Active Site of the NAD+-Reducing Hydrogenase ... - ACS Publications

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Active Site of the NAD+‑Reducing Hydrogenase from Ralstonia eutropha Studied by EPR Spectroscopy Julia Löwenstein,† Lars Lauterbach,‡ Christian Teutloff,† Oliver Lenz,‡ and Robert Bittl*,† †

Fachbereich Physik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: Pulsed ENDOR and HYSCORE measurements were carried out to characterize the active site of the oxygen-tolerant NAD+-reducing hydrogenase of Ralstonia eutropha. The catalytically active Nia-C state exhibits a bridging hydride between iron and nickel in the active site, which is photodissociated upon illumination. Its hyperfine coupling is comparable to that of standard hydrogenases. In addition, a histidine residue could be identified, which shows hyperfine and nuclear quadrupole parameters in significant variance from comparable histidine residues that are conserved in standard [NiFe] hydrogenases, and might be related to the O2 tolerance of the enzyme.

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INTRODUCTION Hydrogenase enzymes are found in many microorganisms that use dihydrogen as an energy source by catalyzing the reversible heterolytic cleavage of this small molecule: H2 ⇌ H+ + H−.1 Hydrogenases are metalloproteins that can be classified with respect to their metal content: two-iron containing [FeFe] hydrogenases, [Fe] hydrogenases carrying an iron-guanylylpyridinol cofactor, and the heterobimetallic [NiFe] hydrogenases.2 Most [NiFe] hydrogenases are inactivated even by traces of O2, while all hydrogenases of the chemolithoautotrophic βproteobacterium Ralstonia eutropha H16 (Re) are O2 tolerant, i.e., able to convert hydrogen also in the presence of oxygen. The inactivation of standard hydrogenases by O2 is caused by the binding of oxygen species to the catalytically active [NiFe] site. For the H2-sensing regulatory hydrogenase (RH), a narrow gas channel is proposed to obstruct O2 approach to the catalytic site,3,4 and thus enabling O2 tolerance. In the periplasmic membrane-bound hydrogenase (MBH), redox dependent morphing of the proximal cluster near the active site is supposed to contribute to reductive removal of oxygen species from the catalytic site,5,6 thereby avoiding enzyme inactivation. Contrary to the situation in RH and MBH, the O2 protection mechanism of the cytoplasmic NAD+-reducing soluble hydrogenase (SH) is still unresolved.7 The enzyme is able to catalyze H2 oxidation under atmospheric O2 concentration,8 and a recent study9 demonstrated that the mechanism of its O2 tolerance relies on its capability to continuously reduce O2 to water and hydrogen peroxide. This capacity might be related to reversible oxygenations of the active site cysteine residues.10 The SH couples H2 oxidation with NAD+ reduction, providing the cell with reducant in the form of NADH.11,12 It is mainly composed of a heterodimeric hydrogenase module, carrying the catalytically active [NiFe] center as well as a [FeS] center, together with a NAD+-reductase heterodimer, containing [FeS] centers and FMN (Figure 1). The SH shares © XXXX American Chemical Society

similarities with the MBH and RH, especially with respect to the active [NiFe] site, which is coordinated by the standard ligand set of one carbonyl and two cyanides.13 Additionally, coordination to the protein matrix by four cysteinyl thiolates, two of which serve as bridging ligands between the nickel and the iron, is proposed.9 The catalytically active center undergoes several redox states in both O2-sensitive and O2-tolerant hydrogenases. It is interesting to note that due to the carbonyl and cyanide ligands2,15 the active site iron stays permanently in the Fe(II) low-spin state, and redox transitions occur only at the Ni ion. Oxidizing conditions lead to the oxygen-containing Niu-A and Nir-B species, both involving paramagnetic Ni(III).16 While the Niu-A species is absent in the O2-tolerant hydrogenases under physiological conditions, the Nir-B state has been observed in the O2-tolerant MBH. Fully oxidized SH shows no significant EPR (electron paramagnetic resonance) signal of these species, but exhibits the CO and CN stretching frequencies of a Nir-Blike state.10,13 A one-electron reduction results in a diamagnetic, EPR-silent, intermediate state, typically designated as Nia-S. Further reduction by one electron results in the catalytically active Nia-C state, which features a bridging hydride (H−) between Ni and Fe3,17−21 (Figure 1). The Nia-C state can be converted by illumination to the Nia-L state (formally Ni(I)), in which the hydride is dissociated. The relevance of the Nia-L state as catalytic intermediate is a subject of current research.22−24 Finally, several diamagnetic ready states are present, designated Nia-SR.10 Special Issue: Wolfgang Lubitz Festschrift Received: April 30, 2015 Revised: July 11, 2015

