Synthetic Active Site Model of the [NiFeSe] Hydrogenase

A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe(‘S2Se2’)(CO)3] (H2‘S2Se2’=1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni(‘S2Se2’)] with [Fe(CO)3bda] (bda=benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe(‘S2Se2’)(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe(‘S2Se2’)(CO)3] with the previously reported thiolate analogue [NiFe(‘S4’)(CO)3] (H2‘S4’=H2xbsms=1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe(‘S2Se2’)(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe(‘S2Se2’)(CO)3] and [NiFe(‘S4’)(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution.


Introduction
The depletion of fossil fuel reserves, the increasing levels of at-mosphericC O 2 ,a nd the need for energy security drive the development of new approaches to produce ar enewable energy vector such as H 2 . [1] Inexpensive,s table, and efficient H 2 generation catalysts are needed to produce sustainable H 2 from water in the long term. [2] Hydrogenases are reversible H 2 productionc atalysts and display remarkably high turnover frequencies of over 10 3 s À1 at as mall overpotential. [3] This incredible activity is achieved using the abundant metalsn ickel and iron in the hydrogenasea ctives ite. [4] [NiFeSe] hydrogenases are as ubclass of the [NiFe] hydrogenases, where as elenocysteine (Sec) residuei st erminally coordinated to the nickel center instead of ac ysteine (Cys) in the enzymea ctive site ( Figure 1). [5] [NiFeSe] hydrogenases have emerged as particularly suitable catalysts for H 2 evolution, [6] because they exhibit high catalytic activities for H 2 generation in the presence of H 2 and fast reactivation from O 2 inactivation when compared with other hydrogenases. [7] These advantageous properties make [NiFeSe] hydrogenases attractive for use in H 2 Os plitting sys-tems, and have allowed for their exploitation in an umber of efficient photocatalytic H 2 production schemes. [7e, 8] As with other selenium containing enzymes, [9] it is stillu nclear what role seleniump lays in the [NiFeSe] hydrogenases. [6] The Sec residue may affect the electronic and steric properties of the bimetallic core at the active site. Crystallographic evidence suggestst hat the Sec residue in the [NiFeSe] hydrogenase behaves as ap roton relay during catalytic H 2 cycling, carrying protons to andf rom the active site. [5] It is the Cys residue in the same positioni nt he [NiFe] hydrogenase that was proposed to be the proton relay ( Figure 1). [10] The unique reactivity of the [NiFeSe] hydrogenase with O 2 may be the reason for its fast reactivation from O 2 inactivation.W hen ac onventional [NiFe] hydrogenase reacts with O 2 ,t he nickel center is oxidized to nickel(III)a nd an oxygen containingl igand takes the bridging position between the nickel andt he iron centers. [11] In the O 2 oxidized [NiFeSe] hydrogenase, however,t he nickel center is not oxidized and no bridging ligand is observed between the two metal centers. [7g, 12] Crystallographic evidence suggests that it is the Sec seleniuma nd, in some cases, Cys sulfur that is oxidizedi n [ NiFeSe] hydrogenases. [12c,d, 13] It has been well-established that the protein structure surrounding an active site affects the reactivity of an enzyme, [14] and biomimetic molecules can be employed to learn about the structural and functional properties of the active site. [15] Our aim is to explore the effect of selenium on the enzyme active site using small molecule model chemistry.

