100 % D2O and 10 % D2O, which are void of solvent exchangeable protons in the region upfield of -1 ppm (Figure S4). Hence, the assignment of the 1H signal ‘g’ at -1.6 ppm to the amine proton of the adt ligand is most likely (Table 1). The other hyperfine shifted signals a, b, c, f, h, i and k have been assigned to β-CH2 protons of the [4Fe-4S]H cluster coordinating cysteines. Based on the chemical shift and line width signals f and h might alternatively correspond to α-CH protons of the [4Fe-4S]H cluster coordinating cysteines. The general pattern of up and down field shifted cysteine 1H signals (Table 1) and their temperature dependence (Figure S3) is related to the spin structure of the reduced [4Fe4S]H sub-cluster consisting of a S=9/2 Fe(II)Fe(III) pair antiferromagnetically coupled to an S=4 Fe(II)Fe(II) pair.25,26 Table 1. 1H NMR spectral parameters for the hyperfine shifted resonances of the hydride state of HydA1-C129A at 298 K. A more detailed depiction of the temperature dependence of the chemical shift can be found in the Supporting Information (Figure S3). Peak label a b c d e f g h i j k l m

Peak assignment β-CH2 β-CH2 β-CH2 adt-CH2 adt-CH2 β-CH2 adt-HN β-CH2 β-CH2 t-H β-CH2 adt-CH2 adt-CH2

Chem. shift (ppm) 66.47 55.13 52.44 46.3 28.36 19.47 -1.64 -2.43 -6.34 -9.59 -11.2 -15.23 -17.59

Width (Hz)* 800 900 1100 1000 400 400 100 300 400 400 500 300 500

Temp. dep.§ C aC C C C aC None pC pC pC pC pC pC

Intensity 10 150 100 10 20 270 150 80 40 10 20 40 20

*full-width at half-maximum, §C = Curie, aC= anti-Curie, pC = pseudo-Curie

To further demonstrate that Hhyd of HydA1-C169A closely resembles HydA1-wildtype, which is less stable, a 1H NMR spectrum of Hhyd in presence of 100 mM sodium dithionite at pH 66 reveals a peak pattern very similar to Hhyd of HydA1-C169A (Figure S5). However the quality of the spectrum is poor because of the increased salt concentration in combination with the extreme instability of Hhyd of HydA1-wildtype at room temperature. Also, only about 80 % of the sample was in the Hhyd state directly after preparation (Figure S6). For a discussion of the stability of Hhyd at room temperature please refer to the Supporting Information 4.2 and Figures S6 and S7. In this communication we have shown for the first time, that the detection of a labile hydride intermediate of an enzyme at ambient conditions is possible by NMR spectroscopy when the experimental conditions are carefully optimized. Detection of the key hydride intermediate of [FeFe] hydrogenases in solution at room temperature opens up a new perspective to study their catalytic mechanism, e.g. through in-situ NMR investigations under (steady-state) turnover conditions or even in-vivo27 as has recently been demonstrated using EPR on [FeFe] hydrogenases.28 Furthermore we provide evidence that the amine proton of the adt ligand can also be identified in the NMR via H/D exchange. The obtained 1H chemical shift provides important information on the molecular and electronic structure which may be used in the design of diiron dithiolate complexes with improved catalytic activity. ASSOCIATED CONTENT

Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Methods; supplementary FTIR spectra; summary of complexes mimicking Hhyd; temperature-dependence of 1H chemical shifts (PDF) AUTHOR INFORMATION

Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors thank Claudio Luchinat and Enrico Ravera (Florence) for many stimulating discussions, Nina Breuer for the preparation of HydA1-C169A and Yvonne Brandenburger for recording FTIR spectra. REFERENCES (1) Lubitz, W.; Ogata, H.; Rüdiger, O.; Reijerse, E. Chem. Rev. 2014, 114, 4081-4148. (2) Knörzer, P.; Silakov, A.; Foster, C. E.; Armstrong, F. A.; Lubitz, W.; Happe, T. J. Biol. Chem. 2012, 287, 1489-1499. (3) Morra, S.; Giraudo, A.; Di Nardo, G.; King, P. W.; Gilardi, G.; Valetti, F. PLoS ONE 2012, 7, e48400. (4) Cornish, A. J.; Gärtner, K.; Yang, H.; Peters, J. W.; Hegg, E. L. . Biol. Chem. 2011, 286, 38341-38347.