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DOI: 10.1021/acs.jpcb.5b04144 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. Composition of the SH (upper left) and intermediates of the proposed catalytic cycle (formal redox states of the metal ions are indicated, adapted from Ogata et al.14). The active site images framed in red also contain the relative position of histidine residue 69. A potential hydrogen bond between His69 and one of the bridging cysteine residues is depicted as a dashed line.

Beyond the first coordination sphere, is the environment comparable to standard hydrogenases? As both Nia-C and Nia-L are paramagnetic and detectable in considerable amounts, they are accessible to different electron paramagnetic resonance (EPR) methods like electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. These techniques provide a tool to examine the surroundings of the catalytic site by monitoring hyperfine couplings between the paramagnetic state and nuclei in its surroundings, e.g., protons or nitrogens. Due to the different gyromagnetic ratios of protons and deuterons, buffer exchange provides direct access to distinguish contributions of exchangeable and nonexchangeable protons. This approach has been successfully used to characterize the Nia-C and Nia-L states in the RH.3 The bridging hydride in Nia-C can be exchanged by 2H− and readily be distinguished from the nonexchangeable covalently bound protons in the environment of the [NiFe] site. On the basis of the previously established fingerprints,13,25,27 in the present study we performed a more detailed characterization of the Nia-C state and the light-induced Nia-L state in the SH. We aimed at elucidation of whether the catalytically active Nia-C state of the O2-tolerant SH differs from that of O2sensitive standard hydrogenases in terms of electronic and geometric structure. Via ENDOR and HYSCORE we therefore gathered data on the proposed bridging hydride in Nia-C of SH and its photodissociation. Furthermore, a histidine residue near the active site was characterized by HYSCORE. In different hydrogenases except regulatory ones,29 such a histidine residue is part of a conserved motif, designated 2L or element 2,30,31 and forms a hydrogen bond to one of the bridging cysteines of the catalytic center.

During H2 conversion, the SH seems to pass through the same active site redox states that have been described for other [NiFe] hydrogenases, including O2-sensitive ones (Figure 1). H2 is believed to be incorporated into the vacant bridge between the Ni and Fe ions of the active site in the Nia-S state, thereby forming the Nia-SR state, which has recently be shown to harbor a bridging hydride and a protonated terminal cysteine.14 The withdrawal of an electron and a proton leads to the formation of the Nia-C state, where the Ni resides in the paramagnetic Ni(III) state. The release of a further electron and a proton closes the cycle and results in the initial Nia-S state (Figure 1). So far, few EPR investigation on the active site of the SH have been successfully performed over the past decades. Erkens et al.25 showed the existence of Nia-C and Nia-L EPR signals at mildly reducing conditions (NADH) and found a relatively small redox potential window of −290 to −325 mV in which these species occurred. For the Desulfovibrio vulgaris Miyazaki F hydrogenase, for example, these states were found to occur in the relatively large range between approximately −300 and −500 mV.26 Van der Linden et al.27 corroborated the existence of the Nia-C signal and found varying amounts in different preparations. Horch et al.13 performed EPR and Fourier transform infrared (FTIR) measurements on whole cells. Besides the confirmation of Nia-C and Nia-L as states in the native environment by in situ EPR, EPR-silent states were found via FTIR spectroscopy, revealing one Nir-B-like state and different Nia-SR states. Karstens et al.28 investigated the impact of replacing cysteine and tryptophan residues near the [4Fe4S] cluster in the SH hydrogenase module. Together with a reduction of catalytic activity a decrease of the Nia-C signal intensity could be observed. Additionally, traces of an EPR signal belonging to a Nir-B species were found in the oxidized enzyme. Magnetic coupling of Ni(III) to a high-potential cluster or nickel remaining in the Ni(II) state are under discussion as reasons for the predominant absence of Nir-B-like EPR signals. Several questions concerning the active site of the SH remain. Does the enzyme perform the same catalytic cycle as other hydrogenases? Does it carry the same bridging ligands?