Structural characterization
Single crystals of [Ni('S 2 Se 2 ')] were grown by liquid diffusion of hexane into as aturated dichloromethane solution of the complex and the X-ray crystal structure is shown in Figure 3A. Complex [Ni('S 2 Se 2 ')] crystallizes in the space group P2 1 /n with two crystallographically independent molecules per asymmetric unit. Selected distances and angles for [Ni('S 2 Se 2 ')] and the previously reported [Ni('S 4 ')] [17a] are summarized in Table 1.  Crystald ata and refinement details for [Ni('S 2 Se 2 ')] are given in the Supporting Information, Ta ble S1. [Ni('S 2 Se 2 ')] contains nickel(II) coordinated to two selenolate and two thioether donors with square-planar geometry around the nickel center. The bond distances are as expected:the nickel selenolate distances of 2.295(8) i n[ Ni('S 2 Se 2 ')] are longer than the nickel thiolate distances of 2.184(3) i n[ Ni('S 4 ')], [17a] as observed with other nickel thiolate/selenolate complexes. [18] The five-membered rings in [Ni('S 2 Se 2 ')] constrain the Se-Ni-S angles negligibly to 89.3(4)8 and the seven-membered ring of the xylenediyl group pushes the S1-Ni-S2 angle to 99.4(2)8 . There is as mall tetrahedral distortion from the square plane around the nickel center with an angle of 11.1 AE18 between the planesS e1-Ni-S1 and Se2-Ni-S2. The corresponding tetrahedral distortion in [Ni('S 4 ')] is smaller at 4.31 AE 48. [17a] Single crystals of [NiFe('S 2 Se 2 ')(CO) 3 ]w ere grown from liquid diffusion of pentane into as aturated dichloromethane solution of the complex. The complex crystallizes in the space group P2 1 /n with ad isordered pentanes olvent molecule in half occupancy (Experimental Section and Supporting Information, Ta ble S1). The X-ray crystal structure of [NiFe('S 2 Se 2 ')(CO) 3 ]i s shown in Figure 3B and it is structurally similar to the previously reported [NiFe('S 4 ')(CO) 3 ]. [15d] Selected bond distances and angles for [NiFe('S 2 Se 2 ')(CO) 3 ]a nd [NiFe('S 4 ')(CO) 3 ] [15d] are shown in Table 2. One of the 'S 2 Se 2 's elenolate donor ligands bridges the nickel and the iron centers, whereas the other remains terminallyc oordinated to the nickel center. One of the three carbonyl ligands takes up ab ridging positionb etween the nickel and the iron centers. One of the 'S 2 Se 2 't hioether donors has become uncoordinated from the nickel center and coordinates insteadt oi ron whilst the other remains coordinated to nickel.T he coordination geometry around the nickel center is distorted tetrahedral and around the iron centeri s square-based pyramidal.
The catalytic activity of the complexes was thus assessed using DMFa sas olvent as the pKa of many organic acids in DMF is significantly higher than in acetonitrile. [24] No catalytic current enhancement was detected using [NiFe('S 2 Se 2 ')(CO) 3 ]o r [NiFe('S 4 ')(CO) 3 ]i nD MF in the presence of acetic acid or benzoic acid, but with the stronger TFA, H 2 production activity was observed (Supporting Information, Figure S20). Ac atalytic wave appeared,w hich showed an increase in current with increasinga cid concentrations, whereas voltammograms on ab are glassy carbon electrode under the same conditions with no complex gave negligible current enhancement (Supporting Information, Figure S21).
However,t he catalytic response does not result from homogeneous catalysis, but as olid deposit on the electrode surface formed by the electrodeposition of either [NiFe('S 2 Se 2 ')(CO) 3 ]o r [NiFe('S 4 ')(CO) 3 ]. Following cyclic voltammetry of the complex in DMFw ith increasing concentrations of TFA( up to 100 mm; Supporting Information, Figure S20) the workinge lectrode was removed from the solution and rinsed with DMF.This electrode was then placed in af resh electrolyte solution (rinse test) containing 100 mm of TFAw ithout any NiFe complex in solution and the same catalytic response was observed as with the complex in solution (Figure 7; SupportingI nformation, Figure S22). Thes tabilityo ft he complexes in TFA/DMF solution in the absence of an applied potential was established using electronic absorption spectroscopy,c onfirming that as olid deposit is formed through electrodeposition (Supporting Information, Figure S23).