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(5) Sommer, C.; Adamska-Venkatesh, A.; Pawlak, K.; Birrell, J. A.; Rudiger, O.; Reijerse, E. J.; Lubitz, W. J. Am. Chem. Soc. 2017, 139, 1440-1443. (6) Pelmenschikov, V.; Birrell, J. A.; Pham, C. C.; Mishra, N.; Wang, H.; Sommer, C.; Reijerse, E.; Richers, C. P.; Tamasaku, K.; Yoda, Y.; Rauchfuss, T. B.; Lubitz, W.; Cramer, S. P. J. Am. Chem. Soc. 2017, 139, 16894-16902. (7) Reijerse, E. J.; Pham, C. C.; Pelmenschikov, V.; GilbertWilson, R.; Adamska-Venkatesh, A.; Siebel, J. F.; Gee, L. B.; Yoda, Y.; Tamasaku, K.; Lubitz, W.; Rauchfuss, T. B.; Cramer, S. P. J. Am. Chem. Soc. 2017, 139, 4306-4309. (8) Schilter, D.; Camara, J. M.; Huynh, M. T.; Hammes-Schiffer, S.; Rauchfuss, T. B. Chem. Rev. 2016, 116, 8693-8749. (9) Ezzaher, S.; Capon, J. F.; Gloaguen, F.; Petillon, F. Y.; Schollhammer, P.; Talarmin, J. Inorg Chem 2007, 46, 3426-3428. (10) Barton, B. E.; Rauchfuss, T. B. Inorg. Chem. 2008, 47, 22612263. (11) van der Vlugt, J. I.; Rauchfuss, T. B.; Whaley, C. M.; Wilson, S. R. J. Am. Chem. Soc. 2005, 127, 16012-16013. (12) Barton, B. E.; Zampella, G.; Justice, A. K.; De Gioia, L.; Rauchfuss, T. B.; Wilson, S. R. Dalton T 2010, 39, 3011-3019. (13) Carroll, M. E.; Barton, B. E.; Rauchfuss, T. B.; Carroll, P. J. J. Am. Chem. Soc. 2012, 134, 18843-18852. (14) Zaffaroni, R.; Rauchfuss, T. B.; Gray, D. L.; De Gioia, L.; Zampella, G. J. Am. Chem. Soc. 2012, 134, 19260-19269. (15) Wang, W. G.; Rauchfuss, T. B.; Zhu, L. Y.; Zampella, G. J. Am. Chem. Soc. 2014, 136, 5773-5782. (16) Greif, A. H.; Hrobarik, P.; Kaupp, M. Chem-Eur J 2017, 23, 9790-9803.

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(17) Hrobarik, P.; Hrobarikova, V.; Meier, F.; Repisky, M.; Komorovsky, S.; Kaupp, M. J. Phys. Chem. A 2011, 115, 56545659. (18) Mulder, D. W.; Guo, Y. S.; Ratzloff, M. W.; King, P. W. J. Am. Chem. Soc. 2017, 139, 83-86. (19) Winkler, M.; Senger, M.; Duan, J. F.; Esselborn, J.; Wittkamp, F.; Hofmann, E.; Apfel, U. P.; Stripp, S. T.; Happe, T. Nat. Commun. 2017, 8, 16115. (20) Mulder, D. W.; Ratzloff, M. W.; Bruschi, M.; Greco, C.; Koonce, E.; Peters, J. W.; King, P. W. J. Am. Chem. Soc. 2014, 136, 15394-15402. (21) Finkelmann, A. R.; Stiebritz, M. T.; Reiher, M. Chem. Sci. 2014, 5, 215-221. (22) Rumpel, S.; Ravera, E.; Sommer, C.; Reijerse, E. J.; Farès, C.; Luchinat, C.; Lubitz, W. J. Am. Chem. Soc. 2017, 140, 131134. (23) Adamska, A.; Silakov, A.; Lambertz, C.; Rüdiger, O.; Happe, T.; Reijerse, E.; Lubitz, W. Angew. Chem. Int. Ed. 2012, 51, 11458-11462. (24) Kaesz, H. D.; Saillant, R. B. Chem. Rev. 1972, 72, 231-281. (25) Bertini, I.; Briganti, F.; Luchinat, C.; Scozzafava, A.; Sola, M. J. Am. Chem. Soc. 1991, 113, 1237-1245. (26) Banci, L.; Bertini, I.; Luchinat, C.; Pierattelli, R.; Shokhirev, N. V.; Walker, F. A. J. Am. Chem. Soc. 1998, 120, 8472-8479. (27) Luchinat, E.; Banci, L. J. Biol. Chem. 2016, 291, 37763784. (28) Mészáros, L. S.; Németh, B.; Esmieu, C.; Ceccaldi, P.; Berggren, G. Angew. Chem. Int. Ed. 2018, doi 10.1002/anie.201710740

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