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EXPERIMENTAL DETAILS Sample Preparation. The SH was purified as described previously.9 For solvent exchange (1H2O to 2H2O) the sample was diluted 5-fold with 50 mM KPO4 in 2H2O (pD 7.0), 5% glycerol. Thereafter, the sample was concentrated by ultrafiltration (Millipore Amicon Ultra-0.5 centrifugal filter concentrators) to the starting volume. The cycle was repeated 4 times to obtain a final concentration of >99% 2H2O. The control B


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The Journal of Physical Chemistry B sample was treated identically, but washed in 1H2O buffer instead. The protein solution (70 μM) was anaerobically reduced with 120 mM NADH under anaerobic conditions and subsequently transferred to EPR quartz capillaries. Finally, the samples were rapidly frozen in liquid nitrogen. Spectroscopy. Continuous-wave (cw) EPR measurements were performed on a home-built X-band spectrometer consisting of a Bruker (Rheinstetten, Germany) ER 041 MR microwave bridge controlled by a Bruker ER 048 R microwave bridge controller, a Bruker E088 100-controlled AEG electromagnet, and a Bruker 4122 SHQE-W1 microwave resonator. Lock-in amplification was done by a Stanford Research Systems SR810 DSP Lock-In Amplifier and the microwave frequency measured by an Agilent 53181A Frequency Counter. Cryogenic temperatures were achieved using an ESR 910 cryostat and regulated by an ITC503S temperature controller, both from Oxford Instruments. Cw spectra were measured at T = 10 K with Pmw = 2 mW, modulation frequency = 100 kHz, and amplitude = 1 mT. The microwave frequency was 9.388 75 and 9.389 15 GHz for the dark-adapted and illuminated sample, respectively. All spectra were normalized to 9.4 GHz for comparability. Q-band pulsed ENDOR studies were carried out on a Bruker BioSpin Elexsys E580 spectrometer equipped with a SuperQFT-U microwave bridge and a home-built Q-band ENDOR resonator. Low temperatures were achieved by a CF935 cryostat, regulated by an ITC503S temperature controller, both from Oxford Instruments. ENDOR experiments were recorded with a Bruker DICE ENDOR accessory and an Amplifier Research 250A250A RF amplifier. The ENDOR spectra were obtained in stochastic mode by a Davies-ENDOR pulse sequence πmw,1 − πrf − (π/2)mw,2 − τ − πmw,2, with the pulse lengths being πmw,1 = 128 ns, πmw,2 = 40 ns, and πrf = 20 μs. The experimental conditions were the following: T = 10 K, shot repetition time (srt) 2 ms, stochastic mode. For X-band HYSCORE spectroscopy a Bruker BioSpin Elexsys E680 spectrometer was employed using a dielectric ring resonator (Bruker ER 4118X-MD4-EN3). The microwave was amplified with a TWT (Applied Systems Engineering, Inc., model 117X) with 1 kW output power. The spectra were recorded at a microwave frequency of 9.742 GHz, employing the HYSCORE pulse sequence π/2 − τ − π/2 − T1 − π − T2 − π/2 with π/2 = 16 ns, π = 30 ns, 127 increments of T1 and T2 of 30 ns starting at 200 and 300 ns for T1 and T2, respectively, τ = 180 ns. A 16-step phase cycle was applied. Measurement conditions were T = 10 K, for the dark-adapted samples srt = 4 ms, and for the illuminated one srt = 2 ms. The HYSCORE baseline-corrected time traces were subjected to Hamming-window apodization, zero-filling, and fast Fourier transformation. Both ENDOR and HYSCORE spectra were simulated using the MATLAB (R2012b, The MathWorks) toolbox EasySpin (version 4.5.0).32,33