Ac omparable catalyst precursor activity was also observed for our previously reported mononuclear nickel thiolate/selenolate complexes. [18] Deposition of ag rowing number of firstrow transition-metal complexes onto electrodes is being reported and the nature of the precursor complex affects the morphology and activity of the resulting heterogeneousc atalyst. [2d, 18, 25] Thus, the composition of the deposit from [Ni-Fe('S 2 Se 2 ')(CO) 3 ]a nd [NiFe('S 4 ')(CO) 3 ]w as characterized to determine the nature of the catalytic species. Ag lassy carbon slide with as urfacea rea of 1.6 cm 2 was modified with the deposit through electrodeposition from as olution of [Ni-Fe('S 2 Se 2 ')(CO) 3 ]o r[ NiFe('S 4 ')(CO) 3 ]( 1mm)i nt he presence of TFA( 10 mm)i nD MF at E appl = À1.75 Vv s. Fc + /Fc for 0.5 h. The modified electrode was then removed from the solution and rinsed with DMF (3 mL) before analysis. Scanning electron microscopy (SEM) analysiso ft he slides treated with either [Ni-Fe('S 2 Se 2 ')(CO) 3 ]o r[ NiFe('S 4 ')(CO) 3 ]r evealed that in both cases the electrode is entirely covered in af ilm of the deposit (Figure 8). Energy-dispersive X-ray spectroscopy (EDX) analysis confirmed that both films consist mainly of nickel and iron ( Figure 8; Supporting Information, Table S3). There is sulfur (4 atom %) and selenium (16 atom %) in the film deposited from [NiFe('S 2 Se 2 ')(CO) 3 ]a nd sulfur (13 atom %) in the film depositedfrom [NiFe('S 4 ')(CO) 3 ]. Low levels of sulfur and selenium rule out the possibility that the bulk of the film material is am etal sulfide or metal selenide. Surface analysisb yX -ray photoelectron spectroscopy (XPS) confirmed that the film sur-  Chem. Eur.J.2015, 21,8096 -8104 www.chemeurj.org face is mostly comprised of nickel and iron, with small amountso fs ulfur and/or selenium (Supporting Information, Ta ble S3). The Ni 2p signals in the XPS spectra of both deposits at 874 and8 56 eV with satellites at 880 and 862 eV correspond to Ni(OH) 2 (Supporting Information, Figure S24). The surfaces were exposed to air before analysiss oi ti sr easonable to assume that significant surface oxidation occurred. As mall nickel(0) peak is visible in the Ni 2p spectrumo ft he deposit from [NiFe('S 2 Se 2 ')(CO) 3 ]a t8 52 eV,which is possibly the catalytically active species. The Fe 2p signals in both deposits at 711 and 724 eV with satellites at 719 and 732 eV show that it is in the form of iron oxide and no resolvable iron(0) signal is observed.
The deposits from [NiFe('S 2 Se 2 ')(CO) 3 ]a nd [NiFe('S 4 ')(CO) 3 ]o n ag lassy carbon disk or fluorine-doped tin oxide (FTO) electrode (electrodeposited at À1.75 Vv s. Fc + /Fc for 0.5 hi n a1m msolution of the complex in DMFc ontaining 10 mm TFA) werea lso shown to be electroactive for H 2 evolutioni n an aqueous pH neutral phosphate solution (Supporting Information, Figure S25). The deposits show comparable activity to other nickel containing H 2 production catalyst films formed from molecular precursors recently reported. [18, 25d] Controlled potentiale lectrolysis of such films on an FTO electrode with asurface area of 1.6 cm 2 confirmed the generation of H 2 (headspace gas chromatography analysis).

Conclusion
As ynthetic structuralm odel of the [NiFeSe] hydrogenase active site has been reported. The complex was synthesized using the nickel precursor complex [Ni('S 2 Se 2 ')],i nw hich the nickel center is surrounded by two selenolate and two thioether donors. Complex [NiFe('S 2 Se 2 ')(CO) 3 ]m imics several of the main structural features of the enzyme active site, including one nickel and one iron centerh eldt ogether by two bridging ligandsa nd as elenolate donort erminally coordinated to the nickel center. Relevant distances anda ngles in [Ni-Fe('S 2 Se 2 ')(CO) 3 ]a gree well with those found in the enzyme. The nickel-selenium distance is 0.1 l ongera nd the nickeliron distance in [NiFe('S 2 Se 2 ')(CO) 3 ]o nly slightly longer than the analogous[ NiFe] hydrogenasem odel complex [NiFe('S 4 ')(CO) 3 ]. The metal-carbonyl bond lengths in the two complexes are almosti dentical. The differences in the spectroscopicp roperties of [NiFe('S 2 Se 2 ')(CO) 3 ]a nd [NiFe('S 4 ')(CO) 3 ]i llustrate the differences in their electronic structures. IR spectroscopy revealed that the carbonyl bands in [NiFe('S 2 Se 2 ')(CO) 3 ]a re all shifted to lower frequencies relative to [NiFe('S 4 ')(CO) 3 ], indicating that the more electron-donatings elenolate groups offer an increasede lectron density at the Fe center.T he signals in the electronic absorption spectrum of [NiFe('S 2 Se 2 ')(CO) 3 ]a re shifted to lower energies than in [NiFe('S 4 ')(CO) 3 ]. Extensive electrochemicals tudies revealed that both NiFe complexes do not behavea sh omogenous catalysts for H 2 evolution, but are molecular precursors for active heterogeneous catalysts, which can be readily electrodeposited onto an electrode surface.