Figure 2. X-band cw EPR of Re SH. Solid lines: experimental data. Dashed lines: simulations. Black traces: spectrum before illumination and simulation of Nia-C state. Blue: spectrum after illumination and simulation of Nia-L. Gray traces: simulations of [2Fe2S] cluster and FMN.

those reported earlier.27,28 The feature at ≈362 mT (g = 1.86) might indicate another [FeS] cluster,34 possibly one of the [4Fe4S] clusters.28 The simulation of [NiFe] in the Nia-C state is depicted below the experimental spectrum as a black dashed line. The Nia-C state is characterized by a rhombic g-matrix g = (2.208, 2.139, 2.016), which is in good agreement with that of Nia-C in other [NiFe] hydrogenases. For those, DFT calculations revealed that this Ni(III) state can be described by an electronic configuration, where the unpaired electron occupies the dz2 orbital of nickel.3,19,29,35 Notably, for the SH we did not observe a splitting of the NiaC signal. In other hydrogenases, such splitting is usually observed at below T = 20 K, and it is characteristic for magnetic interaction between the [NiFe] active site and the iron−sulfur cluster in proximal position to the catalytic center.21,36,37 The absence of a split Nia-C signal can be explained by either an extraordinarily low redox potential of the proposed proximal [4Fe4S] cluster38 or a general diamagnetic character of this cofactor.28,39 Upon white light illumination at 80 K the Nia-C signal is replaced by spectral components with g-values at g = (2.281, 2.105, 2.051) (Figure 2, blue traces). This new feature is generally referred to as Nia-L and has been found in several [NiFe] hydrogenases.20,21,37 The generation of the Nia-L state is interpreted as the light-initiated oxidative splitting of the bridging hydride in the form of a proton, while the two electrons presumably reduce the nickel to Ni(I).35,40 In the course of dark adaption at elevated temperatures (200 K), the SH active site returns to the Nia-C state. A buffer exchange using 2H2O as solvent had no visible effect on the EPR field spectra (Supporting Information Figure 1). Therefore, hyperfine spectroscopy was applied in the following. ENDOR. To probe hyperfine couplings of protons in the environment of the [NiFe] center, pulsed Q-band 1H Davies ENDOR at the gy position of Nia-C was performed (Figure 3). Compared to lower frequency bands, Q-Band ENDOR provides higher sensitivity and a better separation of different nuclei. Due to the large 1H Larmor frequency of νH ≈ 48 MHz at Q-band (34 GHz), the weak coupling limit is fulfilled in most cases; i.e., each proton (I = 1/2) gives rise to two ENDOR lines situated symmetrically around the Larmor frequency of 1H, according to νH ± A/2. Small hyperfine couplings close to the Larmor frequency are due to remote protons that are only

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RESULTS EPR. The 10 K cw-EPR spectra of frozen solution of NADHreduced SH are shown in Figure 2. The black trace indicates the spectrum of the dark-adapted sample. It represents the hydrogenase in the Nia-C state. The spectrum is a superposition of signals from the catalytcally active [NiFe], a FMN, and a [FeS] species. Simulations of a semiquinone form of FMN with g = 2.004 and a [2Fe2S] cluster with g = (2.050, 1.961, 1.927), both partly overlapping with gz of the respective Ni species, are given at the top in Figure 2. These signals are comparable with C


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hyperfine coupling with Ax = 17.5 MHz. This option is depicted as trace b2 in Figure 3. Narrowing down the extent of the exchangeable protons Ax via ENDOR is quite difficult due to the large overlap with several β-protons. These can span a wide range of hyperfine couplings, especially in combination with their orientation toward the g-matrix.3,41−43 Orientation selective ENDOR measurements could presumably resolve this problem. In our case, however, the low EPR signal intensity at gx and gz as well as the overlapping [2Fe2S] cluster signal at gz set limitations to this technique (compare Supporting Information Figures 1 and 2). HYSCORE. To remove the ambiguity in the third hyperfine component of the exchangeable proton, detection of 2H in its own frequency regime is the approach of choice. 2H Mims ENDOR is a technique suitable for the detection of small hyperfine couplings, but in the case at hand, the hyperfine anisotropy is too large for this method to be feasible. Thus, we used HYSCORE, a method that is complementary to ENDOR and particularly well-suited for hyperfine couplings with large anisotropy and, as a 2D method, provides higher resolution. To disentangle the overlap between exchangeable and nonexchangeable protons in the ENDOR experiment, we performed X-band HYSCORE measurements on both SH sample types. Through replacement of 1H2O by 2H2O, only exchangeable protons should be visible in the low-frequency region typical for deuterium. The corresponding signals are unaffected by hyperfine couplings of other protons that can be found around the proton Larmor frequency (≈14 MHz at X-band). Recorded at the gy position of Nia-C, the HYSCORE of the 2 H2O SH sample exhibits strong signals between 0 and 7.5 MHz, which are arranged around the Larmor frequency of 2H at 2.1 MHz (Figure 4a). Strong features (labeled as I) range from (1.8, 3.0) to (3.0, 1.8) MHz, with wings to (0.5, 3.6) and (3.6, 0.5) MHz. They resemble correlations between the single-

Figure 3. Pulsed 1H Davies ENDOR at Q-band at gy of Nia-C and corresponding simulations (gray traces a, b1 and b2, see text). Black trace: sample in 1H2O buffer. Red trace: sample in 2H2O buffer. Blue trace: difference spectrum between 1H2O and 2H2O spectrum, magnified by a factor of 4 for better visibility. Blue highlights: see text.

weakly coupled to the active site. For a direct interpretation in terms of hyperfine couplings, the nuclear Larmor frequency has been subtracted from the RF frequency in Figure 3; i.e., the spectra are centered at 0 MHz. Also, the spectra were symmetrized. The spectra obtained for the SH in either 1H2O- or 2H2Oexchanged buffer readily revealed exchangeable protons, such as the proposed bridging hydride. At around ±8−10 MHz the 1 H2O-containing SH sample reveals hyperfine signals that are clearly different from those of the SH-2H2O spectrum, as is visible in the difference spectrum (Figure 3, blue trace). The observed signals correspond to a coupling of approximately 20 MHz. In the 2H2O-containing SH sample, the corresponding deuteron hyperfine couplings are missing in the frequency range around νH of protons because they are shifted to lower frequencies due to the scaling factor of the gyromagnetic ratios of γ1H/γ2H = 6.51. Two components of the hyperfine matrix A1H for the exchangeable proton are immediately apparent in the difference spectrum. These are the largest component with Az = −20.5(0.2) MHz obvious from the spectral edges at about ±10 MHz and the central component Ay = −19.0(0.2) MHz from the peak at about ±9.5 MHz. The error margins given in parentheses result from simulations. The third, smallest component can only be defined to a certain range of Ax = 14.5−17.5 MHz (highlighted blue in Figure 3). Two different scenarios can be taken into account: (a) One is a rather isotropic hyperfine coupling for the bridging hydride. In this case, the trough in the 7.5 MHz range could be considered as marking the lower bound of the hyperfine matrix and yields a coupling constant Ax = −16.3 MHz, see Figure 3, trace a. (b) The other is a highly anisotropic hyperfine coupling for the bridging hydride. Here, two features in the difference spectrum might be considered as indicative for the third hyperfine component. (b1) The peak at about 7 MHz, which, taken together with the trough in the range around 7.5 MHz, would indicate a different relative sign of the last component relative to the other two components, and a coupling constant Ax = 14.5 MHz (Figure 3, trace b1). (b2) Alternatively, the weak local maximum at about 8.7 MHz might be an indication for the third matrix component resulting in the most anisotropic

Figure 4. X-band HYSCORE. Left side: Sample in 2H2O buffer before [(a) on gy of Nia-C] and after [(b) on gy of Nia-L] illumination. (c) Sample in 1H2O buffer, on gy of Nia-C. Right side: (d−f) corresponding simulations. For explanation of roman numbers and asterisks, see text. D