Analysis of the solid deposits shows that thesef ilms contain nickel and iron with some sulfur and selenium. The deposit is electrocatalytically active for proton reduction in organic solvents with acid or aqueous pH neutral phosphate solution.

Materials and methods
All of the complexes were synthesized using anhydrous anaerobic techniques using aSchlenk line unless otherwise noted. All starting materials were purchased from commercial suppliers in the highest available purity for all analytical measurements and used without further purification. Organic solvents were dried and deoxygenated prior to use. Bis(3-chloro-2,2-methyl-1-thiapropyl)-o-xylene and [Ni('S 4 ')], [17a] [Fe(CO) 3 bda], [26] and [NiFe('S 4 ')(CO) 3 ] [15d] have been synthesized using previously reported procedures. Electrochemistrygrade n-Bu 4 NBF 4 electrolyte was purchased from Sigma Aldrich. The glassy carbon electrodes were cleaned by first cycling at positive potentials in 1 m hydrochloric acid using as ilver wire pseudo reference electrode and then polishing using alumina powder (1 mmdiameter).

Physical measurements
NMR spectra were recorded on aB ruker DPX-400 MHz spectrometer and the spectra were referenced against the solvent peak. The mass spectrum of 'S 2 Se 2 ' pre was recorded by the University of Cambridge Mass Spectrometry Service using aB ruker Bio Apex 4.0 FTICR ESI-MS. The mass spectra of the metal complexes were recorded on aW aters Quattro LC electrospray ionization mass spectrometer.E xpected and experimental isotope distributions of the compounds were compared. Elemental analysis was carried out by the microanalysis service of the Department of Chemistry,U niversity of Cambridge. FTIR spectra were recorded on aT hermoscientific Nicolet iS50 FTIR spectrometer with an ATRs ampling accessory. Electronic absorption spectra were recorded on an Agilent Cary UV-Vis 50 Bio spectrometer.T he SEM images and EDX spectra were recorded using aP hilips XL30 132-10 electron microscope. EDX studies (edax PV7760/68 ME) were run at a1 5kVacceleration voltage, spot size 4.0, and an acquisition time of at least 100 s. The elements were assigned and atomic ratios were identified using the built-in software (EDAX). XPS data were obtained at the National EPSRC XPS User's Service (NEXUS) at Newcastle University,U K, an EPSRC Mid-Range Facility.A nalysis was performed using aK aspectrometer (Thermo Scientific, East Grinstead, UK) utilizing am onochromatic Al Ka X-ray source (1486.6 eV,400 mms pot size, 36 W).

X-ray crystallographic studies
Data were recorded with Mo Ka radiation (l = 0.71073 ) on aN onius Kappa CCD diffractometer fitted with an Oxford Cryosystems Cryostream cooling apparatus. The single crystal was mounted in Paratone No il on the tip of ag lass fiber and kept under as tream of N 2 .S tructure solution was carried out using direct methods and refined by least squares (SHELXL-97) [27] using Chebyshev weights on F o 2 .T he weighted R-factor wR and goodness of fit (GOF) are based on F 2 .C rystal data, data collection parameters, and structure refinement details for the complexes are given in the Supporting Information, Ta ble S1. The structure of complex [Ni('S 2 Se 2 ')] contained two crystallographically independent molecules in the asymmetric unit. Ap oorly resolved pentane solvent molecule co-crystallized with [NiFe('S 2 Se 2 ')(CO) 3 ]a nd it was modeled as one half-weight molecule disordered about an inversion center with geometric restraints and ac ommon isotropic displace-ment parameter for the carbon atoms. Selected bond distances and angles are shown in Ta bles 1a nd 2. The mean bond distances and angles for the discussion in the paper were calculated as follows:f or as ample of n observations x i ,aweighted mean value (x u ) with its standard deviation (s)w as calculated using the following equations: x u = S i x i /n, s = {S i (x i Àx u ) 2 /[n(nÀ1)]} 1/2 .C rystal structure images were created using Ortep 3f or Windows. [28] CCDC 1050563 ([Ni('S 2 Se 2 ')]) and CCDC 1050564 ([NiFe('S 2 Se 2 ')(CO) 3 ]·0.5 C 5 H 12 )c ontain the supplementary crystallographic data for this paper.T hese data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Electrochemical measurements
Voltammograms were recorded at room temperature under inert gas using an IviumStat or CompactStat potentiostat. As tandard three-electrode cell was used for all measurements with ag lassy carbon disk working (3 mm diameter), ap latinum mesh counter, and aA g/Ag + (organic solutions) or Ag/AgCl/KCl (sat) (aqueous solutions) reference electrode. For voltammograms recorded in organic solvents containing n-Bu 4 NBF 4 (0.1 m), the Fc + /Fc couple was used as ar eference. For voltammograms recorded in ap H7 aqueous phosphate solution (0.1 m), potentials were converted to the normal hydrogen electrode (NHE) by adding 0.2 Vt ot he potential against Ag/AgCl/KCl (sat) . [29] Unless otherwise stated, the second of consecutive scans is shown, as currents were diffusionl imited on this scan and all subsequent scans were identical.