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to a nitrogen hyperfine coupling of AN = (1.35, 0.95, 0.65) ± (0.05, 0.10, 0.20) MHz, with the quadrupole parameters being e2Qq/h = 1.65−1.75 MHz and η = 0.8−1. The corresponding simulation is shown in Figure 4f. As pointed out in previous studies42,48 and in agreement with our simulation, the appearance of only the double-quantum transitions between mS manifolds is indicative of a nitrogen for which the exact cancellation condition |A| = 2νN is not fulfilled. Comparison with literature data revealed that the parameters are comparable to those of a histidine-derived terminal imidazole nitrogen forming a hydrogen bond to the sulfur of one of the active site coordinating cysteines49 (see Figure 1 and Table 2).

quantum transitions of the two mS spin manifolds. Beginning near the distinct diagonal signal at 3.2 MHz, weaker cross-peaks (II) spread to (2.8, 4.8) and (4.8, 2.8) MHz, continuing weaker between (2.3, 5.6) to (0.9, 7.0) and (5.6, 2.3) to (7.0, 0.9) MHz, respectively. These signals correspond to a singlequantum transition in one mS manifold correlated to the double-quantum transition in the other mS manifold. A third, less prominent component (labeled as III) is present connecting (3.6, 6.4) with (6.4, 3.6) MHz and mirrors the correlations between the double-quantum transitions of both mS manifolds. Simulations of these 2H2O specific signals of the nonilluminated sample (Figure 4d) show that all essential features can be reproduced with only one hyperfine matrix A2H = (2.4, -2.9, -3.1) ± 0.1 MHz. It is in agreement with previously reported hyperfine couplings for the exchangeable proton in Nia-C of hydrogenases (see Table 1). By simulation, a very

Table 2. Nuclear Quadrupole Interaction Parameters e2Qq/h and η and Isotopic Hyperfine Couplings aiso of Nitrogens Close to the [NiFe] Active Sitea

Table 1. Isotropic Hyperfine Parameters aiso and Anisotropy T for Exchangeable Protons of Nia-C in Different Hydrogenases (in MHz)a H2ase

aiso

Re SH Re RH Aa HaseI Dv Dg

−8.0 −3.5 −1.7 −3.5 −10.7

T 23.5 21.9 14.4 21.9 25.7

−11.0 −7.3 −4.5 −7.3 −11.3

ref −12.5 −14.5 −10.0 −14.5 −14.3

b 3 21 42 17

H2ase

e2Qq/h

η

aiso

ref

Re SH Re RH Q67H Re MBH* Aa HaseI Dv* Dg

1.7 1.79 1.94 1.92 1.90 1.9

0.9 0.53 0.36 0.40 0.37 0.4

0.98 1.7 1.8 1.54 1.57 ≈2

b 29 6 21 46 47

a

Nir-B states are marked by asterisks. All other parameters are derived from the Nia-C state. Values for e2Qq/h and aiso are given in MHz. Abbreviations see Table 1. bThis work.

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For the SH, the values derived by HYSCORE were scaled by γ1H/γ2H = 6.51. Abbreviations: Aa HaseI, Aquifex aeolicus hydrogenase I; Dv, Desulfovibrio vulgaris Miyazaki F; Dg, Desulfovibrio gigas. bThis work, employing Euler angels (12°, 66°, 25°) ± 5° with respect to the g-axes system (see Supporting Information Figure 3). a

DISCUSSION Light Sensitivity and Electronic Structure. The Nia-C state of the NAD+-reducing hydrogenase is sensitive to illumination and thereby converted to Nia-L. The g-values of both states are similar to those in standard hydrogenases. Therefore, we conclude the overall electronic structure of the SHs active site is comparable to standard hydrogenases, too. Our data, obtained on NADH-reduced SH, are in line with experiments done on whole cells13 and support a standard set of diatomic ligands and nickel present in different redox states [Ni(III) for Nia-C and Ni(I) for Nia-L]. Shafaat et al.22 recently discussed Nia-L being a catalytically relevant active species since it is present in some enzymes like Hyd-1 from E. coli without the necessity of explicit illumination.50 In the SH, however, this state can only be trapped upon illumination under cryogenic conditions. Exchangeable Proton. HYSCORE simulations of the dark and light 2H2O as well as the dark 1H2O spectra are in good agreement with the experiments and corroborate the hypothesis of an easily exchangeable proton close to the active site. Regarding the possible simulations of the ENDOR difference spectrum, the parameters corresponding to case b2 are identical with the hyperfine values derived from the 2H HYSCORE, scaled by the 1H to 2H gyromagnetic ratio of 6.51. They agree well with the typical values of a hydride bridging two metals44,45 (Table 1). Case a can be ruled out since the comparatively small anisotropy cannot cause the distinct pattern found for the unilluminated 2H2O HYSCORE (Supporting Information Figure 4). Option b1 can be discarded as Ax is too small. The quadrupole parameters point to a bridging hydride between Fe and Ni. Illumination with white light results in its photodissociation, and subsequent dark adaption leads to the recurrence of the bridging hydride.

small nuclear quadrupole parameter of e2Qq/h ≤ 100 kHz is obtained. This is comparable to the corresponding results obtained for the RH of R. eutropha,3 the [NiFe] hydrogenase of D. vulgaris (Dv) Miyazaki F,42 and the hydrogenase I of A. aeolicus (Aa).21 The quadupole parameters are in the typical range of those for bridging metal hydrides as described by Jarrett et al.44 and Guo et al.45 The features marked by asterisks in Figure 4 are discussed below. Upon illumination the rich signal pattern of the 2H2O sample collapses to a strong signal at the 2H Larmor frequency, as visible from the spectrum taken at the gy position of Nia-L (Figure 4b). Simulation (Figure 4e) results in a very small hyperfine coupling of A < 100 kHz. For a rough estimate of the distance between nickel and hydride, a point-dipole approximation might be appropriate. We assume an anisotropic hyperfine coupling resulting from pure dipole interaction, considering that, in the dark-adapted state, the hyperfine anisotropy of the hydride is already larger than its isotropic value. The approximation indicates a large distance of >4.5 Å between nickel and hydride. This means the hydride has left the bridging position, whereas a Ni−H− distance of 1.6−1.8 Å would be typical.21 Additionally, the HYSCORE spectra at the gy position of NiaC of both the 1H2O- and 2H2O-containing SH samples (Figure 4c,a, respectively) reveal two very weak signals on the diagonal at 1.1 and 1.8 MHz and two strong off-diagonal cross-peaks (marked by asterisks) at (1.85, 3.5) MHz and (3.5, 1.85) MHz. These cross-peaks resemble correlations between the doublequantum transitions of both mS manifolds and can be assigned E


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replacement of His69 as the H-bond donor by His70 might be possible and would explain the unusual quadrupole parameters and the smaller isotropic hyperfine coupling observed here. As noted above, the smaller hyperfine coupling of His69 indicates a larger distance between this residue and the bridging Cys461. This particular structural flexibility might be a prerequisite for the recently proposed reversible sulfoxygenation reactions resulting from H2-cycling in the presence of O2.10 In fact, cysteine sulfoxygenations have been found in a number of enzymes, e.g., nitrile hydratase and NADH peroxidase,55 and in the case of the SH such modifications have already been proposed on the basis of X-ray absorption studies.11 Moreover, investigations on hydrogenase active site model compounds, such as (μ-SRS)[Fe(CO)x]2, led to the conclusion that oxygenation of sulfur species might be related to a reduced oxygen sensitivity, provided that deoxygenation is possible.56,57 Thus, changes beyond the first coordination shell of the SH active site might be involved in reversible chemical and conformational rearrangements at the [NiFe] center inducing O2 tolerance, while key active site elements as the photocleavable bridging hydride remain unaltered.