For deposition of the films for characterization, ag lassy carbon slide (1 cm x1cm x0 .1 cm) was immersed in as olution of [Ni-Fe('S 2 Se 2 ')(CO) 3 ]o r[ NiFe('S 4 ')(CO) 3 ]( 1mm)i nt he presence of TFA (10 mm)i nD MF with n-Bu 4 NBF 4 (0.1 m). An electrode surface area of 1.6 cm 2 was in contact with the electrolyte solution. Ap otential of approximately À1.75 Vv s. Fc + /Fc was applied for 0.5 h. The modified electrode was then removed from the solution and rinsed with DMF (3 mL). Catalytic films for controlled potential electrolysis were deposited from as olution of [NiFe('S 2 Se 2 ')(CO) 3 ]o r[ NiFe('S 4 ')(CO) 3 ]( 1mm)i n the presence of TFA( 10 mm)i nD MF with n-Bu 4 NBF 4 (0.1 m)o n ag lassy carbon or FTO-coated glass electrode (geometric surface area in contact with electrolyte solution of approximately 1.6 cm 2 ) at À1.75 Vv s. Fc + /Fc for 0.5 h. The modified electrode was then removed from the solution, rinsed with DMF (3 mL) and immersed into an aqueous phosphate solution (0.1 m,p H7). Controlled potential electrolysis was carried out in an airtight electrochemical cell containing N 2 with 2% methane as internal standard for gas chromatography (GC) analysis. The headspace gas was analyzed using an Agilent 7890 AG Ce quipped with a5m olecular sieve column, using N 2 carrier gas with af low rate of approximately 3mLmin À1 .T he GC columns were kept at 40 8Ca nd at hermal conductivity detector was used.

Synthesis and characterization
Synthesis of 'S 2 Se 2 ' pre :Asolution of selenourea (1.40 g, 11.4 mmol) in ethanol (25 mL) was added to as olution of bis(3-chloro-2,2methyl-1-thiapropyl)-o-xylene (2.00 g, 5.7 mmol) in ethanol (10 mL) and the colorless solution was refluxed for 30 min, during which time aw hite solid precipitated. The reaction mixture was cooled on ice, the solid product was isolated by filtration, washed with cold ethanol (3 5 mL) and diethyl ether ( (4 mL) was added to as uspension of 'S 2 Se 2 ' pre (200 mg, 335 mmol) in ethanol (4 mL) and the reaction mixture was stirred for 10 min until the white solid had dissolved. The solution was then added to as uspension of [Ni(acac) 2 ]( 86 mg, 335 mmol) in ethanol (20 mL), and the reaction mixture was heated to reflux for 1h,d uring which time ag reen solution had formed. The solvent volume was reduced to 10 mL and the resulting green precipitate was separated by filtration, washed with ethanol (3 3 mL), and dried under vacuum. The product was recrystallized by slow liquid diffusiono fh exane into ad ichloromethane solution of the complex. Yield:1 38 mg, 83 %. 1 (CH 2 Cl 2 ) = 290 nm (e = 17.2 10 3 Lmol À1 cm À1 ). Single crystals for Xray analysis were grown from liquid diffusion of hexane into ad ichloromethane solution of the complex.