Comparison with literature values (Table 1) encourages the assumption that the exchangeable proton in Nia-C and its photodissociation represent a conserved motif in different hydrogenases. Pandelia et al.21 have observed a less strongly bound hydride in hydrogenase I from Aquifex aeolicus (HaseI) and discussed potential relevance for the O2 tolerance of certain membranebound hydrogenases. Our simulation for the SH determined hyperfine coupling parameters, which are more comparable to the oxygen-sensitive hydrogenase of D. gigas and clearly larger than those of HaseI from A. aeolicus, especially concerning the anisotropy. Hence, the O2 tolerance of the SH seems not to be related to a comparatively weak binding of the hydride to the [NiFe] site. Nitrogen Coupling. The HYSCORE data for both SH samples additionally revealed signals that can be simulated as a nitrogen with parameters fitting to the protonated Nτ of a histidine residue. A likely candidate is His69 near the active site (Figure 1). This residue is part of motif 2L, which is conserved in a variety of [NiFe] hydrogenases, except for H2-sensing enzymes such as the RH of R. eutropha31 (see Supporting Information Table 1). Notably, the simulation of our experiment yields quadrupole parameters that significantly differ from those for Nτ observed for the corresponding histidines in [NiFe] hydrogenases (e2Qq/ h ≈ 1.9 MHz and η ≈ 0.4) (Table 2). An exception is the Q67H variant of the RH from R. eutropha, for which Buhrke et al.29 found e2Qq/h = 1.79 MHz and η = 0.53, values that are closer to the ones obtained here. On the basis of DFT calculations, a shorter distance of Nτ to the S of the spincarrying cysteine residue was discussed as a possible reason, in line with the rather large observed isotropic hyperfine coupling. However, we find a rather small isotropic hyperfine coupling, indicative of a larger distance between His69 and the spincarrying sulfur of the Cys residue bridging the Ni and the Fe in the catalytic active site. Nuclear quadrupole coupling parameters of an atom are characteristic for its bonding situation to adjacent atoms. Thus, the protonated Nτ and the unprotonated Nπ of a histidine exhibit similar parameters as the corresponding protonated and unprotonated nitrogens in imidazole, respectively. Additional influence is induced by ligands, where, besides donor characteristics, possible charge delocalization and size of the ligand need to be considered.51 In particular, hydrogen bonding effects are predominantly responsible for variations in the quadrupole coupling constant and asymmetry parameter. For imidazole, the change from the gas phase to the crystal phase results in a large shift of the quadrupole parameters of the protonated nitrogens due to hydrogen bonding to unprotonated nitrogens, i.e., e2Qq/h = 2.524 MHz, η = 0.29452 and e2Qq/h = 1.391 MHz, η = 0.930,53 respectively. Apart from the Nτ−H···S bond length, as considered for the Q67H variant of the RH,29 twists and bending of this particular hydrogen bond also might lead to a significant variation of the quadrupole parameters, as described by Fritscher.54 It is noteworthy that the SH harbors two rather bulky residues in place of smaller ones in other hydrogenases. These are an additional histidine at position 70 adjacent to the conserved His69 and a cysteine residue at position 72. The sites are occupied by alanines in other hydrogenases. Since no structure of the SH is available yet, the structural consequences of these two residues remain a matter of debate but could be substantial, and lead to a modification of the His69-Cys461 H-bond. Even a

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CONCLUSION The active site of the SH was investigated by pulsed ENDOR and HYSCORE. We show the presence of a hydride in the active site present in the catalytically relevant Nia-C state. This hydride undergoes photodissociation upon illumination and has a hyperfine coupling comparable to that of standard [NiFe] hydrogenases. Interestingly, the nuclear quadrupole parameters of a histidine residue close to the active site differ considerably from those of other [NiFe] hydrogenases. This might be related to an increased flexibility of the active site, which was recently supposed to be required for the extraordinary O2 tolerance of the SH.10

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b04144. SI Figure 1, Q-band field-swept echo of the SH; SI Figure 2, Davies ENDOR on gx position of Nia-C; SI Figure 3, orientation selective HYSCORE experiments and simulations including Euler angles; SI Figure 4, HYSCORE simulation for case a; SI Table 1, sequence alignment of different hydrogenases (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Cluster of Excellence UniCat (DFG EXC-314) is gratefully acknowledged. REFERENCES

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